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PTP1B Contributes to the Oncogenic Properties of Colon Cancer Cells through Src Activation
Abstract
Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src.
... In addition, it also has been studied that, higher the expression of PTP1B shows higher risk of developing metastasis and lowers the overall survival in CRC [54]. In colon cancer development, it is reported that Src activity is higher in colon cancer and PTP1B promotes Src activity and thus increases oncogenic properties [56]. Thus, targeting PTP1B might result in inhibition of angiogenesis and induction of apoptosis resulting into beneficial effect in CRC. ...
... A study demonstrated that after SOV treatment led to reduction in glycosylated hemoglobin in the diabetic rats [13]. SOV also exhibits anti-tumorigenic role in different cancers except colon cancer like lung cancer [5,17], pancreatic cancer [18,23], thyroid carcinoma [12], breast cancer [56], hepatocellular carcinoma [51] and oral cancer [21]. Although vanadium compound and specifically sodium orthovanadate is reported to have anti-cancer and hypoglycemic effects, but studies with respect to on colon cancer associated with diabetes mellitus is not reported. ...
Background
The presence of type 2 diabetes mellitus increases the risk of developing the colon cancer. The main objective of this study was to determine the role of sodium orthovanadate (SOV) in colon cancer associated with diabetes mellitus by targeting the competitive inhibition of PTP1B.
Methods
For in vivo study, high fat diet with low dose streptozotocin model was used for inducing the diabetes mellitus. Colon cancer was induced by injecting 1,2-dimethylhydrazine (25 mg/kg, sc) twice a week. TNM staging and immunohistochemistry (IHC) was carried out for colon cancer tissues. In vitro studies like MTT assay, clonogenic assay, rhodamine-123 dye assay and annexin V-FITC assay using flow cytometry were performed on HCT-116 cell line. CAM assay was performed to examine the anti-angiogenic effect of the drug.
Results
Sodium orthovanadate reduces the blood glucose level and tumor parameters in the animals. In vitro studies revealed that SOV decreased cell proliferation dose dependently. In addition, SOV induced apoptosis as depicted from rhodamine-123 dye assay and annexin V-FITC assay using flow cytometry as well as p53 IHC staining. SOV showed reduced angiogenesis effect on eggs which was depicted from CAM assay and also from CD34 and E-cadherin IHC staining.
Conclusions
Our data suggest that SOV exhibits protective role in colon cancer associated with diabetes mellitus. SOV exhibits anti-proliferative, anti-angiogenic and apoptotic inducing effects hence can be considered for therapeutic switching in diabetic colon cancer.
... In this regard, PI3K/Akt axis activation induced by insulin/IGFs has been observed to increase the resistance to 5-FU and cycloheximide cytotoxicity in HT29 colon cancer cell line [81]. In addition, a reduction in Src activity, a tyrosine protein kinase, which activates the PI3K/Akt pathway, leads to inhibition of tumor cell proliferation and metastasis in SW48 and HT-29 colon cancer cell lines [84]. In addition, it has been demonstrated that the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF-1R) overexpression in colonic mucosae leads to Src activation that supports the proliferation of human colorectal carcinoma cell lines [85]. ...
... Adipokines Leptin c-Jun, Akt, and JAK/STAT3 axis IL-6, MMPs, and TGF-β ↑ carcinogenesis metastasis [5,14] Adiponectin AMPK axis inhibition of tumor cell proliferation, angiogenesis, and metastasis [5,64,65] Apelin tumor growth and migration pathways lymph node and distant metastasis [75] Gherlin gherlin gene ↓ increase in TME inflammation [58] Resistin Toll-like receptor 4 ↑ IL-6 and TNF-α ↑ promotion of tumor angiogenesis and metastasis [1,76] Visfatin ERK1/2, p38 MAPK, PI3K/mTOR, JNK, and JAK/STAT axis visfatin/SDF-1/Akt axis support of tumor cell proliferation and metastasis inhibition of 5-FU therapeutic effect [79,80] Insulin PI3K/Akt pathway increase in the resistance to 5-FU and cycloheximide cytotoxicity inhibition of tumor cell proliferation and metastasis [81,84] IFGs Src (tyrosine-protein kinase) carcinogenesis proliferation of tumor cells [85,86] VEGF VEGF gene tumor angiogenesis and metastasis [96] IL-6 STAT3 axis support of tumor growth and metastasis [5,88,89] TNF-α NF-kB, Wnt/β-catenin, ERK1/2 axis carcinogenesis and tumor cell migration [90][91][92] PAI-1 PAI1-tPA axis increase in TGF-β expression in colitis CRC carcinogenesis and metastasis (?) [94] CCL2 CCL2 gene increase in TME macrophages infiltration and induction of tumor progression [14] MMP9 and MMP11 MMP genes induction of tumoral extracellular matrix remodeling and metastasis [1,4,5,28] Metabolites Lactate Pyruvate ATP fatty acids oxidation tumor growth and metastasis [5,28] FFAs FFAs receptors (FABPs and CD36) ...
Colorectal cancer (CRC) is one of the most commonly diagnosed types of cancer, especially in obese patients, and the second cause of cancer-related death worldwide. Based on these data, extensive research has been performed over the last decades to decipher the pivotal role of the tumor microenvironment (TME) and its cellular and molecular components in CRC development and progression. In this regard, substantial progress has been made in the identification of cancer-associated adipocytes’ (CAAs) characteristics, considering their active role in the CCR tumor niche, by releasing a panel of metabolites, growth factors, and inflammatory adipokines, which assist the cancer cells’ development. Disposed in the tumor invasion front, CAAs exhibit a fibroblastic-like phenotype and establish a bidirectional molecular dialogue with colorectal tumor cells, which leads to functional changes in both cell types and contributes to tumor progression. CAAs also modulate the antitumor immune cells’ response and promote metabolic reprogramming and chemotherapeutic resistance in colon cancer cells. This review aims to report recent cumulative data regarding the molecular mechanisms of CAAs’ differentiation and their activity spectrum in the TME of CRC. A better understanding of CAAs and the molecular interplay between CAAs and tumor cells will provide insights into tumor biology and may open the perspective of new therapeutic opportunities in CRC patients.
... Insulin and IGF are overexpressed in obese patients, leading to activation of the PI3K/Akt signaling pathway that leads to increased survival and growth of cells, amplifying carcinogenesis [23]. Src is a protein oncogene (protein tyrosine kinase) that increases cell growth, proliferation, survival, and migration [24][25][26]. It has many domains (SH2, SH3, regulatory tails, etc.) in an inactivated form in normal cells, and tends to induce phosphorylation and activation of the PI3K/Akt pathway when activated, promoting the development of CRC [24][25][26]. ...
... Src is a protein oncogene (protein tyrosine kinase) that increases cell growth, proliferation, survival, and migration [24][25][26]. It has many domains (SH2, SH3, regulatory tails, etc.) in an inactivated form in normal cells, and tends to induce phosphorylation and activation of the PI3K/Akt pathway when activated, promoting the development of CRC [24][25][26]. IGF-1 causes cytoplasmic degradation of P53 (a tumor suppressor gene), which leads to uncontrolled cell proliferation and neoplasia [27]. ...
Obesity has played a crucial role in the pathogenesis of various cancers, including colorectal cancer (CRC). Obesity has shown to increase the blood levels of insulin, insulin-like growth factor-1 (IGF-1), leptin, resistin, inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) which in turn acts via various signaling pathways to induce colonic cell proliferation and in turn CRC development. It has been shown that estrogen can prevent and cause CRC based on which receptor it acts. Obese patients have relatively low levels of ghrelin and adiponectin that inhibit cell proliferation which further adds to their risk of developing CRC. Obesity can alter the microbial flora of the gut in such a way as to favor carcinogenesis. Weight loss and good physical activity have been related to a reduced incidence of CRC; obese individuals should be screened for CRC and counseled about the importance of weight reduction, diet, and exercise. The best way of screening is using BMI and waist circumference (WC) to calculate the CRC risk in obese people. This study has reviewed the association between obesity and its pathophysiological association with CRC development.
... Being an important regulator of cell signaling pathways, PTP1B also regulates the activity of kinase cascades associated with carcinogenesis, and, in particular, is a therapeutic target for HER2-positive cancers of the breast [177], lung [185], prostate [186], stomach [187], and colon [188]. PTP1B stimulates ErbB2-induced oncogenesis at the level of Ras/mitogen-activated protein kinase and PI3/protein kinase B signaling pathways. ...
... As monotherapy in animal models of solid tumors [180,186,[188][189][190][191][192][193][194][195]; as monotherapy in phase 1 clinical study for HER-2 positive metastatic breast cancer (NCT02524951) ...
This review comprehensively describes the recent advances in the synthesis and pharmacological evaluation of steroid polyamines squalamine, trodusquemine, ceragenins, claramine, and their diverse analogs and derivatives, with a special focus on their complete synthesis from cholic acids, as well as an antibacterial and antiviral, neuroprotective, antiangiogenic, antitumor, antiobesity and weight-loss activity, antiatherogenic, regenerative, and anxiolytic properties. Trodusquemine is the most-studied small-molecule allosteric PTP1B inhibitor. The discovery of squalamine as the first representative of a previously unknown class of natural antibiotics of animal origin stimulated extensive research of terpenoids (especially triterpenoids) comprising polyamine fragments. During the last decade, this new class of biologically active semisynthetic natural product derivatives demonstrated the possibility to form supramolecular networks, which opens up many possibilities for the use of such structures for drug delivery systems in serum or other body fluids.
... Studies have shown that PTP1B promotes the proliferation of colon cancer, breast cancer, and lung cancer by phosphorylating and activating Src (Zhu et al., 2007;Arias-Romero et al., 2009;Liu et al., 2015), a tyrosine kinase containing an SH3 domain. Activated Src then further activates the downstream Ras/ERK signaling pathway, thereby promoting tumor cell growth and migration (Bollu et al., 2017). ...
... In addition, PTP1B can also promote tumor cell growth, migration, and invasion by regulating Src activity. For example, PTP1B expression is upregulated in colon cancer, and this high expression can inhibit the phosphorylation of Src Tyr529, which leads to the rapid proliferation of cancer cells in vitro and the acceleration of tumor growth in mice (Zhu et al., 2007). In breast cancer cells, high PTP1B expression is associated with Src activation and increased pseudopodia formation; however, inhibiting PTP1B can delay the formation of breast tumors and reduce the risk of breast cancer lung metastasis through the suppression of Src/Ras/ERK and AKT signaling pathways (Julien et al., 2007;Cortesio et al., 2008;Arias-Romero et al., 2009). ...
Moringa oleifera Lam. is a tropical and subtropical plant that has been used for centuries as both food and traditional medicine. 4-[(α-L-Rhamnosyloxy) benzyl] isothiocyanate (MIC-1) is an active substance in M. oleifera , with anti-cancer activity. However, whether MIC-1 exerts anti-renal cancer effects is unknown. Therefore, the aim of the present study was to evaluate the effects of MIC-1 on the growth and migration of renal cell carcinoma (RCC) cells and to identify the putative underlying mechanism. We found that, among 30 types of cancer cells, MIC-1 exerted the strongest growth inhibitory effects against 786-O RCC cells. In addition, MIC-1 (10 μM) significantly inhibited the growth of five RCC cell lines, including 786-O, OSRC-2, 769-P, SK-NEP-1, and ACHN cells, but was not toxic to normal renal (HK2) cells. Also, MIC-1 suppressed 786-O and 769-P cell migration and invasion abilities, and reduced the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, MIC-1 induced apoptosis and cell cycle arrest, increased Bax/Bcl-2 ratio, and decreased cell cycle-related protein expression in 786-O cells and 769-P cells. Molecular docking and small-molecule interaction analyses with PTP1B both showed that MIC-1 inhibited PTP1B activity by binding to its active site through hydrogen bonding and hydrophobic interactions. Additionally, MIC-1 could suppress the growth and migration of 786-O cells by inhibiting PTP1B-mediated activation of the Src/Ras/Raf/ERK signaling pathway. In vivo experiments further showed that MIC-1 markedly inhibited the growth of xenograft tumors in mice, and greatly increased Bax/Bcl-2 ratio in tumor tissues. In addition, MIC-1 had no effect on the PTP1B-dependent Src/Ras/Raf/ERK signaling pathway in HCT-116 cells, Hep-G2 cells, and A431 cells. Overall, our data showed that MIC-1 could be a promising, non-toxic, natural dietary supplement for the prevention and treatment of renal cancer.
... In addition to its role in regulating insulin, PTP1B has a dual role in cancer development. PTP1B expression was increased in prostate [9], breast [10], ovarian [11], lung [12], colon [13], gastric [14] and pancreatic [15] cancers. In colon cancer, PTP1B has been shown to activate Src by dephosphorylating its negative regulatory residue [13]. ...
... PTP1B expression was increased in prostate [9], breast [10], ovarian [11], lung [12], colon [13], gastric [14] and pancreatic [15] cancers. In colon cancer, PTP1B has been shown to activate Src by dephosphorylating its negative regulatory residue [13]. PTP1B has also been shown to be a positive mediator of ErbB2-induced signals that trigger breast tumorigenesis and to be required for ErbB2 transformation in breast epithelial cells [16]. ...
Previous studies have demonstrated that protein tyrosine phosphatase 1B (PTP1B) can promote tumor progression in breast cancer, colon cancer and prostate cancer. Additionally, PTP1B also acts as a tumor suppressor in esophageal cancer and lymphoma. These findings suggest that PTP1B functions as a double-faceted molecule in tumors. However, the role of PTP1B in malignant melanoma (MM) is still unknown. PTP1B expression in normal and melanoma tissues was evaluated by GEO analysis and immunohistochemistry. The effects of PTP1B on cell migration and invasion were evaluated in melanoma cells with up- and downregulated PTP1B expression. In this study, we initially demonstrated that the expression of PTP1B in malignant melanoma tissue is significantly higher than its expression in benign nevus tissue and indicated poor survival of malignant melanoma patients. In vitro studies have demonstrated that inhibition of PTP1B suppresses and overexpression of PTP1B promotes migration and invasion of melanoma cells. Moreover, we found that PTP1B could interact with Src via coimmunoprecipitation and dephosphorylation of the Tyr530 site. Collectively, our study revealed that PTP1B can promote melanoma cell metastasis by interacting with Src and provides a theoretical basis for future applications of PTP1B inhibitors in the treatment of malignant melanoma.
... In B-cell lymphoma and esophageal cancer, it is a tumor suppressor [26,27]. In contrast, PTPN1 is as a tumor promoter in colorectal, non-small cell lung, breast, gastric, and colon cancer [28][29][30][31][32][33]. The role of PTPN1 in HCC remains unknown. ...
... It is well established that the protein tyrosine phosphatase PTPN1 directly regulates the insulin and the leptin signaling pathway, making it a promising therapeutic target for type II diabetes and obesity [50]. However, PTPN1 plays conflicting roles in different types of tumors [26][27][28][29][30][31][32][33]. Here, we found that PTPN1 was a tumor promoter, and its expression increased in HCC tissues and cell lines. ...
Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers world-wide. miR-125a-5p is a tumor suppressor in HCC and other cancers, but its mechanisms of action during HCC tumorigenesis remain largely unknown. In this study, we found that miR-125a-5p expression was significantly lower in HCC tissues and cell lines than matched normal tissues and liver cells. miR-125a-5p overexpression inhibited HCC cell proliferation and induced apoptosis in vitro and in vivo, while miR-125a-5p knockdown had the opposite effects. In addition, PTPN1 and MAP3K11 were identified as targets of miR-125a-5p. Knocking down PTPN1 and MAP3K11 activated the JNK MAPK signaling pathway to suppress HCC cell proliferation and induce apoptosis. Our findings suggest that miR-125a-5p may be a useful therapeutic target for treatment of HCC patients.
... 10,36,37 For example, Ptpn1 gene is commonly amplified in breast, colon, prostate and gastric cancer. [38][39][40][41] In addition, recent reports indicate that PTP1B overexpression is associated with poor prognosis in several solid tumors, with a high PTP1B expression linked to poor outcome, including shortened progression-free and overall survival. [41][42][43][44] However, the molecular mechanisms that explain the contribution of PTP1B and its downstream targets to carcinogenesis are not entirely understood. ...
PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 in vitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.
... The actions of PTP1B are mediated through its involvement in an abundant list of signalling cascades, of which the most prominent examples are the insulin-, JAK/STAT and leptin pathways [6][7][8]. Increased PTP1B expression and/or activity have been linked to several pathological conditions including cancer, diabetes and, more recently, atherosclerosis [9][10][11][12][13]. ...
Objective
Atherosclerosis is a chronic inflammatory process induced by the influx and entrapment of excess lipoproteins into the intima media of arteries. Previously, our lab demonstrated that systemic PTP1B inhibition protects against atherosclerosis in preclinical LDLR −/− models. Similarly, it was shown that myeloid-specific PTP1B ablation decreases plaque formation and ameliorates dyslipidaemia in the ApoE −/− model of atherosclerosis. We hypothesized that the relevant improvements in dyslipidaemia following modification of PTP1B activation may either result from changes in hepatic cholesterol biosynthesis and/or increased uptake and degradation by liver-resident macrophages. We examined this in animal models and patients with coronary artery disease.
Methods
In this study, we determined the cholesterol-lowering effect of myeloid-PTP1B deletion in mice fed a high-fat high-cholesterol diet and examined effects on total cholesterol levels and lipoprotein profiles. We also determined the effects of PTP1B inhibition to oxLDL-C challenge on foam cell formation and cholesterol efflux in human monocytes/macrophages.
Results
We present evidence that myeloid-PTP1B deficiency significantly increases the affinity of Kupffer cells for ApoB containing lipoproteins, in an IL10-dependent manner. We also demonstrate that PTP1B inhibitor, MSI-1436, treatment decreased foam cell formation in Thp1-derived macrophages and increased macrophage cholesterol efflux to HDL in an AMPK-dependent manner. We present evidence of three novel and distinct mechanisms regulated by PTP1B: an increase in cholesterol efflux from foam cells, decreased uptake of lipoproteins into intra-lesion macrophages in vitro and a decrease of circulating LDL-C and VLDL-C in vivo.
Conclusions
Overall, these results suggest that myeloid-PTP1B inhibition has atheroprotective effects through improved cholesterol handling in atherosclerotic lesions, as well as increased reverse cholesterol transport.
Trial registration Research registry, researchregistry 3235. Registered 07 November 2017, https://www.researchregistry.com/browse-the-registry#home/registrationdetails/5a01d0fce7e1904e93e0aac5/ .
... Pterocarpans are isoflavonoids with inhibitory potency against many enzymes, including Ptp1B. A series of diethyl 2,5-diaminothiophene-3,4dicarboxylate derivatives have been tested for their antiproliferative activity on cell lines and found to inhibit Ptp1B [94]. ...
Loss of function in epigenetic acting genes together with driver alterations in the PIK3CA pathway have been shown significantly associated with poor outcome in cervical squamous cell cancer. More recently, a CoxBoost analysis identified 16 gene alterations and 30 high level activated proteins to be of high interest, due to their association with either good or bad outcome, in the context of treatment received by chemoradiation. The objectives here were to review and confirm the significance of these molecular alterations as suggested by literature reports and to pinpoint alternate treatments options for poor-responders to chemoradiation.
... Furthermore, ITC-α inhibits the growth and migration of renal carcinoma cells by regulating the PTP1B-dependent Src/Ras/Raf/ERK signaling pathway. [8][9][10][11][12][13] Colorectal cancer is the third most common cancer in the Western Hemisphere. Most colorectal cancers are located with or without metastases in the lymph nodes. ...
The diet is gaining relevance within the therapies in degenerative diseases. Recent studies mention that these diets improved the quality of life of patients; this improvement is achieved through functional foods and nutraceuticals. The objective of this research was to study the anticancer properties of 4-(α-l-rhamnosyloxy) benzyl isothiocyanate (ITC-α) with a focus on colorectal cancer using quantum chemistry. Hyperchem software was used as a quantum chemistry simulator. The fundamental basis of quantum calculations was the theory of the electron transfer coefficient (ETC). As a result, almost similar oxidation-reduction interactions are shown in two box-and-whisker diagrams. With this result we can infer that there is homeostasis or chemical balance between ITC-α and the AAs of human proteins. As a general conclusion, we say that ITC-α inhibits the metabolic performance of many proteins and enzymes. KEYWORD: 4-(α-l-rhamnosyloxy) benzyl isothiocyanate, Anticancer properties, Colorectal cancer, Quantum chemistry.
... Dysregulation of different mechanisms can lead to increased activation of SFKs ( Figure 2), including: 1. Dysregulation of phosphatases that target the inactivation loop at the C-terminus (Y 527 for Src or the equivalent peptide QYQ) (Fang et al., 1994;Roskoski, 2005;Zhu et al., 2007;Narla et al., 2018), 2. Downregulation of proteins like CSK or CHK, which normally phosphorylate the inactivation loop , 3. Dysregulation of the expression or localisation of scaffolding or adapter proteins that help position SFKs near their substrates (including CSK) (Oneyama et al., 2008;Chüeh et al., 2021), or 4. Dysregulation of kinases/phosphatases that target phosphorylation sites on scaffolding/adapter proteins recognized by the SH2/ SH3 domains present in SFKs (Narla et al., 2018). In summary, while SFKs are proto-oncogenes that can contribute to cancer progression, their over-activation is typically not caused by direct mutations in SFKs themselves, but rather by dysregulation of upstream proteins in their signalling pathway. ...
Cancer progression and metastasis are processes heavily controlled by the integrin receptor family. Integrins are cell adhesion molecules that constitute the central components of mechanosensing complexes called focal adhesions, which connect the extracellular environment with the cell interior. Focal adhesions act as key players in cancer progression by regulating biological processes, such as cell migration, invasion, proliferation, and survival. Src family kinases (SFKs) can interplay with integrins and their downstream effectors. SFKs also integrate extracellular cues sensed by integrins and growth factor receptors (GFR), transducing them to coordinate metastasis and cell survival in cancer. The non-receptor tyrosine kinase CSK is a well-known SFK member that suppresses SFK activity by phosphorylating its specific negative regulatory loop (C-terminal Y527 residue). Consequently, CSK may play a pivotal role in tumour progression and suppression by inhibiting SFK oncogenic effects in several cancer types. Remarkably, CSK can localise near focal adhesions when SFKs are activated and even interact with focal adhesion components, such as phosphorylated FAK and Paxillin, among others, suggesting that CSK may regulate focal adhesion dynamics and structure. Even though SFK oncogenic signalling has been extensively described before, the specific role of CSK and its crosstalk with integrins in cancer progression, for example, in mechanosensing, remain veiled. Here, we review how CSK, by regulating SFKs, can regulate integrin signalling, and focus on recent discoveries of mechanotransduction. We additionally examine the cross talk of integrins and GFR as well as the membrane availability of these receptors in cancer. We also explore new pharmaceutical approaches to these signalling pathways and analyse them as future therapeutic targets.
... Y530 of Src is phosphorylated by CSK, a ubiquitously expressed protein kinase. To counteract against CSK and activate Src, phosphorylated Y530 is dephosphorylated by several protein tyrosine phosphatases including PTP-α, PTP-λ, SHP-1, SHP-2, and PTP1B (Zhu et al., 2007;Caner et al., 2021). ...
The Src family kinases (SFK) plays an important role in multiple signal transduction pathways. Aberrant activation of SFKs leads to diseases such as cancer, blood disorders, and bone pathologies. By phosphorylating and inactivating SFKs, the C-terminal Src kinase (CSK) serves as the key negative regulator of SFKs. Similar to Src, CSK is composed of SH3, SH2, and a catalytic kinase domain. However, while the Src kinase domain is intrinsically active, the CSK kinase domain is intrinsically inactive. Multiple lines of evidence indicate that CSK is involved in various physiological processes including DNA repair, permeability of intestinal epithelial cells (IECs), synaptic activity, astrocyte-to-neuron communication, erythropoiesis, platelet homeostasis, mast cell activation, immune and inflammation responses. As a result, dysregulation of CSK may lead to many diseases with different underlying molecular mechanisms. Furthermore, recent findings suggest that in addition to the well-established CSK-SFK axis, novel CSK-related targets and modes of CSK regulation also exist. This review focuses on the recent progress in this field for an up-to-date understanding of CSK.
... Within the last decades, PTP-1B arose as one of the newly pursued anticancer therapeutic targets for illustrating significant positive regulation of the erythroblastic oncogene B-2 (ErbB-2, a.k.a. human epidermal growth factor receptor-2 (HER-2)) and cellular sarcoma (Scr)-induced cellular signaling [23][24][25][26]. Major challenges are considered for developing hPTP-1B inhibitors since the active site bears high charge density as well as being conserved across the PTPase family that would raise issues of off-target effects [27,28]. ...
... Within the last decades, PTP-1B arose as one of the newly pursued anticancer therapeutic targets for illustrating significant positive regulation of the erythroblastic oncogene B-2 (ErbB-2, a.k.a. human epidermal growth factor receptor-2 (HER-2)) and cellular sarcoma (Scr)-induced cellular signaling [23][24][25][26]. Major challenges are considered for developing hPTP-1B inhibitors since the active site bears high charge density as well as being conserved across the PTPase family that would raise issues of off-target effects [27,28]. ...
Cancer is the leading cause of death globally, with an increasing number of cases being annually reported. Nature-derived metabolites have been widely studied for their potential programmed necrosis, cytotoxicity, and anti-proliferation leading to enrichment for the modern medicine , particularly within the last couple of decades. At a more rapid pace, the concept of multi-target agents has evolved from being an innovative approach into a regular drug development procedure for hampering the multi-fashioned pathophysiology and high-resistance nature of cancer cells. With the advent of the Red Sea Penicillium chrysogenum strain S003-isolated indole-based alkaloids, we thoroughly investigated the molecular aspects for three major metabolites: meleagrin (MEL), roque-fortine C (ROC), and isoroquefortine C (ISO) against three cancer-associated biological targets Cdc-25A, PTP-1B, and c-Met kinase. The study presented, for the first time, the detailed molecular insights and near-physiological affinity for these marine indole alkaloids against the assign targets through molecular docking-coupled all-atom dynamic simulation analysis. Findings highlighted the superiority of MEL's binding affinity/stability being quite in concordance with the in vitro anti-cancer activity profile conducted via sulforhodamine B bioassay on different cancerous cell lines reaching down to low micromolar or even nanomolar potencies. The advent of lengthy structural topologies via the metabolites' extended tetracyclic cores and aromatic imidazole arm permitted multi-pocket accommodation addressing the selectivity concerns. Additionally, the presence decorating polar functionalities on the core hydrophobic tetracyclic ring contributed compound's phar-macodynamic preferentiality. Introducing ionizable functionality with more lipophilic characters was highlighted to improve binding affinities which was also in concordance with the conducted drug-likeness/pharmacokinetic profiling for obtaining a balanced pharmacokinetic/dynamic profile. Our study adds to the knowledge regarding drug development and optimization of marine-Citation: Bogari, H.A.; Elhady, S.S.; Darwish, K.M.; Refaey, M.S.; Mohamed, R.A.; Abdelhameed, R.F.A.; Almalki, A.J.; Aldurdunji, M.M.; Lashkar, M.O.; Alshehri, S.O.; et al. Molecular and Biological Investigation of Isolated Marine Fungal Metabolites as Anticancer Agents: A Multi-Target Approach. Metabolites 2023, 13, 162. https://
... For instance, PDGFR binds to Src SH2 domain while ß3 subunit of integrins binds to Src SH3 domains (reviewed in [15]). The disassembly of intramolecular contacts allows autophosphorylation at Tyr416 [16] and dephosphorylation at Tyr527, which can be catalysed by the receptor protein tyrosine phosphatase α (PTPRA) [17], the nonreceptor tyrosine phosphatase SHP-1 (PTPN6) [18] or PTP1B [19]. Src adopts an open conformation in which the Src SH3 domain interacts with the proline rich region and the Src SH2 domain with the phosphotyrosine site, both at the target substrate. ...
Metabolism must be tightly regulated to fulfil the dynamic requirements of cancer cells during proliferation, migration, stemness and differentiation. Src is a node of several signals involved in many of these biological processes, and it is also an important regulator of cell metabolism. Glucose uptake, glycolysis, the pentose-phosphate pathway and oxidative phosphorylation are among the metabolic pathways that can be regulated by Src. Therefore, this oncoprotein is in an excellent position to coordinate and finely tune cell metabolism to fuel the different cancer cell activities. Here, we provide an up-to-date summary of recent progress made in determining the role of Src in glucose metabolism as well as the link of this role with cancer cell metabolic plasticity and tumour progression. We also discuss the opportunities and challenges facing this field.
... Notably, we demonstrated for the first time, that low nasal washings expression of mir-34c-3p correlates with orbital and intracranial disease extension; interestingly, miR-34c-3p is predicted to target Protein Tyrosine Phosphatase PTP4A1 and PTP4A2, two closely related family members of PTP4A3, whose gains were more frequently reported in ITACs with advanced stages and intracranial invasion [33]. Gains of PTPN1 were also more frequently associated to intracranial extension [33] and the interaction between PTPN1, also known as PTP1B, and miR-34c, experimentally demonstrated in glioma [48], has been associated with increased proliferation and metastasis in colon and lung cancer [49,50]. Since intracranial invasion and local recurrence are the most common causes of death in ITAC [39], low mir-34c levels in nasal washings appears of potential prognostic relevance in these patients. ...
Introduction
Sinonasal intestinal-type adenocarcinomas (ITACs) are rare and aggressive tumors, closely related to professional exposure to wood dusts or leather. Here we explored the role of non-coding RNAs controlling MUC2 in liquid biopsies and tumors from ITAC patients with the aim of identifying biomarkers and molecular mechanisms to improve early diagnosis, prognosis, and therapeutic approaches for this rare cancer.
Methods
MiR-34c-3p, lncRNA AF147447 and MUC2 were measured in tumors and normal mucosa, in nasal washings (NW) from the affected and non-affected nostril and in plasma from 17 ITAC patients. The Apparent Diffusion Coefficient (ADC) was also evaluated by Magnetic Resonance Imaging.
Results
MiR-34c was higher in ITACs compared to the corresponding normal mucosa (p = 0.021). Differentiated tumors exhibited higher miR-34c levels (p = 0.025) and lower ADC values (p<0.001) compared to mucinous ones and these parameters were also inversely correlated (r = 0.87; p = 0.001). High MUC2 tumor expression was associated with orbital extension (p = 0.010). Low miR-34c levels in NW were associated with orbital (p = 0.009) and intracranial (p = 0.031) extension and with advanced TNM stage (p = 0.054). Functional analysis identified Wnt, Focal adhesion, MAPK and inflammatory signalings among the pathways most enriched in mir-34c targets.
Discussion
Our results suggest measuring miR-34c in NW as a biomarker for early diagnosis and monitoring of ITAC patients and for the surveillance of wood and leather exposed workers. Further research on the involvement of miR-34c regulated pathways in ITAC tumorigenesis may also allow the development of new therapeutic approaches for this rare cancer.
... Recent evidence suggests that PTP plays an essential role in the development and progression of different types of cancer [10,32,33]. For example, Ptpn1 gene is commonly amplified in breast, colon, prostate and gastric cancer [34][35][36][37]. In addition, recent reports indicate that PTP1B overexpression is associated with poor prognosis in several solid tumors, with a high PTP1B expression linked to poor outcome, including shortened progression-free and overall survival [37][38][39][40]. ...
Protein tyrosine phosphatase 1B (PTP1B) plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify possible molecular targets of PTP1B that mediate its positive role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify the Cyclin-dependent kinase 3 (Cdk3) as a novel PTP1B substrate. Molecular docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at Tyrosine residue 15 and interacts with it in the nuclear envelope of HEK293-T cells and the nucleus and cytoplasm of human glioblastoma (GB) cells. Finally, we found that the pharmacological inhibition of PTP1B leads to a cell cycle progression delay with the diminished activity of Cdk3, the consequent hypophosphorylation of Rb, and the down-regulation of E2F and its target genes Cdk1, Cyclin A, and Cyclin E1. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells and suggest new therapeutic strategies for treating these tumors.
... 15,16 Specifically, PTP1B promotes CC through activation of the nonreceptor tyrosine kinase, Src. 17 Lately, PTP1B is shown to promote colorectal cancer (CRC) via PITX-1/p120RasGAP axis as well. 18 Although PTP1B is presumed to be a probable candidate to predict treatment outcome in CC and might be a therapeutic target in obesity and linked CC, no therapeutic inhibitor of PTP1B is available yet. ...
Protein tyrosine phosphatase 1B (PTP1B) has emerged as one of the links between obesity and colon cancer (CC). Anti-obesity and anti-colon cancer attributes of sweet potato (Ipomoea batatas) reported sparsely. Here, we aimed to study the potential of PTP1B as a target in CC, particularly in obese population. Expression and genomic alteration frequency of PTPN1 (PTP1B) checked in CC. Interacting partners of PTP1B through STRING and hub genes through Cytoscape (MCODE) were identified. Hub genes were subjected to functional enrichment analyses (via Metascape), differential gene expression, CNV and SNV analyses (GSCA database). Cancer-related pathways and associated immune infiltrates of the hub genes were checked too. 11 sweet potato-derived compounds selected through drug likeness and toxicity filters were explored via molecular docking (AutoDock Vina) to reveal interactions with PTP1B. Genomic alteration frequency of the PTPN1 was highest in CC compared to all other TCGA cancers and a high expression (RNA and protein) also observed in CC, correlated to a poor overall survival. Further, PTP1B and related proteins were enriched in different biological processes and signalling pathways related to carcinogenesis including epithelial-mesenchymal-transition. Overall, PTP1B identified as a potential target in obesity-linked CC and sweet potato might exert its protective action by targeting the PTP1B. Sweet potato compounds (e.g., pelargonidin and luteolin) interacted with the catalytic P loop and the WPD loop of the PTP1B. Further, MD simulation study ascertained that luteolin has the highest affinity against the PTP1B whereas, pelargonidin and quercetin showed good binding affinity too, thus can be explored further.
... Insulin/IGFs system promotes the proliferation of colorectal cancer cells via the activation of PI3K/Akt signaling pathway [73]. In colorectal carcinomas, IGF-binding protein 1 stimulates tyrosine-protein kinase Src, which in turn regulates PI3K/Akt signaling, leading to the proliferation and migration of colorectal cancer cells [74]. Thus, insulin/IGFs system has an essential role in the pathogenesis of obesity-associated CRC. ...
Colorectal cancer (CRC) is the third leading cause of cancer death worldwide. Obesity has been proven to be closely related to colorectal carcinogenesis. This review summarized the potential underlying mechanisms linking obesity to CRC in different aspects, including energy metabolism, inflammation, activities of adipokines and hormones. Furthermore, the potential therapeutic targets of obesity-associated CRC were predicted using network-based target analysis, with total predicted pathways not only containing previously reported pathways, but also putative signaling pathways pending for investigation. In addition, the current conventional therapeutic treatment options, plus the potential use of herbs and natural products in the management of obesity-associated CRC were also discussed. Taken together, the aim of this review article is to provide strong theoretical basis for future drug development, particularly herbs and natural products, in obesity-associated CRC.
... Although PTP1B can act as a tumor suppressor, and its deletion in mice promotes increased development of acute leukemia with age [77], PTP1B predominantly functions as an oncoprotein, and its inhibition or deletion can attenuate oncogene-induced tumorigenesis. Indeed, PTP1B has been shown to function as an oncoprotein in breast [39,40], prostate [78,79], gastric [80], and colon [37] cancers, as well as squamous cell carcinoma [81]. In many cases, including breast cancers, PTP1B levels are increased and this correlates with advancement of the disease and worsened prognoses [39,40]. ...
Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Here, using Drosophila as a model organism, we have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. We demonstrate that a Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, we show that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK–STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, we show that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS–MAPK and JAK–STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis.
... PTP1B, being a negative regulator of insulin and leptin signaling is considered as a promising therapeutic target for treatment of type 2 diabetes and obesity [19]. Overexpression of this phosphatase was also found to contribute to tumorigenesis of cells [20][21]. Megakaryocyte protein tyrosine phosphatase MEG-2 which involved in regulating of hematopoietic signaling and blood glucose homeostasis is of interest as a therapeutic target for treatment of type 2 diabetes and myeloproliferative disorders [23][24][25]. PTPβ, also called as vascular endothelialprotein tyrosine phosphatase (VE-PTP), downregulates Tie2 signaling and often associated with development of variety endothelial dysfunctions. ...
Thiazole-containing derivatives of rhodanine-3-alkanoic acids with propanoic or undecanoic acid groups were synthesized and evaluated as inhibitors of some protein tyrosine phosphatases and glutathione S-transferases. The rhodanines bearing longer carboxylated N-alkyl chain were found to inhibit PTP1B, MEG1, MEG2, and VE-PTP as well as GST from equine liver and GSTA1-1 with IC50 values in the low micromolar range. The inhibitory effect on protein tyrosine phosphatase activity depends on substituent at position 2 of the thiazole ring. The best compound showed a competitive type of VE-PTP inhibition. In case of GST from equine liver, the inhibition was of mixed or non-competitive type with respect to glutathione or CDNB substrate, respectively. Possible binding modes of the inhibitors were discussed based on molecular docking calculations.
... Subconfluent cells in tissue culture were lysed in RIPA lysis buffer supplemented with phosphatase inhibitors and protease inhibitors (Zhu et al., 2007). Lysates were clarified by centrifugation at 10,000 g for 20 min. ...
Src is an important oncogene that plays key roles in multiple signal transduction pathways. Csk-homologous kinase (CHK) is a kinase whose molecular roles are largely uncharacterized. We previously reported expression of CHK in normal human colon cells, and decreased levels of CHK protein in colon cancer cells leads to the activation of Src (Zhu et al., 2008). However, how CHK protein expression is downregulated in colon cancer cells has been unknown. We report herein that CHK mRNA was decreased in colon cancer cells as compared to normal colon cells, and similarly in human tissues of normal colon and colon cancer. Increased levels of DNA methylation at promotor CpG islands of CHK gene were observed in colon cancer cells and human colon cancer tissues as compared to their normal healthy counterparts. Increased levels of DNA methyltransferases (DNMTs) were also observed in colon cancer cells and tissues. DNA methylation and decreased expression of CHK mRNA were inhibited by DNMT inhibitor 5-Aza-CdR. Cell proliferation, colony growth, wound healing, and Matrigel invasion were all decreased in the presence of 5-Aza-CdR. These results suggest that increased levels of DNA methylation, possibly induced by enhanced levels of DNMT, leads to decreased expression of CHK mRNA and CHK protein, promoting increased oncogenic properties in colon cancer cells.
... Conversely, protein tyrosine phosphatase PTP1B (encoded by the PTPN1 gene) acts as an upstream activator of SFKs by dephosphorylating this C-terminal tail tyrosine [18]. Expression of PTP1B is increased in CRC and reported to contribute to CRC development and progression [6,19]. In addition to phosphorylation, direct binding of CHK/MATK also suppresses the activity of all SFKs via a non-catalytic mechanism [17,20,21]. ...
Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.
... Recent studies have also reported that PTP1B is involved in the occurrence and development of various tumors. For example, PTP1B was found to promote the carcinogenicity of colon cancer by activating Src [25]. In prostate cancer, PTP1B affected the invasion of cancer cells [26]. ...
Glioma is one of the most pervasive and invasive primary malignancies in the central nervous system. Due to its abnormal proliferation, glioma remains hard to cure at present. Protein tyrosine phosphatase 1B (PTP1B) has been proved to be involved in the process of proliferation in many malignancies. However, whether PTP1B is involved in the proliferation of glioma and how it acts are still unclear. In this study, the PTP1B expressions in glioma tissues and cells were determined by quantitative real-time PCR and western blot analysis. The effects of PTP1B on the proliferation characteristics of glioma were explored using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation assay, and tumor xenografts in mice. We found that the protein and mRNA levels of PTP1B in glioma tissues were significantly higher than those in paired nontumor tissues. MTT and clone formation assays showed that PTP1B is closely related to human glioma cell proliferation. In addition, TargetScan revealed that miR-34c regulates PTP1B. Mechanistically, we proved that miR-34c negatively regulates PTP1B and then participates in the regulation of glioma cell proliferation in vivo. Collectively, these results suggested that miR-34c inhibits the proliferation of human glioma cells by targeting PTP1B, which will provide a potential target for the treatment of glioma.
... The upregulation of PTP1B in the hippocampus is further responsible for neuroinflammation and neuronal loss. Studies also reported enhanced expression of PTP1B in adipocyte, hepatocyte cell lines, and mouse hypothalamus as well as in high-fat diet-induced obesity in the animal model [3] . Thus, PTP1B plays a diverse role in neuroinflammation, diabetes, obesity, and cancer. ...
Neuroinflammation is considered as the pathological hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). Under inflammatory conditions, activation of microglial cells results in the increased expression of Protein tyrosine phosphatase 1B (PTP1B) levels, enhancing pro-inflammatory response. Computation based in silico docking studies were done using maestro interface to identify the inhibitors of PTP1B. A library of 1941 phytomolecules from Zinc database was screened for evaluation of their binding potential with PTP1B. Phytomolecules with better activity and binding were shortlisted after HTVS, SP, and XP mode of docking. The refining of the hit molecules was done by the analysis of the pharmacokinetic properties and toxicity prediction using QikProp tool. The molecules with better binding and pharmacokinetic properties were selected for WaterMap analysis and Molecular dynamics simulation study. The top three compounds were selected for further studies, including QikProp analysis, water map analysis, and molecular dynamics study. Compounds ZINC000001785780, ZINC000013543704, ZINC000040874044 were found to be potent inhibitors against PTP1B based on the docking score and binding interactions. Among the selected molecules, ZINC000013543704 showed better stability in the molecular dynamics simulation study and WaterMap study. The identified hit could be a potent inhibitor of PTP1B that further requires experimental validation using in vitro and in vivo experimental models.
... Src also plays a critical role in the transformation and growth of CRC cells. Knockdown or inhibition of Src inhibited cell metastasis and proliferation in human cancer cells SW480 and HT29 [45,46]. Phosphorylated IR (pIR) was highly expressed in low-grade colorectal adenocarcinoma, which indicated activation of IR is an early event in CRC tumorigenesis [47]. ...
The incidence of obesity and colorectal cancer (CRC) has risen rapidly in recent decades. More than 650 million obese and 2 billion overweight individuals are currently living in the world. CRC is the third most common cancer. Obesity is regarded as one of the key environmental risk factors for the pathogenesis of CRC. In the present review, we mainly focus on the epidemiology of obesity and CRC in the world, the United States, and China. We also summarize the molecular mechanisms linking obesity to CRC in different aspects, including nutriology, adipokines and hormones, inflammation, gut microbiota, and bile acids. The unmet medical needs for obesity-related CRC are still remarkable. Understanding the molecular basis of these associations will help develop novel therapeutic targets and approaches for the treatment of obesity-related CRC.
... This is the first human study to use the Illumina Infinium MethylationEPIC BeadChip array to assess differential DNAm in relation to smoking, which led to novel insights. For instance, some new genes that warrant further investigation include a zinc finger protein (ZNF83), with a role of coordinating zinc ions (Cassandri et al. 2017), PTPN1, which is involved in oncogenic transformations (Zhu et al. 2007), and RAB32, a member of the RAS oncogene family, whose overexpression has been shown in 20% to 25% of all human tumors and up to 90% in pancreatic cancer (Downward 2003). For former smoking, however, we only found six significant CpGs, which indicates that current smoking is more relevant for methylation changes than former smoking. ...
Background:
The epigenetic effects of individual environmental toxicants in tobacco remain largely unexplored. Cadmium (Cd) has been associated with smoking-related health effects, and its concentration in tobacco smoke is higher in comparison with other metals.
Objectives:
We studied the association of Cd and smoking exposures with human blood DNA methylation (DNAm) profiles. We also evaluated the implication of findings to relevant methylation pathways and the potential contribution of Cd exposure from smoking to explain the association between smoking and site-specific DNAm.
Methods:
We conducted an epigenome-wide association study of urine Cd and self-reported smoking (current and former vs. never, and cumulative smoking dose) with blood DNAm in 790,026 CpGs (methylation sites) measured with the Illumina Infinium Human MethylationEPIC (Illumina Inc.) platform in 2,325 adults 45-74 years of age who participated in the Strong Heart Study in 1989-1991. In a mediation analysis, we estimated the amount of change in DNAm associated with smoking that can be independently attributed to increases in urine Cd concentrations from smoking. We also conducted enrichment analyses and in silico protein-protein interaction networks to explore the biological relevance of the findings.
Results:
At a false discovery rate (FDR)-corrected level of 0.05, we found 6 differentially methylated positions (DMPs) for Cd; 288 and 17, respectively, for current and former smoking status; and 77 for cigarette pack-years. Enrichment analyses of these DMPs displayed enrichment of 58 and 6 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes gene sets, respectively, including biological pathways for cancer and cardiovascular disease. In in silico protein-to-protein networks, we observed key proteins in DNAm pathways directly and indirectly connected to Cd- and smoking-DMPs. Among DMPs that were significant for both Cd and current smoking (annotated to PRSS23, AHRR, F2RL3, RARA, and 2q37.1), we found statistically significant contributions of Cd to smoking-related DNAm.
Conclusions:
Beyond replicating well-known smoking epigenetic signatures, we found novel DMPs related to smoking. Moreover, increases in smoking-related Cd exposure were associated with differential DNAm. Our integrative analysis supports a biological link for Cd and smoking-associated health effects, including the possibility that Cd is partly responsible for smoking toxicity through epigenetic changes. https://doi.org/10.1289/EHP6345.
... PTP1B-induced Fyn C-terminal peptide (FCP) phosphatase activity enhances Src activity by inhibiting the phosphorylation of Tyr530 in Src. PTP1B inhibitors decrease the metastatic potential and tumor growth by inhibiting Src activation [58]. Additionally, six PTP1B truncation mutants were identified in 2-21% of patients with CRC. ...
Src family kinases (SFKs) are non-receptor kinases that play a critical role in the pathogenesis of colorectal cancer (CRC). The expression and activity of SFKs are upregulated in patients with CRC. Activation of SFKs promotes CRC cell proliferation, metastases to other organs and chemoresistance, as well as the formation of cancer stem cells (CSCs). The enhanced expression level of Src is associated with decreased survival in patients with CRC. Src-mediated regulation of CRC progression involves various membrane receptors, modulators, and suppressors, which regulate Src activation and its downstream targets through various mechanisms. This review provides an overview of the current understanding of the correlations between Src and CRC progression, with a special focus on cancer cell proliferation, invasion, metastasis and chemoresistance, and formation of CSCs. Additionally, this review discusses preclinical and clinical strategies to improve the therapeutic efficacy of drugs targeting Src for treating patients with CRC.
... It was reported to act as tumour suppressor or promoter according to the tissue in which it is expressed and the involved substrates in its signalling pathway (Lessard et al. 2010). It was reported to act as a tumour promoter in breast, prostate, colon and lung cancers (Lessard et al. 2012; Liu et al. 2015;Liu et al. 2016;Zhu et al. 2007). Therefore, inhibition or downregulation of PTP1B can contribute to the suppression of carcinogenesis. ...
Sixteen new analogues were synthesized from ricinine and tested alongside with seven known analogues for their cytotoxic activity against oral cancer (SAS cells) and normal epithelial cells (L132 cells). In contrast to 5-FU, the synthesized ricinine analogues did not show toxicity to normal cells. However, some of them inhibited the proliferation of oral cancer cells at 25 µM as evident from the MTT assay results. Ricinine analogue (19) was shown to be the most active derivative (69.22% inhibition). Potential targets involved in the oral cancer inhibitory activity of compound 19 were investigated using in-silico studies and western blot analysis. PTP1B was predicted to be a target for ricinine using reverse docking approach. This prediction was confirmed by western blot analysis that revealed the downregulation of PTP1B protein by compound 19. Moreover, it showed downregulation of COX-2 which is also extensively expressed in oral cancer.
... The overexpression of PTP1B is strictly correlated with development of both insulin and leptin resistance, which are important pathogenic mechanisms underlying the onset and progression of type 2 diabetes mellitus (T2DM) and obesity [5][6][7]. In accord with this hypothesis, it PTP1B expression is significantly associated with the progression of several human tumor types, such as colon and breast cancers, and, accordingly, the inhibition of the enzyme results in better prognostic outcome [20][21][22][23][24]. ...
Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin and leptin signalling and is crucially involved in the development of type 2 diabetes mellitus, obesity, cancer and neurodegenerative diseases. Pursuing our efforts to identify PTP1B inhibitors endowed with drug-like properties, we designed and evaluated 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as a novel class of non-carboxylate PTP1B inhibitors. In agreement with our design, kinetic studies demonstrated that selected compounds 7 act as reversible, non-competitive inhibitors of the target enzyme at low micromolar concentrations. Accordingly, molecular docking experiments suggested that these inhibitors can fit an allosteric site of PTP1B that we previously individuated. Moreover, cellular assays demonstrated that compound 7e acts as a potent insulin-sensitizing agent in human liver HepG2 cells. Taken together, our results showed that these non-competitive PTP1B inhibitors can be considered promising lead compounds aimed to enhance druggability of the target enzyme and identify novel antidiabetic drugs.
... Many studies have shown that PTP1B can act as an activator of Src kinase, increase tumorigenicity and promote tumor progression of colon cancer cells (18) and pancreatic cancer (19). ...
... Furthermore, high expression of PTPN1 predicted poor prognosis of patients with GBM, therefore, indicating that PTPN1 may serve as a prognostic marker and a putative target in the treatment of glioma. A previous study indicated that PTPN1 overexpression may activate Src by reducing phosphorylation at tyrosine 530 (29). Src activation has been reported to be associated with proliferation, survival and metastasis in cancer cells, by stimulating multiple signaling pathways, including the Src/PI3K/Akt and Src/MAPK/ERK signaling pathways (30). ...
Glioma is the most common primary brain tumor and is characterized by a poor prognosis. Protein tyrosine phosphatase 1B (PTPN1), as a non‑transmembrane protein tyrosine phosphatase, has been reported to serve a critical role in different diseases, including cancer. However, the role of PTPN1 in the progression of glioma remains unclear. The present study investigated the expression and clinicopathological characteristics of PTPN1 by analyzing the data from The Cancer Genome Atlas and 136 patients with glioma. It was indicated that PTPN1 was overexpressed in glioma tissues and served as a predictor for poor prognosis in patients with glioma. In addition, a series of in vitro experiments were performed to examine the underlying mechanism of PTPN1 overexpression and the clinical prognosis in patients with glioma. Knockdown of PTPN1 by small interfering RNA suppressed proliferation of glioma cells, including SF295 and A172. In addition, cell mobility was also inhibited by PTPN1 knockdown, downregulating the expression of matrix metallopeptidase 2 (MMP‑2) and MMP‑9. As indicated by western blot analysis, the mitogen‑activated protein kinase (MAPK)/extracellular‑signal‑regulated kinase (ERK) signaling pathway and the phosphatidylinositol 3‑kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was regulated by PTPN1, while knockdown of PTPN1 significantly suppressed the MAPK/ERK and PI3K/AKT pathways, in addition to the downstream oncogenic transcription factor MYC Proto‑Oncogene. In conclusion, it was demonstrated that PTPN1 is upregulated in glioma tissue and the overexpression of PTPN1 predicted the poor prognosis of patients with glioma. PTPN1 promotes the progression of glioma by activating the MAPK/ERK and PI3K/AKT pathways.
... Given that Src positively regulates mTORC1 activity, we asked whether mTORC1 activity is higher in cells having endogenous activation of Src. To test this possibility, we investigated Src-mTORC1 signaling in HT-29 and Caco-2 colon cancer cells which have higher Src activity compared to normal colon cells [28][29][30][31] . We observed a significant increase in mTORC1 activity in both Caco-2 and HT-29 cells compared to the normal colon epithelial cell line FHC 28 under amino acid starvation (Fig. 5a, b), similar to cells transfected with active Src constructs (Fig. 1g). ...
The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell survival and autophagy, and its activity is regulated by amino acid availability. Rag GTPase-GATOR1 interactions inhibit mTORC1 in the absence of amino acids, and GATOR1 release and activation of RagA/B promotes mTORC1 activity in the presence of amino acids. However, the factors that play a role in Rag-GATOR1 interaction are still poorly characterized. Here, we show that the tyrosine kinase Src is crucial for amino acid-mediated activation of mTORC1. Src acts upstream of the Rag GTPases by promoting dissociation of GATOR1 from the Rags, thereby determining mTORC1 recruitment and activation at the lysosomal surface. Accordingly, amino acid-mediated regulation of Src/mTORC1 modulates autophagy and cell size expansion. Finally, Src hyperactivation overrides amino acid signaling in the activation of mTORC1. These results shed light on the mechanisms underlying pathway dysregulation in many cancer types.
... Recent researches revealed to activate c-Src by the dephosphorylation of inhibitory pTyr-530 [23]. This c-Src-mediated activation is required for the transformation of the MCF10A by the ErbB2 thereby contributing to the tumor development SW48 colon cancer in mammals [24,25]. P62Dok is a known substrate which p120RasGAP which shows negative regulatory functions on the Ras-Erk1/2 pathway [26]. ...
The enzyme protein tyrosine phosphatase (PTP) is responsible for the regulation of cellular functions including cell growth, replication, and signal transduction. Dysregulation of this enzyme leads to various diseases including Type II diabetes and cancers as well. The PTP enzyme functions as a promising drug target for these diseases, leading to new innovations in developing new drug targets in the field of clinical studies and pharmacology. The PTP is considered as the next generation drug targets. The protein tyrosine drug targets can be targeted to cure the diseases caused due to its dysregulations. However, due to its complex structure and highly conserved active sites are the major challenges which block this strategy. Moreover, two enzyme proteins PTP A and PTP B of PTP enzyme family are essential for the survival of Mycobacterium in host macrophages and cause infection resulting in chronic tuberculosis. The novel drug-like properties of L335-M34 and L01Z08 compounds are selective inhibitors of this enzyme responsible for the tuberculosis virulence in mammals. Some of the commercial inhibitors such as ertiprotafib, arylbenzonaphthofurans, and arylbenzonaphthothiophenes have also proven to inhibit the enzyme’s virulence. This review summarizes the latest innovations to lead a map for developing new innovative drugs against the various classes of target enzymes of tyrosine phosphatase.
... For example, PTP1B acts as a tumor suppressor in leukemia and lymphoma but can be as an oncogene in breast cancer and non-small cell lung cancer. It has been reported that PTP1B acts as an oncogene through interacting with several oncogenic substrates such as Src [63,64], ERK1/2 [65], p62dok [66,67], human epidermal growth factor receptor 2 (HER2) [68,69], and p130Cas [70]. Conversely, PTP1B can also act as a tumor suppressor by negatively regulating several oncogenic kinases such as Bcr-Abl [71], JAK-STAT [72,73], and β-catenin [74]. ...
The protein tyrosine phosphatase (PTP) family is involved in multiple cellular functions and plays an important role in various pathological and physiological processes. In many chronic diseases, for example cancer, PTP is a potential therapeutic target for cancer treatment. In the last two decades, dozens of PTP inhibitors which specifically target individual PTP molecules were developed as therapeutic agents. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is the second most lethal cancer worldwide due to a lack of effective therapies. Recent studies have unveiled both oncogenic and tumor suppressive functions of PTP in HCC. Here, we review the current knowledge on the involvement of PTP in HCC and further discuss the possibility of targeting PTP in HCC.
... Inhibition of PTP1B has been shown to increase the sensitivity of insulin receptor (IR), thus demonstrating a promising approach in the treatment of insulin resistance-related diseases such as type 2 diabetes [17] and obesity [18]. These findings have resulted in PTP1B being pursued as a potentially attractive therapeutic target for the treatment of many diseases, including obesity, diabetes [16], breast cancer [19], colon cancer [20], and neurodegenerative disorders [21]. ...
Catalytic proteins such as human protein tyrosine phosphatase 1B (PTP1B), with conserved and highly polar active sites, warrant the discovery of druggable non-active sites, such as allosteric sites, and potentially, therapeutic small molecules that can bind to these sites. Catalyzing the dephosphorylation of numerous substrates, PTP1B is physiologically important in intracellular signal transduction pathways in diverse cell types and tissues. Aberrant PTP1B is associated with obesity, diabetes, cancers, and neurodegenerative disorders. Utilizing clustering methods (based on root mean square deviation, principal component analysis, non-negative matrix factorization, and independent component analysis), we have examined multiple PTP1B structures. Using the resulting representative structures in different conformational states, we determined consensus clustroids and used them to identify both known and novel binding sites, some of which are potentially allosteric. We report several lead compounds that could potentially bind to the novel PTP1B binding sites and can be further optimized. Considering the possibility for drug repurposing, we discovered homologous binding sites in other proteins, with ligands that could potentially bind to the novel PTP1B binding sites. This article is protected by copyright. All rights reserved.
The prevalence of obesity has increased significantly worldwide, and this trend is likely to continue in the coming years. There is substantial evidence that obesity plays a crucial role in the development of colorectal cancer. Epidemiological data have consistently demonstrated a correlation between obesity and colorectal cancer. Insulin resistance, hyperinsulinemia, chronic inflammation, altered levels of growth factors, adipocytokines, and various hormones are plausible biological mechanisms. In addition, obesity has been shown to have an impact on recurrence, treatment success, and overall survival. There are some reports, although the evidence is not conclusive, that weight loss and lifestyle changes such as dietary modification and physical activity can reduce the risk of colorectal cancer. The understanding that obesity is a potentially modifiable risk factor that can affect the incidence and prognosis of colorectal cancer is crucial knowledge that can have an impact on the prevention and treatment of the condition.
Background:
Metformin (MET), a worldwide used drug for treating type 2 diabetes but not metabolized by humans, has been found with the largest amount in the aquatic environment. Two MET chlorination byproducts, including Y and C, were transformed into drinking water during chlorination. However, the potential toxicity of the byproducts in hepatotoxicity and reproduction toxicity remains unclear.
Methods:
The TOPKAT database predicted the toxicological properties of metformin disinfection by-products. The targets of metformin disinfection by-products were mainly obtained from the PharmMapper database, and then the targets of hepatotoxicity and reproductive toxicity were screened from GeneCards. The overlapping targets of toxic component targets and the hepatotoxicity or reproduction toxicity targets were regarded as the key targets. Then, the STRING database analyzed the key target to construct a protein-protein interaction network (PPI) and GO, and KEGG analysis was performed by the DAVID platform. Meanwhile, the PPI network and compound-target network were constructed by Cytoscape 3.9.1. Finally, Discovery Studio 2019 software was used for molecular docking verification of the two toxic compounds and the core genes.
Results:
Y and C exhibited hepatotoxicity, carcinogenicity, and mutagenicity evaluated by TOPKAT. There were 22 potential targets relating to compound Y and hepatotoxicity and reproduction toxicity and 14 potential targets relating to compound C and hepatotoxicity and reproduction toxicity. PPI network analysis showed that SRC, MAPK14, F2, PTPN1, IL2, MMP3, HRAS, and RARA might be the key targets; the KEGG analysis indicated that compounds Y and C caused hepatotoxicity through Hepatitis B, Pathways in cancer, Chemical carcinogenesis-reactive oxygen species, Epstein-Barr virus infection; compound Y and C caused reproduction toxicity through GnRH signaling pathway, Endocrine resistance, Prostate cancer, Progesterone-mediated oocyte maturation. Molecular docking results showed that 2 compounds could fit in the binding pocket of the 7 hub genes.
Conclusion:
This study preliminarily revealed the potential toxicity and possible toxicity mechanism of metformin disinfection by-products and provided a new idea for follow-up research.
Since tyrosine phosphorylation is reversible and dynamic in vivo, the phosphorylation state of proteins is controlled by the opposing roles of protein tyrosine kinases (PTKs) and protein tyrosine phosphatase (PTPs), both of which perform critical roles in signal transduction. Of these, intracellular non-receptor PTPs (PTPNs), which belong to the largest class I cysteine PTP family, are essential for the regulation of a variety of biological processes, including but not limited to hematopoiesis, inflammatory response, immune system, and glucose homeostasis. Additionally, a substantial amount of PTPNs have been identified to hold crucial roles in tumorigenesis, progression, metastasis, and drug resistance, and inhibitors of PTPNs have promising applications due to striking efficacy in antitumor therapy. Hence, the aim of this review is to summarize the role played by PTPNs, including PTPN1/PTP1B, PTPN2/TC-PTP, PTPN3/PTP-H1, PTPN4/PTPMEG, PTPN6/SHP-1, PTPN9/PTPMEG2, PTPN11/SHP-2, PTPN12/PTP-PEST, PTPN13/PTPL1, PTPN14/PEZ, PTPN18/PTP-HSCF, PTPN22/LYP, and PTPN23/HD-PTP, in human cancer and immunotherapy and to comprehensively describe the molecular pathways in which they are implicated. Given the specific roles of PTPNs, identifying potential regulators of PTPNs is significant for understanding the mechanisms of antitumor therapy. Consequently, this work also provides a review on the role of non-coding RNAs (ncRNAs) in regulating PTPNs in tumorigenesis and progression, which may help us to find effective therapeutic agents for tumor therapy.
In vivo, tyrosine phosphorylation is a reversible and dynamic process governed by the opposing activities of protein tyrosine kinases and phosphatases. Defective or inappropriate operation of these proteins leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases, including cancers. PTP1B, a non-transmembrane phosphatase, is generally considered a negative regulator of the metabolic signaling pathways and a promising drug target for type Ⅱ diabetes and obesity. Recently, PTP1B is also attracting considerable interest due to its important function and therapeutic potential in other diseases. An increasing number of studies have indicated that PTP1B plays a vital role in the initiation and progression of cancers and could be a target for new cancer therapies. Following recent advances in the aspects mentioned above, this review is focused on the major functions of PTP1B in different types of cancer and the underlying mechanisms behind these functions, as well as the potential pharmacological effects of PTP1B inhibitors in cancer therapy.
The main post-translational reversible modulation of proteins is phosphorylation and dephosphorylation, catalyzed by protein kinases (PKs) and protein phosphatases (PPs) which is crucial for homeostasis. Imbalance in this crosstalk can be related to diseases, including cancer. Plenty of evidence indicates that protein tyrosine phosphatases (PTPs) can act as tumor suppressors and tumor promoters. In gastric cancer (GC), there is a lack of understanding of the molecular aspects behind the tumoral onset and progression. Here we describe several members of the PTP family related to gastric carcinogenesis. We discuss the associated molecular mechanisms which support the down or up modulation of different PTPs. We emphasize the Helicobacter pylori (H. pylori) virulence which is in part associated with the activation of PTP receptors. We also explore the involvement of intracellular redox state in response to H. pylori infection. In addition, some PTP members are under influence by genetic mutations, epigenetics mechanisms and miRNA modulation. The understanding of multiple aspects of PTPs in GC may provide new targets and perspectives on drug development.
While protein tyrosine kinases (PTKs) play an initiative role in growth factor-mediated cellular processes, protein tyrosine phosphatases (PTPs) negatively regulates these processes, acting as tumor suppressors. Besides selective tyrosine dephosphorylation of PTKs via PTPs may affect oncogenic pathways during carcinogenesis. The PTP family contains a group of dual-specificity phosphatases (DUSPs) that regulate the activity of Mitogen-activated protein kinases (MAPKs), which are key effectors in the control of cell growth, proliferation and survival. Abnormal MAPK signaling is critical for initiation and progression stages of carcinogenesis. Since depletion of DUSP-MAPK phosphatases (MKPs) can reduce tumorigenicity, altering MAPK signaling by DUSP-MKP inhibitors could be a novel strategy in anti-cancer therapy. Moreover, Cdc25A is, a DUSP and a key regulator of the cell cycle, promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases (CDK). Cdc25A-CDK pathway is a novel mechanism in carcinogenesis. Besides the mammalian target of rapamycin (mTOR) kinase inhibitors or mammalian target of rapamycin complex 1 (mTORC1) inhibition in combination with the dual phosphatidylinositol 3 kinase (PI3K)/mTOR or AKT kinase inhibitors are more effective in inhibiting the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and cap-dependent translation. Dual targeting of the Akt and mTOR signaling pathways regulates cellular growth, proliferation and survival. Like the Cdc2-like kinases (CLK), dual-specific tyrosine phosphorylation-regulated kinases (DYRKs) are essential for the regulation of cell fate. The crosstalk between dual-specific phosphatases and dual- specific protein kinases is a novel drug target for anti-cancer therapy. Therefore, the focus of this chapter involves protein kinase modules, critical biochemical checkpoints of cancer therapy and the synergistic effects of protein kinases and anti-cancer molecules.
Colon cancer is a major health issue and number of cases are increasing every year. Diabetes mellitus is also a significant health issue that is growing day by day worldwide having negative influences on the survival of individuals. Research has shown a strong relationship between the two malignant diseases. The risk of colon cancer with patients who have type 2 diabetes mellitus has spiked by 30%. The scientific research suggests insulin has a major role in the spread of cancer and the condition unifying between the two diseases is hyperinsulinemia. Several anti-diabetic agents are used for the treatment of type 2 diabetesmellitus. However, their mechanism of action against cancer activity is a question and only a few agents have shown positive signs of action in colon cancer associated with type 2 diabetesmellitus. Hence, the identification of targets, which is common for both colon cancer, associated with type 2 diabetesmellitus has become an urgent requirement. Novel targets such as Liver X receptors, Histone deacetylase inhibitors (HDACi), Glucose Transporters (GLUTs), Peroxisome proliferator activator receptors (PPARs), Dipeptidyl peptidase-IV inhibitors (DPP4i), Cyclin-dependent kinase 4 inhibitors (CDK4i), Estrogen receptors,Mechanistic target of rapamycin (mTOR), Insulin-like growth factor receptors (IGF) are some of the targets which are common for both, type 2 diabetesmellitus and colon cancer. This current review gives an overview of the targets (using one worm) which are common for both viz. diabetes mellitus and colon cancer (two fish).
Pioneering experiments performed by Harold Varmus and Mike Bishop in 1976 led to one of the most influential discoveries in cancer research and identified the first cancer-causing oncogene called Src. Later experimental and clinical evidence suggested that Src kinase plays a significant role in promoting tumor growth and progression and its activity is associated with poor patient survival. Thus, several Src inhibitors were developed and approved by FDA for treatment of cancer patients. Tumor microenvironment (TME) is a highly complex and dynamic milieu where significant cross-talk occurs between cancer cells and TME components, which consist of tumor-associated macrophages, fibroblasts, and other immune and vascular cells. Growth factors and chemokines activate multiple signaling cascades in TME and induce multiple kinases and pathways, including Src, leading to tumor growth, invasion/metastasis, angiogenesis, drug resistance, and progression. Here, we will systemically evaluate recent findings regarding regulation of Src and significance of targeting Src in cancer therapy.
Kinases and phosphatases are major players in a variety of cellular events, including cell signaling. Aberrant activity or mutations in kinases and phosphatases can lead to diseases, such as cancer, diabetes, and Alzheimer's. Compared to kinases, phosphatases are understudied, which is partly a result of the limited methods to identify substrates. As a solution, we developed a proteomics-based method called Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS) that previously identified substrates of Ser/Thr phosphatases using small molecule inhibitors. Here, for the first time, K-BIPS was applied to identify substrates of a tyrosine phosphatase, Protein Tyrosine Phosphatase 1B (PTP1B), using siRNA knockdown conditions. Eight possible substrates of PTP1B were discovered in HEK293 cells, including the known substrate Pyruvate Kinase. In addition, L-lactate Dehydrogenase (LDHA) was validated as a novel PTP1B substrate. With the ability to use knockdown conditions with Ser/Thr or Tyr phosphatases, K-BIPS represents a general discovery tool to explore phosphatases biology by identifying unanticipated substrates.
Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Protein tyrosine phosphatase 1B (PTP1B) is a member of protein tyrosine phosphatases (PTPs) family. In our previous work, PTP1B was found to be overexpressed in ESCC tissues and made contributions to the the cell migration and invasion as well as lung metastasis of ESCC. In this study, we explored the underlying molecular mechanisms. PTP1B enhanced cell migration and invasion by promoting epidermal growth factor receptor (EGFR) expression in ESCC, which was relied on phosphatase activity of PTP1B. Using GST-pulldown combined with LC/MS/MS, we found that nonmuscle myosin IIA (MYH9) was a novel substrate of PTP1B in ESCC cells. PTP1B dephosphorylated MYH9 at Y1408, by which PTP1B up-regulated EGFR expression and enhanced cell migration and invasion in ESCC. In conclusion, our study first reported that PTP1B was the positive regulator of EGFR by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion, which revealed the regulatory mechanism of PTP1B-MYH9-EGFR axis in ESCC.
Background:
Dose rate variation is a critical factor affecting radionuclide therapy (RNT) efficacy. Relatively few studies to date have investigated the dose rate effect in RNT. Therefore, the aim of this study was to benchmark 90Y RNT (at different dose rates) against external beam radiotherapy (EBRT) in vitro and compare cell kill responses between the two irradiation processes.
Results:
Three human colorectal carcinoma (CRC) cell lines (HT29, HCT116, SW48) were exposed to 90Y doses in the ranges 1-10.4 and 6.2-62.3 Gy with initial dose rates of 0.013-0.13 Gy/hr (low dose rate, LDR) and 0.077-0.77 Gy/hr (high dose rate, HDR), respectively. Results were compared to a 6-MV photon beam doses in the range from 1-9 Gy with constant dose rate of 277 Gy/hr. The cell survival parameters from the linear quadratic (LQ) model were determined. Additionally, Monte Carlo simulations were performed to calculate the average dose, dose rate and the number of hits in the cell nucleus. For the HT29 cell line, which was the most radioresistant, the α/β ratio was found to be ≈ 31 for HDR-90Y and ≈ 3.5 for EBRT. LDR-90Y resulting in insignificant cell death compared to HDR-90Y and EBRT. Simulation results also showed for LDR-90Y, for doses ≲ 3 Gy, the average number of hits per cell nucleus is ≲ 2 indicating insufficiently delivered lethal dose. For 90Y doses [Formula: see text] 3 Gy the number of hits per nucleus decreases rapidly and falls below ≈ 2 after ≈ 5 days of incubation time. Therefore, our results demonstrate that LDR-90Y is radiobiologically less effective than EBRT. However, HDR-90Y at ≈ 56 Gy was found to be radiobiologically as effective as acute ≈ 8 Gy EBRT.
Conclusion:
These results demonstrate that the efficacy of RNT is dependent on the initial dose rate at which radiation is delivered. Therefore, for a relatively long half-life radionuclide such as 90Y, a higher initial activity is required to achieve an outcome as effective as EBRT.
There are a large number of new structure compounds with good pharmacological activity in the natural plants, can be applied to the treatment of human diseases. Finding active ingredients from the plants is one of the important ways to develop new drugs. Triterpenes are widespread in plants, and lupenone belongs to lupane type triterpenoids. Lupenone is very common natural ingredient distributed in multi-family plants including Asteraceae, Balanophoraceae, Cactaceae, Iridaceae, Musaceae, Urticaceae, Leguminosae, Bombacaceae, etc., but its distribution has no regular. The consumption of lupenone in vegetarian diet is high in human life. Pharmacological screening of lupenone revealed various pharmacological activities including anti-inflammatory, anti-virus, anti-diabetes, anti-cancer, improving Chagas disease without major toxicity. Based on these important pharmacological activities, this review provides detailed account of pre-clinical studies conducted to determine the utility of lupenone as a therapeutic and chemopreventive agent for the treatment of various diseases.
Protein tyrosine phosphorylation is critical for proper function of cells and organisms. Phosphorylation is regulated by the concerted but generically opposing activities of tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs), which ensure its proper regulation, reversibility, and ability to respond to changing physiological situations. Historically, PTKs have been associated mainly with oncogenic and pro-tumorigenic activities, leading to the generalization that protein dephosphorylation is anti-oncogenic and hence that PTPs are tumor-suppressors. In many cases PTPs do suppress tumorigenesis. However, a growing body of evidence indicates that PTPs act as dominant oncogenes and drive cell transformation in a number of contexts, while in others PTPs support transformation that is driven by other oncogenes. This review summarizes the known transforming and tumor-promoting activities of the classical, tyrosine specific PTPs and highlights their potential as drug targets for cancer therapy.
Continuous epithelial-like cell lines derived from normal adult rat liver and hepatocarcinomas were evaluated for their growth in soft agar and five properties of the cell membrane as markers for neoplastic transformation. A correlation of these properties was made to the tumorigenicity of the lines in nude mice. Growth in soft agar was a specific and sensitive marker, whereas the data on uptake of 2-deoxy-D-glucose were consistent, with high uptake being a specific but clearly not a sensitive marker. Agglutination and hemadsorption mediated by concanavalin A, multinucleation in the presence of cytochalasin B, and the cell membrane activity of adenosine triphosphatase did not correlate with tumorigenicity of the other markers for transformation. In addition, it is shown that Mycoplasma infection does not alter any of these properties but that infection can be eliminated by passage of cells through nude mice.
The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.
We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three-dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI-MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking.
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The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from human colon carcinoma tissues and tumor-derived cell lines was found to be elevated over that from normal colon tissues or cultures of normal colon mucosal cells. The elevated pp60c-src protein kinase activity in tumor tissues and in cultured colon carcinoma cells does not appear to result solely from an increase in the abundance of the c-src-encoded protein, suggesting that the specific activity of the pp60c-src tyrosine phosphotransferase is enhanced. These results raise the possibility that activation of the pp60c-src protein kinase may contribute to the genesis of human colon tumors.
We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.
The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.
NIH 3T3 mouse cells were transfected with plasmids that induce efficient expression of either (i) the Rous sarcoma virus v-src gene, (ii) the chicken c-src gene, or (iii) a recombinant gene combining the 5' portion of c-src with the 3' end of v-src. Focus formation in tissue culture and formation of large colonies in soft agar did not occur in cells transfected with c-src. Cells transfected with c-src expression plasmids did not form foci but were isolated using a coselectable biological marker. They display morphological and substrate-independent growth characteristics intermediate between those of normal and v-src-transformed mouse cells, and lysates from these cells have enhanced in vitro tyrosine kinase activity. Transfection with the c-src-v-src recombinant induced focus formation with an efficiency similar to that obtained with a v-src expression plasmid. These results imply that v-src-induced transformation does not result just from overexpression of an essentially normal cellular protein but, at least in part, depends on the mutations distinguishing the cellular and viral proteins.
We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.
Normal human melanocytes, and some human melanoma cell lines, contain c-SRC which is constitutively activated by hypophosphorylation of tyrosine 530. We investigated the possibility that the activation of c-SRC in melanocytes might be attributable to elevated levels of tyrosine 530-directed protein tyrosine phosphatase activity in these cells. We found three times more of this phosphatase activity in cell extracts from melanocytes compared to fibroblasts. The majority of the tyrosine 530-dephosphorylating activity was present in the particulate fraction of cell homogenates, where c-SRC is also located. Treatment of melanocytes with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, caused inactivation of c-SRC. From these results, we conclude that activation of c-SRC in human melanocytes may be attributed to an elevated level of protein tyrosine phosphatase activity directed against tyrosine 530.
Activation of the tyrosine kinase of the c-src gene product, pp60c-src, has been shown to occur in nearly every primary colorectal carcinoma, and is found as early as in polyps of high malignant potential. However, no studies have addressed potential pp60c-src changes which occur during progression. To examine this question, we have studied kinase activity and protein levels in 7 colonic polyps, 19 primary lesions, and 19 liver metastases relative to normal colonic mucosa. Significant increases in tyrosine kinase activity were seen as early as in colonic polyps of high malignant potential. Further increases were observed in activity and level in primary tumors. However, the greatest increases in activity and protein levels were observed in liver metastases. Additionally, six metastatic lesions were obtained in which synchronous primary tumor was resected. In each of these liver metastases, pp60c-src activity and level were significantly increased relative to the corresponding primary tumor, as well as to normal colonic mucosa. Our results demonstrate that progression of colon primary tumors to liver metastases correlates with increased pp60c-src kinase activity and protein level.
Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.
The Src family of kinases are held in an inactive state by interaction of their SH2 domain with a C-terminal phosphotyrosine. Dephosphorylation of this site can reactivate Src; however, recent evidence suggests that activation can also occur without dephosphorylation. In this study, platelet-derived growth factor receptor phosphorylation of Src on Tyr-213 specifically blocked binding of its SH2 domain to a phosphopeptide corresponding to the C-terminal regulatory sequence, while binding to other sequences, such as the platelet-derived growth factor receptor or a peptide from the epidermal growth factor receptor, was unaffected. Consequently, Src was activated over 50-fold. This is the first demonstration of regulation of a SH2 domain specificity by post-translational modification and is likely to be a general mechanism for regulation of all Src-like kinases.
Treatment of quiescent NIH3T3 cells with PDGF BB results in the transient activation and hyperphosphorylation of the protein-tyrosine kinase, c-Src. These effects correlate with novel serine and tyrosine phosphorylations in the N-terminal non-catalytic region of the molecule, which contains an SH3 and SH2 domain. In this study, a site of PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the SH3 domain; Tyr 138 is exposed on the SH3 peptide binding surface. This same site is phosphorylated in vitro by the PDGF receptor when purified baculovirus-expressed c-Src is complexed with the activated receptor. Phosphorylation of Tyr 138 required association of c-Src with the PDGF receptor via its SH2 domain. When a c-Src Phe 138 mutant was stably expressed in Src- mouse fibroblasts, it was activated to the same extent as wild type c-Src following PDGF stimulation, indicating that phosphorylation of this site is not required for PDGF-mediated activation. However, Tyr 138 phosphorylation was found to diminish SH3 domain peptide ligand binding ability in vitro.
We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot
bind p130Cas(PA-PTP1B), causes substantial tyrosine dephosphorylation of p130Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290–31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B,
inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130Cas and two proteins that are associated with p130Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation
suppression by PTP1B requires a functional SH3 ligand and suggest that p130Cas may represent an important physiological target of PTP1B in cells.
Protein tyrosine phosphatase (PTP) 1B has long been known to regulate cell proliferation negatively, but the mechanism by which this inhibition occurs is poorly defined. We have shown previously that PTP1B binds to, and dephosphorylates, p130(Cas) (Crk-associated substrate) [1], a protein that is thought to play a role in integrin signaling [2,3]. In this report, we present evidence that PTP1B interferes specifically with cell-adhesion-stimulated, but not growth-factor-stimulated, signaling pathways. In rat fibroblasts that overexpress PTP1B, the activation of mitogen-activated protein (MAP) kinase by growth factors was not affected, but activation by cell adhesion was markedly impaired. The inhibition of adhesion-dependent MAP kinase activation by PTP1B required an intact proline-rich region in the carboxyl terminus of PTP1B, a region we have shown to mediate binding to the Src-homology 3 (SH3) domain of p130Cas [1]. Overexpression of wild-type PTP1B, but not of a proline-to-alanine mutant form (PA-PTP1B) that is unable to bind or dephosphorylate p130Cas, interfered with cell spreading, cytoskeletal architecture, and the formation of focal adhesion complexes. Cells overexpressing wild-type PTP1B also displayed markedly reduced migration in response to a fibronectin gradient, whereas cells expressing the PA-PTP1B mutant migrated normally. These data indicate that PTP1B exerts its inhibitory effects via proline-dependent interactions with one or more critical components of the adhesion-dependent signaling apparatus, and suggest that one of these components may be p130Cas.
To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in beta1-integrin- mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with beta1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.
The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.
Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases.
c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src
expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast
cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the
carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S
cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed
against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the
c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments
identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines
with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells.In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate
that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell
lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.
The protein tyrosine phosphatase PTP1B is responsible for negatively regulating insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase (IRK) activation segment. Here, by integrating crystallographic, kinetic, and PTP1B peptide binding studies, we define the molecular specificity of this reaction. Extensive interactions are formed between PTP1B and the IRK sequence encompassing the tandem pTyr residues at 1162 and 1163 such that pTyr-1162 is selected at the catalytic site and pTyr-1163 is located within an adjacent pTyr recognition site. This selectivity is attributed to the 70-fold greater affinity for tandem pTyr-containing peptides relative to mono-pTyr peptides and predicts a hierarchical dephosphorylation process. Many elements of the PTP1B-IRK interaction are unique to PTP1B, indicating that it may be feasible to generate specific, small molecule inhibitors of this interaction to treat diabetes and obesity.
Previous biochemical evidence has yielded conflicting models for the role of protein tyrosine phosphatase-1B (PTP-1B) in the regulation of integrin signaling. Thus, to establish the physiological relevance for such a role, we employed a genetic approach by generating embryonic fibroblasts from PTP-1B knockout mice. Both primary fibroblasts and their derived cell lines were used in this study. Immortalization of wild-type primary cells with the SV40 Large T antigen resulted in a dramatic increase in the endogenous expression of PTP-1B, suggesting a role during transformation. Moreover, the absence of PTP-1B in the transformed cell lines led to a more pronounced effect on different pathways of fibronectin-mediated signaling compared with the untransformed state. Specifically, p130(Cas) phosphorylation, Erk activation as well as cell spreading were delayed in PTP-1B-deficient cells, compared with their wild-type counterparts. Interestingly, this attenuation in integrin-mediated events closely resembles that of Src-deficient fibroblasts. Indeed, PTP-1B deficient, transformed fibroblasts held in suspension do exhibit a hyperphosphorylation of the inhibitory site (Tyr-527) of Src, compared with their wild-type counterparts. These results establish PTP-1B as a positive physiological regulator of integrin signaling in transformed cells, acting upstream of Src Tyr-527 dephosphorylation that leads to several adhesion-dependent events.
During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) α is enhanced, and its inhibitory binding to
Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in
mitosis. We show here that these effects are abrogated by mutation of Ser180 and/or Ser204, the sites of protein kinase C-mediated phosphorylation within PTPα. Moreover, either a Ser-to-Ala substitution or serine
dephosphorylation specifically eliminated the ability of PTPα to dephosphorylate and activate Src even during interphase.
This explains why the substitutions eliminated PTPα transforming activity, even though PTPα interphase dephosphorylation of
nonspecific substrates was only slightly decreased. This occurred without change in the phosphorylation of PTPα at Tyr789, which is required for “phosphotyrosine displacement” during Src dephosphorylation. Thus, in addition to increasing PTPα
nonspecific catalytic activity, Ser180 and Ser204 phosphorylation (along with Tyr789 phosphorylation) regulates PTPα substrate specificity. This involves serine phosphorylation-dependent differential modulation
of the affinity of Tyr(P)789for the Src and Grb2 SH2 domains. The results suggest that protein kinase C may participate in the mitotic activation of PTPα
and Src and that there are intramolecular interactions between the PTPα C-terminal and membrane-proximal regions that are
regulated, at least in part, by serine phosphorylation.
Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fisher's exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (P < 0.01, Fisher's exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.
Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.
Protein tyrosine phosphatase (PTP) 1B has been implicated as a negative regulator of multiple signaling pathways downstream of receptor tyrosine kinases. Inhibition of this enzyme was initially thought to potentially lead to increased oncogenic signaling and tumorigenesis. Surprisingly, we show that platelet-derived growth factor-stimulated extracellular-regulated kinase signaling in PTP1B-deficient cells is not significantly hyperactivated. Moreover, these cells exhibit decreased Ras activity and reduced proliferation by way of previously uncharacterized pathways. On immortalization, PTP1B-deficient fibroblasts display increased expression of Ras GTPase-activating protein (p120RasGAP). Furthermore, we demonstrate that p62Dok (downstream of tyrosine kinase) is a putative substrate of PTP1B and that tyrosine phosphorylation of p62Dok is indeed increased in PTP1B-deficient cells. Consistent with the decreased Ras activity in cells lacking PTP1B, introduction of constitutively activated Ras restored extracellular-regulated kinase signaling and their proliferative potential to those of WT cells. These results indicate that loss of PTP1B can lead to decreased Ras signaling, despite enhanced signaling of other pathways. This finding may in part explain the absence of increased tumor incidence in PTP1B-deficient mice. Thus, PTP1B can positively regulate Ras activity by acting on pathways distal to those of receptor tyrosine kinases.
Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the beta-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.
Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic
target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple
signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance
of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B
in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading
and migration to fibronectin, increases Tyr527 phosphorylation in Src, and decreases phosphorylation of FAK, p130Cas, and ERK1/2. In addition, PTP1B “substrate-trapping” mutants bind Tyr527-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes
integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr527 and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130Cas, and that the proline-rich motif PPRPPK (residues 309–314) in PTP1B is essential for the complex formation. We suggest that
the specificity of PTP1B for Src pTyr527 is mediated by protein-protein interactions involving the docking protein p130Cas with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr527 motif.
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.
The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates
a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein α-actinin at Tyr-12. Here we report that
protein-tyrosine phosphatase 1B (PTP 1B) is an α-actinin phosphatase. PTP 1B-dependent dephosphorylation of α-actinin was
seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type
α-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the
α-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered
by PTP 1B. In addition, we found that phosphorylated α-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation
at Tyr-397 in FAK triggered by wild-type α-actinin and PTP 1B caused a significant increase in cell migration. We propose
that phosphorylated α-actinin disrupts the FAK·Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel
feedback loop involving phosphorylated α-actinin and PTP 1B that regulates FAK·Src interaction and cell migration.
We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three-dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI-MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking.
The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.
We have previously shown that overexpressed chicken pp60c-src has retarded mobility, novel serine/threonine phosphorylation, and enhanced kinase activity during NIH 3T3 cell mitosis. Here we show that novel mitotic phosphorylations occur at Thr 34, Thr 46, and Ser 72. The possibility, previously raised, that Ser 17 is dephosphorylated during mitosis is excluded. The phosphorylated sites lie in consensus sequences for phosphorylation by p34cdc2, the catalytic component of maturation promoting factor (MPF). Furthermore, highly purified MPF from metaphase-arrested Xenopus eggs phosphorylated both wild-type and kinase-defective pp60c-src at these sites. Altered phosphorylation alone is sufficient to account for the large retardation in mitotic pp60c-src electrophoretic mobility: phosphorylation of normal pp60c-src by MPF retarded mobility and dephosphorylation of mitotic pp60c-src restored normal mobility. These results suggest that pp60c-src is one of the targets for MPF action, which may account in part for the pleiotropic changes in protein phosphorylation and cellular architecture that occur during mitosis.
Elevated levels of pp60c-src tyrosine kinase activity have been implicated in both tumorigenesis and cell differentiation. We have found a 2- to 4-fold elevation in pp60c-src specific activity in certain human melanoma cell lines compared to human foreskin fibroblasts. This activation of pp60c-src did not appear to be related to melanoma tumor progression, because when normal human epidermal melanocytes were examined, it was found that they contained pp60c-src having a 7-fold elevation in specific activity compared to pp60c-src from human fibroblasts. It was determined that pp60c-src from melanocytes was not the neuronal form, pp60c-src+. Melanocyte pp60c-src exhibited a reduced level of phosphorylation on its carboxyl-terminal regulatory site, tyrosine 530, which might be responsible for its elevated specific activity. These results suggest that, in melanocytes, regulation of tyrosine 530 phosphorylation-dephosphorylation favors activation of pp60c-src. This activation may be involved in the growth, differentiation, or function of human melanocytes.
We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).
Oncogenesis by avian sarcoma virus is attributable to a single viral gene (src) which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. A closely related protein, pp60proto-src, occurs in uninfected cells from a wide variety of vertebrate species and is presumed to be the product of a cellular gene that served as progenitor for src. We explored the location of these proteins within the cell by using immunoprecipitation to analyze subcellular fractions prepared from avian sarcoma virus-transformed rat and chicken cells and from uninfected rat cells. We found that both pp60src and pp60proto-src were associated with the plasma membrane as active protein kinases and could be recovered efficiently only by disrupting the membranes with nonionic detergent. Our findings, in conjunction with those of other investigators, indicate that both proteins are embedded in the membrane by means of a hydrophobic domain(s); available evidence indicates that pp60src is not exposed on the surface of the cell but is accessible at the cytoplasmic aspect of the plasma membrane. These conclusions lend credence to two current speculations. First, pp60src and pp60proto-src may have similar or even identical functions. Second, neoplastic transformation may originate from derangements in the plasma membrane or its affiliated structures.
The retroviral oncogene v-src arose by transduction of the cellular gene c-src. The similarity between these genes raised the possibility that c-src might be able to elicit neoplastic growth. We explored this by constructing a chimeric plasmid that allows the expression of chicken c-src. A rat cell line containing ten times the normal intracellular level of pp60c -src was isolated after transfecting rat-2 cells with the chimeric DNA. These cells produce the protein encoded by c-src ( pp60c -src) in quantities at least three times greater than required to achieve transformation by the product of v-src ( pp60v -src). The cells remain phenotypically normal, contain actin cables, and do not grow in soft agar. However, transfection of the cell line containing elevated cells of pp60c -src or Rat-2 cells with a molecular clone of v-src produces cells that exhibit properties of biologically transformed cells: round morphology, disrupted actin cables, and ability to grow in soft agar.
The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genetic and biochemical data have suggested that this negative regulation requires an intact Src homology 2 (SH2) domain. Since SH2 domains recognize phosphotyrosine, it is possible that these two non-catalytic domains associate, and thereby repress c-Src kinase activity. Consistent with this model, an isolated Src SH2 domain expressed in bacteria as a GST fusion protein bound in vitro to a synthetic phosphotyrosine-containing peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the tail peptide, and was modified by both the length and sequence of the peptide. Competition experiments indicated only a moderate binding affinity between the Src SH2 domain and the phosphorylated tail. A distinct phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail phosphorylation site for binding to the SH2 domain. Indeed, this activation was competitively inhibited by purified bacterial Src SH2 domain. These data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactive form.
In greater than 80% of colon tumors and established cell lines, the specific activities of the protein tyrosine kinases pp60(c-src) and pp62(c-yes) are increased with respect to normal colonic epithelial cells. However, no mutations in either gene have been identified in colon tumors. Therefore, the possible biological consequences of activations of these protein tyrosine kinases in colon tumors have been unclear. To determine if pp60(c-src) activation affects growth and tumorigenicity of established colon tumor cell lines, an antisense expression vector that specifically reduces pp60(c-src) expression was constructed. The vector was transfected into HT 29 cells, an established colon tumor cell line in which both pp60(c-src) and pp62(c-yes) are activated. Two stable subclones were isolated in which pp60(c-src) but not pp62(c-yes) expression and activity were reduced. These established cell lines proliferated more slowly than parental cells proportionately to reduction in pp60(c-src) expression. When injected into nude mice, antisense transfected cells formed slow-growing tumors; however, the rate of tumor growth was reduced far greater than would be predicted from decreased proliferation rates in tissue culture. In contrast, stable subclones transfected with a comparable "sense" expression vector were unaltered in growth rates in tissue culture and in nude mice with respect to parental HT 29 cells. These data demonstrate that the activation of pp60(c-src) alone contributes to the tumorigenicity of HT 29 cells, a cell line widely used as a model for biological properties of colon carcinoma. Furthermore, because pp60(c-src) and pp62(c-yes) appear redundant to the growth regulation of normal colonic epithelial cells, the data suggest that src-specific inhibitors might be of therapeutic value for colon cancer.
The nonreceptor tyrosine kinase, c-src, and the steroid hormone,1,25-dihydroxyvitamin D3(1,25(OH)2D3), are essential to development of the osteoclast phenotype. On the other hand, functional relationships between the activities of c-src and 1,25(OH)2D3 are as yet unknown. To determine if 1,25(OH)2D3 modulates c-src in osteoclastogenesis, we tested the steroid's effect on avian marrow-derived osteoclast precursors. We find c-src mRNA and immunoprecipitable c-src protein (pp60c-src) unaltered by 72 h exposure of these cells to 1,25(OH)2D3 (10(-11) to 10(-9) M). Despite no quantitative change in pp60c-src, in vitro kinase assay of the immune complex reveals 1,25(OH)2D3 dose-dependently accelerates the catalytic activity of pp60c-src, enhancing its autophosphorylation and phosphorylation of exogenous substrate. This observation represents the first documentation, in nontransformed cells, of humoral induction of pp60c-src kinase. Consistent with the fact pp60c-src is activated by dephosphorylation of tyrosine 527 (Y527), the phosphotyrosine content of the pp60c-src immunoprecipitate, measured by immunoblot, is decreased by 1,25(OH)2D3. Alternatively, mRNA and protein levels of c-src kinase (CSK), which inactivates pp60c-src by phosphorylating Y527, are not altered by the steroid. In contrast, 1,25(OH)2D3 enhances mRNA and especially protein levels of avian protein tyrosine phosphatase lambda (PTP lambda), an enzyme specifically activating pp60c-src by dephosphorylating Y527 [Fang et al. (1994): J Biol Chem 269:20194-20200]. Thus, treatment of avian osteoclast precursors with 1,25(OH)2D3 accelerates the catalytic activity of pp60c-src independent of protein expression. Activation of the kinase may occur via the Y527 dephosphorylating enzyme PTP, expression of which, we show for the first time, is regulated.
Whereas genetic paradigms are now defined for the development of human colon cancer, little is known regarding the mechanisms that regulate development of the metastatic phenotype. Recent reports have indirectly linked the expression and activation of c-Src to the process of human colon cancer metastasis. Whereas v-Src, a highly activated mutational derivative of c-Src, has been shown to induce metastasis, normal c-Src has not been tested for this property. We hypothesized that c-Src overexpression in the milieu of a poorly metastatic cancer cell might permit the development of a highly metastatic cell. Two poorly metastatic human colon cancer cell lines were stably transfected with expression vectors encoding normal human c-Src. Clones producing 4-10-fold more c-Src than controls were injected s.c. and intrasplenically into the nude mouse to assess primary tumor growth and liver metastatic potential. Whereas metastatic potential was unaffected, primary tumor growth in vivo was significantly enhanced by c-Src overexpression. No effects on rates of tumor cell proliferation were seen in vitro. Our findings suggest that normal c-Src may be necessary but is insufficient for the induction of the metastatic phenotype.
The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.
Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.
Since the discovery of the v-src and c-src genes and their products, much progress has been made in the elucidation of the structure, regulation, localization, and function of the Src protein. Src is a non-receptor protein tyrosine kinase that transduces signals that are involved in the control of a variety of cellular processes such as proliferation, differentiation, motility, and adhesion. Src is normally maintained in an inactive state, but can be activated transiently during cellular events such as mitosis, or constitutively by abnormal events such as mutation (i.e. v-Src and some human cancers). Activation of Src occurs as a result of disruption of the negative regulatory processes that normally suppress Src activity, and understanding the various mechanisms behind Src activation has been a target of intense study. Src associates with cellular membranes, in particular the plasma membrane, and endosomal membranes. Studies indicate that the different subcellular localizations of Src could be important for the regulation of specific cellular processes such as mitogenesis, cytoskeletal organization, and/or membrane trafficking. This review will discuss the history behind the discovery and initial characterization of Src and the regulatory mechanisms of Src activation, in particular, regulation by modification of the carboxy-terminal regulatory tyrosine by phosphatases and kinases. Its focus will then turn to the different subcellular localizations of Src and the possible roles of nuclear and perinuclear targets of Src. Finally, a brief section will review some of our present knowledge regarding Src involvement in human cancers.
We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.
Coordinated tyrosine phosphorylation is essential for signalling pathways regulated by insulin and leptin. Type 2 diabetes and obesity are characterised by resistance to hormones insulin and leptin, possibly due to attenuated or diminished signalling from the receptors. Pharmacological agents capable of inhibiting the negative regulator(s) of the signalling pathways are expected to potentiate the action of insulin and leptin and therefore be beneficial for the treatment of Type 2 diabetes and obesity. A large body of data from cellular, biochemical, mouse and human genetic and chemical inhibitor studies have identified protein tyrosine phosphatase 1B (PTP1B) as a major negative regulator of both insulin and leptin signalling. In addition, evidence suggests that insulin and leptin action can be enhanced by the inhibition of PTP1B. Consequently, PTP1B has emerged as an attractive novel target for the treatment of both Type 2 diabetes and obesity. The link between PTP1B and diabetes and obesity has led to an avalanche of research dedicated to finding inhibitors of this phosphatase. With the combined use of structure and medicinal chemistry, several groups have demonstrated that it is feasible to obtain small-molecule PTP1B inhibitors with the requisite potency and selectivity. The challenge for the future will be to transform potent and selective small molecule PTP1B inhibitors into orally available drugs with desirable physicochemical properties and in vivo efficacies.
Although initially viewed as housekeeping enzymes, research over the last 15 years has revealed that the protein tyrosine phosphatases (PTPs) are critical regulators of tyrosine phosphorylation-dependent signaling events and may represent novel targets for therapeutic intervention in a variety of human diseases. In this review I will describe some of the key advances in the characterization of the structure, regulation and function of the prototypic PTP, PTP1B, and illustrate how our understanding of the properties of this enzyme has revealed principles that apply to the PTP family as a whole.
Receptor PTPalpha is a widely expressed transmembrane enzyme enriched in brain. PTPalpha knockout (PTPalpha(-/-)) mice are viable and display no gross abnormalities. Brain and embryo derived fibroblast src and fyn activity is reduced to <50% in PTPalpha(-/-) mice. These protein kinases are implicated in multiple aspects of neuronal development and function. However, the effect of the loss of function of the PTPalpha gene on behavior has yet to be investigated. PTPalpha(-/-) and WT mice were tested for anxiety, swimming ability, spatial learning, cued learning, locomotor activity, and novel object recognition (NOR). PTPalpha(-/-) mice were indistinguishable from WT in swimming ability, cued learning and novel object recognition. Knockout mice showed decreased anxiety without an increase in head dips and stretch-attend movements. During Morris water maze (MWM) learning, PTPalpha(-/-) mice had increased latencies to reach the goal compared to WT on acquisition, but no memory deficit on probe trials. On reversal learning, knockout mice showed no significant effects. PTPalpha(-/-) mice showed decreased exploratory locomotor activity, but responded normally to a challenge dose of D-methamphetamine. The data suggest that PTPalpha serves a regulatory function in learning and other forms of neuroplasticity.
As in other fields of biomedical research, the use of gene-targeted mice by homologous recombination in embryonic stem cells has provided important findings on the function of several members of the protein tyrosine phosphatase (PTP) family. For instance, the phenotypic characterization of knockout mice has been critical in understanding the sites of action of the related PTPs protein tyrosine phosphatase 1B (PTP1B) and T-cell-PTP (TC-PTP). By their increased insulin sensitivity and insulin receptor hyperphosphorylation, PTP1B null mice demonstrated a clear function for this enzyme as a negative regulator of insulin signaling. As well, TC-PTP has also been recently involved in insulin signaling in vitro. Importantly, the high identity in their amino acid sequences suggests that they must be examined simultaneously as targets of drug development. Indeed, they possess different as well as overlapping substrates, which suggest complementary and overlapping roles of both TC-PTP and PTP1B. Here, we review the function of PTP1B and TC-PTP in diabetes, obesity, and processes related to cancer.