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A compendium of ERK targets
Abstract
The RAF-MEK-ERK cascade is one of the most studied signaling pathways as it controls many vital cellular programs. There has been an immense amount of effort to determine ERK target proteins that are involved in regulating these programs. Classical biochemical and genetic approaches have identified hundreds of direct ERK substrates, and with the advent of phospho-proteomic technologies, numerous studies have expanded the number of ERK target proteins. Here, we compile a comprehensive ERK target phospho-site archive in which we gathered information from various research studies in a database to form a searchable compendium of ERK targets. This article is protected by copyright. All rights reserved.
... ERK1/2 are activated by various extracellular triggers such as GPCRs, integrins, and receptor tyrosine kinases, and are responsible for the induction of cellular responses such as proliferation, differentiation, and cell survival. The cascade is involved in the development and progression of many diseases including cancer, heart failure, developmental diseases, and autoimmune diseases, but are also vital for many physiological effects such as protection from cell death [34][35][36][37][38][39][40][41]. Despite the many triggers that activate this central signaling cascade, it is unclear how ERK1/2 can transmit specific and controlled cellular responses. ...
... The activation of these substrates may vary depending on the type of extracellular stimuli, the availability of the scaffold proteins, and on their subcellular localization. Substrates of ERK1/2 have been identified, for example, in the cytoplasm, mitochondria, endoplasmatic reticulum, and particularly in the nucleus [35,38,42]. Thus, a tight control and understanding of the activating and modifying signals is central to direct the effects of ERK1/2 to the desired cellular outcome. ...
... The phosphorylation of the effector kinases ERK1 and ERK2 at the Thr and Tyr residues within the Thr-X-Tyr motif of their activation loop (Thr183 and Thr185 in mouse ERK2) leads to their activation and the subsequent phosphorylation of a vast array of substrates localized in all cellular compartments. These substrates include protein kinases, cell signaling, receptors, cytoskeletal proteins, and nuclear transcriptional regulators [38,57,58]. Several mechanisms are thought to contribute to the specificity of the signaling scenario despite the numerous direct and indirect targets of ERK1/2. ...
Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity.
... Autophosphorylation by the insulin receptor tyrosine kinase is accompanied by tyrosine phosphorylation of receptor substrates, such as insulin receptor substrate (IRS) and Src homology 2 domain-containing transforming proteins (SHC) proteins. Phosphorylation of IRS allows binding of phosphatidylinositol-3-kinase (PI3K) and synthesis of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), which eventually leads to the phosphorylation and activation of the serine/threonine-specific protein kinase B (AKT). Upon activation, AKT interacts with several substrates which mediate anabolic effects of insulin; these include glucose uptake, glycogen synthesis, de novo lipogenesis, and protein synthesis [2]. ...
... Additional pathways triggered by the activated insulin receptor comprise phosphorylation of SHC, followed by activation of the Rat sarcoma (Ras)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway. The terminal kinase ERK is a mitogen-activated kinase promoting cell proliferation and further cellular activities including protein synthesis [3]. Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase 4 and subsequent hydrogen peroxide-mediated inhibition of phosphatase and tensin homolog (PTEN), which is an important negative regulator of PI3K signaling [4] (Fig. 1). ...
Background:
Insulin shares a limited physiological concentration range with other endocrine hormones. Not only too low, but also too high systemic insulin levels are detrimental for body functions.
Main body:
The physiological function and clinical relevance of insulin are usually seen in association with its role in maintaining glucose homeostasis. However, insulin is an anabolic hormone which stimulates a large number of cellular responses. Not only too low, but also excess insulin concentrations are detrimental to the physiological balance. Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening the efficiency of insulin signaling ("insulin resistance"), this is not the case for most other hormonal actions of insulin, including the promotion of protein synthesis, de novo lipogenesis, and cell proliferation; the inhibition of lipolysis, of autophagy-dependent cellular turnover, and of nuclear factor E2-related factor-2 (Nrf2)-dependent antioxidative; and other defense mechanisms. Hence, there is no general insulin resistance but selective impairment of insulin signaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthase (eNOS). Because of the largely unrestricted insulin signaling, hyperinsulinemia increases the risk of obesity, type 2 diabetes, and cardiovascular disease and decreases health span and life expectancy. In epidemiological studies, high-dose insulin therapy is associated with an increased risk of cardiovascular disease. Randomized controlled trials of insulin treatment did not observe any effect on disease risk, but these trials only studied low insulin doses up to 40 IU/day. Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes from Mendelian randomization studies comparing individuals with genetically controlled low or high insulin production.
Conclusions:
The detrimental actions of prolonged high insulin concentrations, seen also in cell culture, argue in favor of a lifestyle that limits circadian insulin levels. The health risks associated with hyperinsulinemia may have implications for treatment regimens used in type 2 diabetes.
... That activated Braf can phenocopy mutant Kras and, together with Tp53 mutations, drive full development of metastatic PDAC in mouse models, which supports the key role of the MAPK cascade in driving Kras-dependent PDAC growth (9). While the substrates of RAF and MEK kinases are highly restricted, ERK1/2 serine/threonine kinase activation can cause direct or indirect phosphorylation of a diverse spectrum of more than one thousand proteins (10). Since ERK substrates include other protein kinases (e.g., ribosomal S6 kinases 1-4, MAPKinteracting kinase 2, and mitogen-and stress-activated kinases 1/2), ERK activation can regulate a highly diverse phosphoproteome (11). ...
... Nevertheless, many of the 28 KRAS-regulated kinases are not known to be directly associated with ERK signaling. Only eight (CKIIε, CDK1, CHK1, ERK1, ephrin type-A receptor 2, SRPK2, TRAF and NCIK-interacting protein kinase, and TTK) are listed in a recent compilation of direct or indirect ERK substrates from 14 different phosphoproteomic studies (10). However, it is still possible that some of the other 20 KRAS-regulated kinases are ERK substrates. ...
Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared to WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.
... MAPK cascades play a central role in human cancer and are hyperactivated in a large variety of tumors [36,37]. At the end of the MAPK cascade, ERK kinases translocate to the nucleus and phosphorylate a large spectrum of substrates, mostly transcription factors that are involved in a variety of processes, such as proliferation, survival, and differentiation, in a highly contextdependent manner [38,39]. It has been reported that the extracellular signal-regulated kinases ERK-1 and ERK-2 are evolutionarily conserved, ubiquitous serine-threonine kinases that regulate cellular signaling under both normal and pathological conditions [40]. ...
Ankyrin repeat and fibronectin type III domain containing 1 (ANKFN1) is reported to be involved in human height and developmental abnormalities, but the expression profile and molecular function of ANKFN1 in hepatocellular carcinoma (HCC) remain unknown. This study aimed to evaluate the clinical significance and biological function of ANKFN1 in HCC and investigate whether ANKFN1 can be used for differential diagnosis in HCC. Here, we showed that ANKFN1 was upregulated in 126 tumor tissues compared with adjacent nontumorous tissues in HCC patients. The upregulation of ANKFN1 in HCC was associated with cirrhosis, alpha-fetoprotein (AFP) levels and poor prognosis. Moreover, silencing ANKFN1 expression suppressed HCC cell proliferation, migration, invasion, and metastasis in vitro and subcutaneous tumorigenesis in vivo. However, ANKFN1 overexpression promoted HCC proliferation and metastasis in an orthotopic liver transplantation model and attenuated the above biological effects in HCC cells. ANKFN1 significantly affected HCC cell proliferation by inducing G1/S transition and cell apoptosis. Mechanistically, we demonstrated that ANKFN1 promoted cell proliferation, migration, and invasion via activation of the cyclin D1/Cdk4/Cdk6 pathway by stimulating the MEK1/2-ERK1/2 pathway. Moreover, ANKFN1-induced cell proliferation, migration, and invasion were partially reversed by ERK1/2 inhibitors. Taken together, our results indicate that ANKFN1 promotes HCC cell proliferation and metastasis by activating the MEK1/2-ERK1/2 signaling pathway. Our work also suggests that ANKFN1 is a potential therapeutic target for HCC.
... In particular, activating mutations of the MAPK/ERK signaling pathway are the key for melanoma development and progression and are typically associated with a worse prognosis [5]. The activation of this pathway, in fact, promotes cell detachment from the extracellular matrix and cellular motility and activates a number of transcription factors and nuclear proteins implicated in cell cycle regulation [6]. ...
Understanding the role of mitogen-activated protein kinase (MAPK) pathway-activating mutations in the development and progression of melanoma and their possible use as therapeutic targets has substantially changed the management of this neoplasm, which, until a few years ago, was burdened by severe mortality. However, the presence of numerous intrinsic and extrinsic mechanisms of resistance to BRAF inhibitors compromises the treatment responses’ effectiveness and durability. The strategy of overcoming these resistances by combination therapy has proved successful, with the additional benefit of reducing side effects derived from paradoxical activation of the MAPK pathway. Furthermore, the use of other highly specific inhibitors, intermittent dosing schedules and the association of combination therapy with immune checkpoint inhibitors are promising new therapeutic strategies. However, numerous issues related to dose, tolerability and administration sequence still need to be clarified, as is to be expected from currently ongoing trials. In this review, we describe the clinical results of using BRAF inhibitors in advanced melanoma, with a keen interest in strategies aimed at overcoming resistance.
... The RAF-MEK-ERK cascade regulates important physiological functions primarily through the activity of ERK, which phosphorylates a large number of substrates that perform diverse molecular functions ( Table 1). Upstream of ERK, RAF and MEK kinases are activated in a context dependent manner for initiating cell-specific outcomes [54,55]. ...
The RAF-MEK-ERK signaling cascade is a well-characterized MAPK pathway involved in cell proliferation and survival. The three-layered MAPK signaling cascade is initiated upon RTK and RAS activation. Three RAF isoforms ARAF, BRAF and CRAF, and their downstream MEK1/2 and ERK1/2 kinases constitute a coherently orchestrated signaling module that directs a range of physiological functions. Genetic alterations in this pathway are among the most prevalent in human cancers, which consist of numerous hot-spot mutations such as BRAFV600E. Oncogenic mutations in this pathway often override otherwise tightly regulated checkpoints to open the door for uncontrolled cell growth and neoplasia. The crosstalk between the RAF-MEK-ERK axis and other signaling pathways further extends the proliferative potential of this pathway in human cancers. In this review, we summarize the molecular architecture and physiological functions of the RAF-MEK-ERK pathway with emphasis on its dysregulations in human cancers, as well as the efforts made to target the RAF-MEK-ERK module using small molecule inhibitors.
... Direct ERK interactors identified for both mouse and human. Targets were primarily obtained from a compendium of ERK targets [26]. ...
Integration of cellular responses to extracellular cues is essential for cell survival and adaptation to stress. Extracellular signal-regulated kinase (ERK) 1 and 2 serve an evolutionarily conserved role for intracellular signal transduction that proved critical for cardiomyocyte homeostasis and cardiac stress responses. Considering the importance of ERK1/2 in the heart, understanding how these kinases operate in both normal and disease states is critical. Here, we review the complexity of upstream and downstream signals that govern ERK1/2-dependent regulation of cardiac structure and function. Particular emphasis is given to cardiomyocyte hypertrophy as an outcome of ERK1/2 activation regulation in the heart.
... Two motifs have been described, the D-and F-motifs, that can cooperate to enhance the substrate affinity of ERK and to set phosphorylation kinetics [7]. ERK1/2 phosphorylate more than 600 proteins, leading to responses such as cell cycle progression, proliferation, cytokinesis, transcription, differentiation, senescence, cell death, migration, formation of GAP junctions, actin and microtubule networks, neurite extension, cell adhesion and motility, survival and apoptosis [8]. To ensure that these cell responses are adaptive to stimuli in space and time, a fine regulation of MAPK signaling is thus necessary. ...
Protein phosphorylation represents a rapid and reversible post-translational regulation that enables a fast control of protein activation that play key roles in cell signaling. For instance, Mitogen Activated Protein Kinase (MAPK) pathways are activated upon sequential phosphorylations, resulting in phosphorylation of cytosol and nuclear targets. We focus here on MAPK ERK1/2 signaling that accounts for diverse cellular responses such as cell cycle progression, proliferation, differentiation, senescence, migration, formation of GAP junctions, cell adhesion, cell motility, survival and apoptosis. We review the role of protein phosphorylation in MAPK ERK1/2 activation, in its regulation in time and space and how its dysregulation can lead to tumorigenesis.
... 6,10,28,29 The ERK regulates multifold proteins via phosphorylation of mainly Ser/Thr-Pro (S/T-P) residues. 30,31 The current study demonstrated that the ERK signaling pathway was activated in LSECs following their stimulation with ox-LDL. Interestingly, the results indicated that PD98059 inhibited the ox-LDL-mediated activation of the ERK signaling pathway. ...
Background and aim:
Both type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) are closely associated with elevated levels of low-density lipoprotein cholesterol (LDL-C) and its oxidized form (ox-LDL). This study aimed to investigate the regulation of sortilin in liver tissue and its potential implications for lipid metabolism.
Methods:
Sixty male Wistar rats were randomly divided into four groups: control group (n=15), ox-LDL group (n=15), PD98059 group (n=15), and ox-LDL + PD98059 group (n=15). Liver sinusoidal endothelial cells (LSECs) were extracted from liver tissue of the control group and were identified using an anti-CD31 antibody. Lipid droplet accumulation was observed by Oil red O (ORO) and hematoxylin-eosin (HE) staining. The protein expression level were detected by immunohistochemical staining, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blot. Histopathologic examinations were performed by Gomori methenamine silver (GMS) staining.
Results:
Ox-LDL group exhibited increased lipid droplet accumulation. Further, ox-LDL activated the extracellular signal-regulated kinase (ERK)-mediated downregulation of sortilin expression, whereas blocking of ERK signaling by PD98059 increased sortilin protein expression. Consistently, HE staining showed that the structure of the hepatocytes was loose and disordered in arrangement, with lipid droplets present in the cytoplasm of the ox-LDL group. However, PD98059 significantly improved the integration of the scaffold structure. GMS staining showed that the ox-LDL group had darker and more obvious fragmented silver nitrate deposits in the basement membrane and sinus space.
Conclusions:
Sortilin can protect LSECs from injury and maintain integration of the liver scaffold structure in ox-LDL-induced lipid-injured liver.
... Remarkably, agents that trigger differentiation of neuroblastoma or pheochromocytoma require the activation of several signaling pathways including ERK (Zogovic et al., 2015. ERK proteins can in turn phosphorylate more than six hundred different targets, including transcription factors, in a context dependent manner (Unal et al., 2017), which explains their wide-range of effects. Importantly, our in silico analysis showed that high FASN expression was found in patients with low TRKA levels in all three cohorts. ...
Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status.
... The ERK cascade is involved in physiological responses, various physiological functions and diseases [13]. ERK1/2 can act on hundreds of targets [14]. Therefore, inhibition of the activity of ERK has a profound effect on cellular senescence and aging. ...
Drugs or compounds have been shown to promote longevity in various approaches. We used Drosophila to explore novel natural compounds can be applied to anti-aging. Here we reported that a flavonoid named Dihydromyricetin can increase stress that tolerance and lipid levels, slow down gut dysfunction and extend Drosophila lifespan. Dihydromyricetin can also lessen pERK and pAKT signaling, consequently activating FOXO and AOP to modulate longevity. Our results suggested that DHM could be used as an effective compound for anti-aging intervention, which could likely be applied to both mammals and humans.
... Activated ERK can translocate to the nucleus to phosphorylate and regulate transcription factors resulting in changes in gene expression 19 . Some ERK substrates are also found in the cytoplasm and other organelles 20 . ...
In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.
... Many of those targets contain a D domain and a DEF motif that improves the efficacy and specificity of the interaction with ERK1/2's CD/CRS (common docking motif/cytosolic retention sequence) domain [109]. In 2017, based on various phospho-proteomic analyses, Ünal et al. reviewed 2507 ERK1/2-targets, of which 659 were direct and 1848 were indirect targets [110]. Direct targets are mainly involved in cell-cycle regulation and cell-signaling, for example, the well-known ERK1/2 targets, the Ets family of transcription factors-ELK-1, ELK-4, Ets-2-but also MYC, phospholipase A2, p90 RSK , paxillin, and calnexin [67,[111][112][113]. ...
Neuroblastoma is the most common extra-cranial solid tumor in children, representing approximately 8% of all malignant childhood tumors and 15% of pediatric cancer-related deaths. Recent sequencing and transcriptomics studies have demonstrated the RAS-MAPK pathway’s contribution to the development and progression of neuroblastoma. This review compiles up-to-date evidence of this pathway’s involvement in neuroblastoma. We discuss the RAS-MAPK pathway’s general functioning, the clinical implications of its deregulation in neuroblastoma, and current promising therapeutics targeting proteins involved in signaling.
... Tumour growth was monitored twice weekly via calliper measurement and tumour volume calculated using the equation: 3.14 x length x width 2 /6000. Growing tumours were randomised and recruited onto study when they reached an average of ~0.4cm3 for target engagement studies and ~0.2cm 3 for efficacy studies. AZD0364 was formulated in 10% DMSO & 90% of 40% kleptose, selumetinib was formulated in 0.5% Methocel (hydroxypropyl methocellulose)/0.1% Polysorbate 80. ...
The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signalling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF mutant melanoma, with limited effect of single agent pathway inhibitors in KRAS mutant tumours. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signalling in KRAS mutant tumours and achieve meaningful clinical benefit. Here we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF mutant and KRAS mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose and time-dependent modulation of ERK1/2-dependent signalling biomarkers resulting in tumour regression in sensitive BRAF and KRAS mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor selumetinib (AZD6244, ARRY142886) enhances efficacy in KRAS mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signalling pathway that is not achievable with single agent treatment. The AZD0364 and selumetinib combination also results in significant tumour regressions in multiple KRAS mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS mutant tumours.
... RAF dimers phosphorylate and activate MEK, which in turn phosphorylate and activate ERK. ERK has >650 targets both in the cytosol and nucleus [41]. also as switches when the affinity of PPIs is dynamically controlled (Figure 2). ...
Macromolecular protein assemblies govern many cellular processes and are disturbed in many diseases including cancer. Often seen as static molecular machines, protein complexes involved in signal transduction networks exhibit intricate dynamics that are critical for their function. Using the RAS-RAF-MEK-ERK pathway as example we discuss recent progress in our understanding of protein complex dynamics achieved through mathematical modelling, computational simulations and structural studies. The emerging picture highlights that both spatial and temporal dynamics cooperate to enable correct signal processing and the fine tuning of timing, duration and strengths of signalling. These dynamic processes are subverted by oncogenic mutations and contribute to tumorigenesis and drug resistance.
... The ERK pathway participates in the phosphorylation over 250 cellular substrates [43] thereby regulating various cellular processes such as cell adhesion, cell cycle progression, migration, survival, differentiation, metabolism, proliferation, and transcription [44]. Its activation was reported to induce cell cycle entry and G1 progression by upregulating proliferative transcription factors [45] and downregulating antiproliferative genes [46]. ...
Previously, we demonstrated the in vitro anti-tumor effects of desethylamiodarone (DEA) in bladder and cervix cancer cell lines. In the present study, we intended to establish its potentiality in B16-F10 metastatic melanoma cells in vitro and in vivo. We assessed cell proliferation, apoptosis and cell cycle by using sulforhodamine B assay, Muse™ Annexin V & Dead Cell and Muse® Cell Cycle assays, respectively. We determined colony formation after crystal violet staining. For studying mechanistic aspects, immunoblotting analysis was performed. We used a C57BL/6 experimental lung metastasis model for demonstrating in vivo anti-metastatic potential of DEA. DEA inhibited in vitro proliferation and colony formation, and in vivo lung metastasizing properties of B16-F10 cells. It arrested the cells in G0/G1 phase of their cycle likely via p21 in a p53-dependent fashion, and induced caspase mediated apoptosis likely via inversely regulating Bcl-2 and Bax levels, and reducing Akt and ERK1/2 activation. In this study, we provided in vitro and in vivo experimental evidences for DEA's potentiality in the therapy of metastatic melanomas. Since DEA is the major metabolite of amiodarone, a worldwide used antiarrhythmic drug, safety concerns could be resolved more easily for it than for a novel pharmacological agent.
... Protein kinases ERK1/2 are central signaling kinases that control cell proliferation, survival, differentiation and apoptosis. ERK1/2 phosphorylates multiple substrates localized in different subcellular compartments and activates the expression of many regulatory proteins involved in the cell cycle and apoptosis, including p21 and survivin (4)(5)(6)(7). ...
Background/aim:
Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4.
Materials and methods:
REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting.
Results:
VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells.
Conclusion:
Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.
... Based on parallel findings it is assumed that this is involved in the activation of various mitogen-activated protein kinases (MAPKs), which results in immunostimulatory or immunosuppressive effects depending on the frequency, dose and the duration of exposure to the mycotoxin (11)(12)(13)(14). Having identified the negative impact of DON on co-stimulatory molecules for T cells (see above), we hypothesized that DON might also influence the expression of transcription factors which are frequent targets of MAPK signaling (15). ...
Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 μM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.
... ERK1/2 then activate or inactivate a variety of proteins via phosphorylation in different subcellular compartments. While a number of ERK1/2 substrates have been identified to date (reviewed in [3,4]), their catalogue is likely to expand further due to continuing efforts in phosphoproteomics approaches that search for candidate ERK1/2 substrates (reviewed in [5,6]). The Raf/MEK/ERK pathway is regulated by a complex network of regulators, including additional small GTPases, phosphatases, scaffolds, and other kinases, which affects the magnitude, duration, and subcellular compartmentalization of the pathway activity. ...
In response to extracellular stimuli, the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway regulates diverse cellular processes. While mainly known as a mitogenic signaling pathway, the Raf/MEK/ERK pathway can mediate not only cell proliferation and survival but also cell cycle arrest and death in different cell types. Growing evidence suggests that the cell fate toward these paradoxical physiological outputs may be determined not only at downstream effector levels but also at the pathway level, which involves the magnitude of pathway activity, spatial-temporal regulation, and non-canonical functions of the molecular switches in this pathway. This review discusses recent updates on the molecular mechanisms underlying the pathway-mediated growth inhibitory signaling, with a major focus on the regulation mediated at the pathway level.
... These intracellular cascades lead to the phosphorylation and activation of serine/threonine-specific protein kinase B (AKT). There are three isoforms of AKT, and AKT2 is the most important isoform for glucose homeostasis [6]. Several substances interact, and via the PI3K/AKT pathway, anabolic May 15, 2021 ...
Insulin has complex effects on cell growth, metabolism and differentiation, and these effects are mediated by a cell-surface bound receptor and eventually a cascade of intracellular signaling events. Among the several metabolic and growth-promoting effects of insulin, insulin resistance is defined as an attenuated effect of insulin on glucose metabolism, primarily the limited export of blood glucose into skeletal muscle and adipose tissue. On the other hand, not all the signaling pathways and insulin-responsive tissues are equally affected, and some effects other than the metabolic actions of insulin are overexpressed. Ovaries and the adrenal glands are two examples of tissues remaining sensitive to insulin actions where insulin may contribute to increased androgen secretion. Polycystic ovary syndrome (PCOS) is the most common form of androgen excess disorder (AED), and its pathogenesis is closely associated with insulin resistance. Patients with idiopathic hirsutism also exhibit insulin resistance, albeit lower than patients with PCOS. Although it is not as evident as in PCOS, patients with congenital adrenal hyperplasia may have insulin resistance, which may be further exacerbated with glucocorticoid overtreatment and obesity. Among patients with severe insulin resistance syndromes, irrespective of the type of disease, hyperinsulinemia promotes ovarian androgen synthesis independently of gonadotropins. It is highly debated in whom and how insulin resistance should be diagnosed and treated among patients with AEDs, including PCOS. It is not suitable to administer an insulin sensitizer relying on only some mathematical models used for estimating insulin resistance. Instead, the treatment decision should be based on the constellation of the signs, symptoms and presence of obesity; acanthosis nigricans; and some laboratory abnormalities such as impaired glucose tolerance and impaired fasting glucose.
... Additionally, MCM7 itself is a known MAPK target 20 . Furthermore, MCM3 and the ATR kinase, which phosphorylates MCM3 upon replication stress, have been identified as (indirect) ERK targets 52 . Thus, it remains to be determined to what extend activated RAS-MEK-ERK signaling in colorectal cancer cells impinges on both expression and phosphorylation-dependent activation of replication complex members to foster origin firing. ...
To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.
... ERK1 is phosphorylated at Thr202 and Tyr204 and ERK2 at Thr183 and Tyr185 [34,35]. ERK phosphorylates a wide spectrum of substrates in the cytoplasm, organelles, and nucleus [36]. Ras-Raf-ERK-mediated proliferative functions are mainly an outcome of the stimulation-induced translocation of ERK to the nucleus [37]. ...
The central protein in the oncogenic circuitry is the Ras GTPase that has been under intense scrutiny for the last four decades. From its discovery as a viral oncogene and its non–oncogenic contribution to crucial cellular functioning, an elaborate genetic, structural, and functional map of Ras is being created for its therapeutic targeting. Despite decades of research, there still exist lacunae in our understanding of Ras. The complexity of the Ras functioning is further exemplified by the fact that the three canonical Ras genes encode for four protein isoforms (H-Ras, K-Ras4A, K-Ras4B, and N-Ras). Contrary to the initial assessment that the H-, K-, and N-Ras isoforms are functionally similar, emerging data are uncovering crucial differences between them. These Ras isoforms exhibit not only cell–type and context-dependent functions but also activator and effector specificities on activation by the same receptor. Preferential localization of H-, K-, and N-Ras in different microdomains of the plasma membrane and cellular organelles like Golgi, endoplasmic reticulum, mitochondria, and endosome adds a new dimension to isoform-specific signaling and diverse functions. Herein, we review isoform-specific properties of Ras GTPase and highlight the importance of considering these towards generating effective isoform-specific therapies in the future.
... Components of this pathway are frequently mutated in human cancer and developmental disorders, resulting in constitutive activation of the ERK1 and ERK2 MAPKs (Bardwell, 2020;Tidyman & Rauen, 2016). Activated MAPKs phosphorylate numerous targets, including many transcription factors (Unal et al, 2017;Yoon & Seger, 2006;Zeke et al, 2016). ...
There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.
... The ERK signaling pathway is a paracrine activated cascade by which an extracellular growth factor, such as the fibroblast growth factor (FGF), activates the Ras-Raf-MEK-ERK cascade ( Fig 3A). The cascade consists of a chain of serial phosphorylations that result in the double phosphorylation of ERK and its shuttling to the nucleus, where it affects the expression of more than 600 direct targets [37]. Even though the pathway is relatively direct, the dynamics of ERK localization and activity can be very complex: ERK is able to display a large variety of dynamic responses in different cellular contexts, even when presented with similar extracellular signals. ...
Mounting evidence shows that oscillatory activity is widespread in cell signaling. Here we review some of this recent evidence, focusing on both the molecular mechanisms that potentially underlie such dynamical behavior, and the potential advantages that signaling oscillations might have in cell function. The biological processes considered include tissue maintenance, embryonic development and wound healing. With the aid of mathematical modeling, we show that a common principle, namely delayed negative feedback, underpins this wide variety of phenomena.
... ERK also feedback inhibits the pathway directly by phosphorylating negative regulatory sites on RAF and RTKs, and indirectly by activating the transcription of negative regulators, including the dual-specificity phosphatase (DUSP) proteins, which modulate ERK activity, and the Sprouty (SPRY) proteins, which inhibit RTKs. Thus, the suppression of ERK activity mediated by MEK inhibition relieves this naturally occurring negative feedback, leading to renewed upstream signaling [52,53]. ...
Activating mutations in KRAS are present in 25% of human cancers. When mutated, the KRAS protein becomes constitutively active, stimulating various effector pathways and leading to the deregulation of key cellular processes, including the suppression of apoptosis and enhancement of proliferation. Furthermore, mutant KRAS also promotes metabolic deregulation and alterations in the tumor microenvironment. However, some KRAS mutant cancer cells become independent of KRAS for their survival by activating diverse bypass networks that maintain essential survival signaling originally governed by mutant KRAS. The proposed inducers of KRAS independency are the activation of YAP1 and/or RSK-mTOR pathways and co-mutations in SKT11 (LKB1), KEAP1, and NFE2L2 (NRF2) genes. Metabolic reprogramming, such as increased glutaminolysis, is also associated with KRAS autonomy. The presence or absence of KRAS dependency is related to the heterogeneity of KRAS mutant cancers. Epithelial-to-mesenchymal transition (EMT) in tumor cells is also a characteristic phenotype of KRAS independency. Translationally, this loss of dependence is a cause of primary and acquired resistance to mutant KRAS-specific inhibitors. While KRAS-dependent tumors can be treated with mutant KRAS inhibitor monotherapy, for KRAS-independent tumors, we need an improved understanding of activated bypass signaling pathways towards leveraging vulnerabilities, and advancing therapeutic options for this patient subset.
... Functionally, since MAPK/ERK1/2 target over 600 substrates within the cell, they serve as a central hub that governs fundamental cellular behaviors including cell survival, differentiation, proliferation, growth, migration, and metabolism [31,32]. These cellular responses are critical for efficient organ regeneration, implying substantial involvement of the MAPK/ERK pathway in regeneration. ...
Damage to organs by trauma, infection, diseases, congenital defects, aging, and other injuries causes organ malfunction and is life-threatening under serious conditions. Some of the lower order vertebrates such as zebrafish, salamanders, and chicks possess superior organ regenerative capacity over mammals. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), as key members of the mitogen-activated protein kinase (MAPK) family, are serine/threonine protein kinases that are phylogenetically conserved among vertebrate taxa. MAPK/ERK signaling is an irreplaceable player participating in diverse biological activities through phosphorylating a broad variety of substrates in the cytoplasm as well as inside the nucleus. Current evidence supports a central role of the MAPK/ERK pathway during organ regeneration processes. MAPK/ERK signaling is rapidly excited in response to injury stimuli and coordinates essential pro-regenerative cellular events including cell survival, cell fate turnover, migration, proliferation, growth, and transcriptional and translational activities. In this literature review, we recapitulated the multifaceted MAPK/ERK signaling regulations, its dynamic spatio-temporal activities, and the profound roles during multiple organ regeneration, including appendages, heart, liver, eye, and peripheral/central nervous system, illuminating the possibility of MAPK/ERK signaling as a critical mechanism underlying the vastly differential regenerative capacities among vertebrate species, as well as its potential applications in tissue engineering and regenerative medicine.
... ERKs are located at the end of the MAPKs signaling pathway, which is a central pathway that transmits a variety of extracellular signals. A number of studies demonstrated that ERK1/2 has more than 600 substrates, including transcription factors, protein kinases and phosphatases, apoptosis regulators, etc. 17,42 . The diversity of substrates allows ERK1/2 to mediate a variety of cellular processes in the MAPK pathway, such as regulating cell proliferation, differentiation, angiogenesis, chromatin remodeling, etc. 43,44 . ...
The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is widely activated by a variety of extracellular stimuli, and its dysregulation is associated with the proliferation, invasion, and migration of cancer cells. ERK1/2 is located at the distal end of this pathway and rarely undergoes mutations, making it an attractive target for anticancer drug development. Currently, an increasing number of ERK1/2 inhibitors have been designed and synthesized for antitumor therapy, among which representative compounds have entered clinical trials. When ERK1/2 signal transduction is eliminated, ERK5 may provide a bypass route to rescue proliferation, and weaken the potency of ERK1/2 inhibitors. Therefore, drug research targeting ERK5 or based on the compensatory mechanism of ERK5 for ERK1/2 opens up a new way for oncotherapy. This review provides an overview of the physiological and biological functions of ERKs, focuses on the structure-activity relationships of small molecule inhibitors targeting ERKs, with a view to providing guidance for future drug design and optimization, and discusses the potential therapeutic strategies to overcome drug resistance.
... Early growth response 1 is one of several genes regulated by Elk-1 and related E twenty-six domain transcription factors. Many of these are downstream substrates of ERK1/2 (Buchwalter et al., 2004;Ünal et al., 2017) and their expression is regulated by interventions that change ERK1/2 activity. In HEK293β2 cells and all epithelial cell models, MEK1/2 inhibitors reduced constitutive Elk-1 phosphorylation and repressed EGR1 gene expression confirming that basal Elk-1-EGR1 signaling was maintained by ERK1/2. ...
Chronic use of β2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. Herein, we tested the hypothesis that β2-adrenoceptor agonists, including formoterol, promote biased, β-arrestin 2 (βArr2)-dependent activation of the mitogen-activated protein (MAP) kinases, ERK1/2, in human airway epithelial cells and, thereby, effect changes in gene expression that could contribute to their adverse clinical outcomes. Three airway epithelial cell models were used: the BEAS-2B cell line, human primary bronchial epithelial cells (HBEC) grown in submersion culture and HBEC that were highly differentiated at an air-liquid interface. Unexpectedly, treatment of all epithelial cell models with formoterol decreased basal ERK1/2 phosphorylation. This was mediated by cAMP-dependent protein kinase and involved the inactivation of C-rapidly-activated fibrosarcoma, which attenuated down-stream ERK1/2 activity, and the induction of dual-specificity phosphatase-1. Formoterol also inhibited the basal expression of early growth response-1, an ERK1/2-regulated gene that controls cell growth and repair in the airways. Neither carvedilol, a β2-adrenoceptor agonist biased towards βArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells although β2-adrenoceptor desensitization was compromised in ARRB2-deficient cells. Collectively, these results contest the hypothesis that formoterol activates ERK1/2 in airway epithelia by nucleating a βArr2 signaling complex; instead, they indicate that β2-adrenoceptor agonists inhibit constitutive ERK1/2 activity in a cAMP-dependent manner. These findings are the antithesis of results obtained using acutely challenged native and engineered HEK293 cells, which have been used extensively to study mechanisms of ERK1/2 activation, and highlight the cell-type-dependence of β2-adrenoceptor-mediated signaling. Significance Statement It has been proposed that the adverse-effects of β2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of β-arrestin 2-dependent activation of ERK1/2. This study shows that β2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream Ras-C-Raf complex formation and inducing DUSP1. Moreover, these effects were PKA-dependent suggesting that β2-adrenoceptor agonists were not biased toward β-arrestin 2 and acted via canonical, cAMP-dependent signaling.
In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.
KRAS is widely mutated in human cancers, resulting in nearly unchecked tumour proliferation and metastasis. No therapies have been developed for targeting KRAS-mutant tumours. Herein, we observed that KRAS-mutant stomach/colorectal tumour cells were hypersensitive to the MEK1/2 kinase inhibitor trametinib, which elicits strong apoptotic responses. Genome-wide screening revealed that TP53 is critical for executing trametinib-induced apoptosis of KRAS-mutant tumours, as validated by TP53 knockout and rescue experiments. Mechanistically, p53 physically associates with phosphorylated ERK2 in the presence of mutant KRAS, which inactivates p53 by preventing the recruitment of p300/CBP. Trametinib disrupts the p53-ERK2 complex by inhibiting ERK2 phosphorylation, allowing the recruitment of p300/CBP to acetylate p53 protein; acetylated p53 activates PUMA transcription and thereby promotes the apoptosis of KRAS-mutant tumours. Our study unveils an important role of the ERK2-p53 axis and provides a potential therapeutic strategy for treating KRAS- mutant cancer via ERK2 inhibition.
Les « processing bodies » ou p-bodies sont des structures sans membranes (MLO : Membraneless Organelles) formées par un processus connu sous le nom de séparation de phase liquide-liquide (LLPS). Les p-bodies sont des agrégats de protéines et d’ARNs localisés dans le cytoplasme des cellules. Ils ont été caractérisés comme étant un lieu de stockage des ARNs réprimés, puisque les ARNs qui y sont accumulés, sont stables et non traduits. Pour autant, l’absence de membrane rend leur contenu rapidement mobilisable par les cellules, suggérant un rôle important dans les réponses immédiates au stress. Au cours de ma thèse, je me suis intéressée, à la fois au mécanisme de formation et de dissolution des p-bodies. Tout d’abord, j’ai mis en évidence l’effet de la dissolution des p-bodies sur les ARNm. L’induction de la dissolution des p-bodies conduit à une augmentation de la traduction des ARNm qui y sont stockés et réprimés tels que les oncogènes KRAS et NRAS. Il semble alors, que ce réservoir d’ARNm constitue un mécanisme dynamique d’adaptation au stress cellulaire, et en particulier pour le développement de cellules résistantes aux MEKi. Ensuite, afin de mieux comprendre la formation des p-bodies, j’ai étudié la localisation de l’exoribonucléase 1 (XRN1). Cette enzyme comme son nom l’indique est impliquée dans la dégradation des ARNs, mais est retrouvée accumulée dans les p-bodies sous forme inactive. Dans ce contexte, afin de déterminer le rôle de la protéine XRN1 dans les p-bodies, je me suis intéressée aux régions importantes pour son adressage dans les p-bodies. Ainsi, j’ai pu montrer que les zones permettant l’accumulation de XRN1 dans les p-bodies se trouvent dans la partie C-terminale de la protéine au niveau des acides aminés 1406-1468 et 1607-1706. Ces résultats indiquent donc que ces deux régions conservées chez les vertébrés sont indispensables à la localisation de la protéine de façon indépendante de la partie catalytique située en N-terminal. En conclusion, j’ai, au cours de ma thèse, travaillé sur deux aspects des p-bodies : la séquestration protéique et la séquestration des ARNs. Ces travaux permettent de mieux comprendre le rôle joué par p-bodies dans des processus physiologiques ou physiopathologiques tels que le cancer.
Activating mutations in RAS genes are the most common genetic driver of human cancers. Yet, drugging this small GTPase has proven extremely challenging and therapeutic strategies targeting these recurrent alterations have long had limited success. To circumvent this difficulty, research has focused on the molecular dissection of the RAS pathway to gain a more-precise mechanistic understanding of its regulation, with the hope to identify new pharmacological approaches. Here, we review the current knowledge on the (dys)regulation of the RAS pathway, using melanoma as a paradigm. We first present a map of the main proteins involved in the RAS pathway, highlighting recent insights into their molecular roles and diverse mechanisms of regulation. We then overview genetic data pertaining to RAS pathway alterations in melanoma, along with insight into other cancers, that inform the biological function of members of the pathway. Finally, we describe the clinical implications of RAS pathway dysregulation in melanoma, discuss past and current approaches aimed at drugging the RAS pathway, and outline future opportunities for therapeutic development.
The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases.
Significance
Label-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.
Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown. Here, we investigated the regulators of MVB fate by analyzing the effects of signaling inhibitors on EV secretion from cancer cells engineered to secrete luciferase-labeled EVs. Inhibition of the oncogenic MEK/ERK pathway suppressed EV release and activated lysosome formation. MEK/ERK-mediated lysosomal inactivation impaired MVB degradation, resulting in increased EV secretion from cancer cells. Moreover, MEK/ERK inhibition prevented c-MYC expression and induced the nuclear translocation of MiT/TFE transcription factors, thereby promoting the activation of lysosome-related genes, including the gene encoding a subunit of vacuolar-type H+ -ATPase, which is responsible for lysosomal acidification and function. Furthermore, c-MYC upregulation was associated with lysosomal genes downregulation in MEK/ERK-activated renal cancer cells/tissues. These findings suggest that the MEK/ERK/c-MYC pathway controls MVB fate and promotes EV production in human cancers by inactivating lysosomal function.
Mutational activation of the KRAS oncogene is found in ~ 95% of pancreatic ductal adenocarcinoma (PDAC), the major form of pancreatic cancer. With substantial experimental evidence that continued aberrant KRAS function is essential for the maintenance of PDAC tumorigenic growth, the National Cancer Institute has identified the development of effective anti-KRAS therapies as one of four major initiatives for pancreatic cancer research. The recent clinical success in the development of an anti-KRAS therapy targeting one specific KRAS mutant (G12C) supports the significant potential impact of anti-KRAS therapies. However, KRASG12C mutations comprise only 2% of KRAS mutations in PDAC. Thus, there remains a dire need for additional therapeutic approaches for targeting the majority of KRAS-mutant PDAC. Among the different directions currently being pursued for anti-KRAS drug development, one of the most promising involves inhibitors of the key KRAS effector pathway, the three-tiered RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. We address the promises and challenges of targeting ERK MAPK signaling as an anti-KRAS therapy for PDAC. In particular, we also summarize the key role of the MYC transcription factor and oncoprotein in supporting ERK-dependent growth of KRAS-mutant PDAC.
The RAS-RAF-MEK-ERK pathway is the most well-studied of the mitogen-activated protein kinase (MAPK) cascades and is critical for cell proliferation, differentiation, and survival. Abnormalities in regulation resulting from mutations in components of this pathway, particularly in upstream proteins RAS and RAF, are responsible for a significant fraction of human cancers and nearly all cutaneous melanomas. Activation of receptor tyrosine kinases by growth factors and various extracellular signals to the sequential activation of RAS, RAF, MEK, and finally ERK, which activates numerous transcription factors and facilitates oncogenesis in the case of aberrant pathway activation. While extensive studies have worked to elucidate the activation mechanisms and structural components of upstream MAPK components, comparatively less attention has been directed towards the kinases, MEK and ERK, due to the infrequency of oncogenic activating mutations in these kinases. However, acquired drug resistance has become a major issue in the treatment of RAS- and RAF- mutated cancers. Targeting the terminal kinases in the MAPK cascade has shown promise for overcoming many of these resistance mechanisms and improving treatment options for patients with MAPK-aberrant cancers. Here, we will describe the role of MEK and ERK in MAPK signaling and summarize the current understanding of their interaction and activation mechanisms. We will also discuss existing targeted approaches for MEK and ERK, and the benefits of alternative strategies. Areas requiring further exploration will be highlighted to guide future research endeavors and aid in the development of alternative therapeutic strategies to combat surmounting drug resistance in treating MAPK-mediated cancers.
Leptin is known to selectively suppress neural and taste cell responses to sweet compounds. The sweet suppressive effect of leptin is mediated by the leptin receptor Ob‐Rb, and the ATP‐gated K⁺ (KATP) channel expressed in some sweet‐sensitive, T1R3‐positive taste cells. However, the intracellular transduction pathway connecting Ob‐Rb to KATP channel remains unknown. Here we report that phosphoinositide 3‐kinase (PI3K) mediates leptin’s suppression of sweet responses in T1R3‐positive taste cells. In in situ taste cell recording, systemically administrated leptin suppressed taste cell responses to sucrose in T1R3‐positive taste cells. Such leptin’s suppression of sucrose responses was impaired by co‐administration of PI3K inhibitors (wortmannin or LY294002). In contrast, co‐administration of signal transducer and activator of transcription 3 (STAT3) inhibitor (Stattic) or Src homology region 2 domain‐containing phosphatase‐2 (SHP2) inhibitor (SHP099) had no effect on leptin’s suppression of sucrose responses, although STAT3 and SHP2 were expressed in T1R3‐positive taste cells. In peeled tongue epithelium, phosphatidylinositol (3,4,5)‐trisphosphate (PIP3) production and phosphorylation of AKT by leptin were immunohistochemically detected in some T1R3‐positive taste cells but not in GAD67‐positive taste cells. Leptin‐induced PIP3 production was suppressed by LY294002. Thus, leptin suppresses sweet responses of T1R3‐positive taste cells by activation of Ob‐Rb ‐ PI3K ‐ KATP channel pathway.
We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.
The RAS-regulated RAF–MEK1/2–ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed “paradox breakers” which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins
The extracellular signal-regulated kinase (ERK) MAP kinase is utilized downstream of Ras>Raf>MEK signaling to control activation of a wide array of targets. Activation of ERK is elevated in Ras-driven tumors and RASopathies, and is thus a target for pharmacological inhibition. Regulatory mechanisms of ERK activation has been studied extensively in vitro and in cultured cells but little in living animals. We used CRISPR to tag the 3′ end of the C. elegans ERK-encoding gene, mpk-1. Endogenous MPK-1 protein is ubiquitously expressed with elevated expression in certain tissues. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During developmental patterning of the six vulval precursor cells, MPK-1 is necessary and sufficient for the central cell, P6.p, to assume 1° fate. We observed MPK-1 to be recruited to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into regulation of MPK-1 activation within a complex intercellular signaling network.
A common bridge between a linear cytoplasmic signal and broad nuclear regulation is the family of MAP kinases which can translocate to the nucleus upon activation by the cytoplasmic signal. One pathway which functions to activate the ERK family of MAP kinases is the Ras signaling pathway which functions at multiple times and locations during the development of Caenorhabditis elegans including the development of the excretory cell, germ cells, male tail, and vulva. It has been most extensively characterized during the development of the vulva which is formed from the vulval precursor cells (VPCs), a set of six equivalent, epithelial cells designated P3.p – P8.p. Although LIN-1 appears to be a primary target of ERK MAP kinase during vulval development, it is likely that other developmentally important molecules are also regulated by ERK-mediated phosphorylation. The identification of physiological substrates of MAP kinases has been aided by the identification of docking site domains in substrate proteins that contribute to high-affinity interactions with kinases.
Our laboratory has identified the C. elegans protein, T08D10.1/NFYA-1, as a potential ERK MAP kinase substrate in this manner, and we have initiated a characterization of its role during Ras-mediated development. T08D10.1 possesses significant homology to the CCAAT-box DNA-binding domain of the vertebrate nuclear transcription factor-Y, alpha (NF-YA) family of proteins. NF-Y proteins act as part of a complex to regulate the transcription of a large number of genes, in particular, genes that function in the G1/S cell cycle transition. T08D10.1/NFYA-1 is predicted to code for a protein containing multiple potential phosphorylation sites for ERK MAP kinase and a D-domain docking site. We demonstrate through biochemical analysis of purified NFYA-1 protein that it can act in vitro as a high affinity substrate for activated ERK MAP kinase. Growth factor activation of the Ras pathway in a tissue culture system has negligible effect on the protein's transactivation potential, however, the DNA-binding activity of the protein is reduced after treatment with activated ERK-MAP kinase. We demonstrate through mutant analysis that nfya-1 acts to inhibit vulval development and functions downstream or in parallel to let-60/ras. Both the NF-Y complex and the Ras signaling pathway play a fundamental role in cell proliferation and oncogenesis and the connection between the two is an important insight into the mechanisms of cell fate specification and cellular response.
The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.
RAS is the most frequently mutated oncogene in human cancer. Scientists attempted for decades to target this protein or its pathways, however, all the attempts failed and RAS was labeled as “undruggable”. With KRAS-G12C covalent inhibitors entering clinical trials, the myth of this “undruggable” RAS is fading away. In 2021, the Food and Drug Administration (FDA) approved the use of Sotorasib (Lumakras) for the treatment of adult patients with KRAS-G12C mutated locally advanced or metastatic NSCLC, following at least one prior systemic therapy. However, and as every other drug, KRAS-G12C inhibitors are facing intrinsic and acquired resistances. In order to overcome these resistances, researchers are now working on combination strategies. Furthermore, studies are currently ongoing to better elucidate the status of KRAS-G12C as a predictive and prognostic tool and to strengthen its role in the field of personalized medicine.
Low miR-1, miR-7, miR-17, miR-26a, miR-29a, miR-30a, miR-34a, miR-124, miR-133, miR-143-145, miR-150, miR-378, miR-455, lincRNA-p21, and MEG3 are related to cancer. In contrast, high miR-9, miR-21, miR-130, miR-155, miR-221, HOTAIR, H19, LINC-ROR, MALAT1, MIAT, NEAT1, TUG1, UCA1, XIST, ZFAS1, ANRIL, ciRs-7, and PVT1 are related to cancer. High and low let-7, miR-146a, miR-181, miR-223, and GAS5 are associated with cancer. These non-coding RNA profiles allow a metabolic reprogramming in tumors to retain stemness and epithelial-mesenchymal transition, insulin sensitized state and angiogenesis. It also explains the shift from OXPHOS to glycolysis, the immune escape, and the prevention of apoptosis in tumors.
Le carcinome hépatocellulaire (CHC) est le principal cancer primitif du foie, il présente un pronostic vital sombre, notamment du fait du peu d’options thérapeutiques disponibles pour les stades avancés. L’analyse génomique des tumeurs a permis sa classification moléculaire en identifiant les principaux oncogènes et suppresseurs de tumeurs impliqués dans son développement. Ces études ont également défini la diversité des bases génétiques du CHC, et donc une importante hétérogénéité inter-tumorale. Mon travail de thèse aborde la question de l'hétérogénéité tumorale sous un angle différent : en me concentrant sur l’étude de la coopération oncogénique et de l’hétérogénéité intra-tumorale fonctionnelle, j'ai mis en évidence une hétérogénéité quantitative, qui a pour origine des variations de l'intensité du signal oncogénique, ce qui influence les interactions de la cellule cancéreuse avec son micro-environnement.Pour étudier la dynamique des populations cellulaires au cours de la croissance tumorale, nous avons développé un modèle murin de CHC qui combine des injections intra-hépatiques et un suivi du lignage cellulaire. Les allogreffes orthotopiques de cellules marquées génèrent des tumeurs hépatiques dont la croissance est analysée notamment en termes de composition clonale et d’invasion métastatique. Le résultat principal de cette étude est la mise en évidence d’un avantage sélectif octroyé par un niveau d’activation optimal d’une voie de signalisation majeure : la voie des MAPK Erk. L'intensité optimale de cette signalisation est définie aussi bien par des mécanismes intrinsèques de la réponse cellulaire (cell-autonomous) que par des interactions avec le microenvironnement. L'analyse approfondie du stroma tumoral, qui est différent pour la tumeur primaire et les sites métastatiques, m'a permis d'identifier les cellules stromales qui seraient les médiateurs de la sélection clonale et de proposer les bases moléculaires de mécanismes qui en seraient responsables.
Melanoma is the cause of most deaths from skin cancer. The extracellular signal-regulated kinase 1/2 (ERK1/2) pathway has been reported to participate in progression of melanoma in fair skinned populations. ERK1/2 is found in both the cytoplasm and nucleus of cells, and phosphorylated ERK1/2 has been implicated in tumor progression. We investigated the relation between melanoma progression and expression of cytoplasmic and nuclear phosphorylated ERK1/2. We examined 34 surgically resected melanomas and investigated their clinicopathologic characteristics. We found immunostaining of phosphorylated ERK1/2 in all melanomas and faint staining in benign nevi. We found expression of cytoplasmic phosphorylated ERK1/2 in most melanomas; however, nuclear phosphorylated ERK1/2 expression was found in only five melanomas. Expression of cytoplasmic phosphorylated ERK1/2 was related to the tumor stage in melanoma. Nine of 10 cases of distant metastasis were positive for cytoplasmic phosphorylated ERK1/2. Our findings suggest that phosphorylated ERK1/2 expression is relevant to clinical pathology and that in melanoma patients, phosphorylated ERK1/2 expression is found in both the cytoplasm and nucleus. Our findings suggest that cytoplasmic phosphorylated ERK1/2 participates in progression of melanoma and that it could be a useful target for clinical treatment of melanoma.
Macrophages are transcriptionally highly dynamic cell type, rapidly adapting to a changing environment to execute innate immune functions. Activation of macrophages with lipopolysaccharides (LPS), a major component of the outer membrane of most Gram-negative bacteria, induces rapid transcriptional changes and within a few hours transcription of several hundred genes is altered. Within these genes are tRNAs, which are synthesised by RNA Polymerase (Pol) III, and whose expression is rapidly upregulated in response to LPS. However, the mechanisms that govern Pol III activation are not fully elucidated.
LPS engage the Toll-like receptor (TLR) 4 and induce various signalling pathways, including mitogen-activated protein kinases (MAPK). MAPKs are serine/threonine kinases that catalyse the phosphorylation of transcription factors, protein kinases, and many other substrates including functional proteins, play a central role in mediating cellular responses to extracellular signals, including inflammatory cues.
Here we show that ERK and p38 MAP kinases contribute to the activation of Pol III in macrophages stimulated with LPS. We also demonstrate that MAP kinases effector MSK1/2 kinases are involved in tRNA upregulation. Our data show that ERK, p38, and MSK kinases are required for upregulation of Pol III activity in macrophages stimulated by LPS. The possible modes of their action are discussed.
RAS proteins (HRAS, KRAS, NRAS) participate in many physiological signal transduction processes related to cell growth, division, and survival. The RAS proteins are small (188/189 amino acid residues) and they function as GTPases. These proteins toggle between inactive and functional forms; the conversion of inactive RAS-GDP to active RAS-GTP as mediated by guanine nucleotide exchange factors (GEFs) turns the switch on and the intrinsic RAS-GTPase activity stimulated by the GTPase activating proteins (GAPs) turns the switch off. RAS is upstream to the RAS-RAF-MEK-ERK and the PI3-kinase-AKT signaling modules. Importantly, the overall incidence of RAS mutations in all cancers is about 19% and RAS mutants have been a pharmacological target for more than three decades. About 84% of all RAS mutations involve KRAS. Except for the GTP/GDP binding site, the RAS proteins lack other deep surface pockets thereby hindering efforts to identify high-affinity antagonists; thus, they have been considered to be undruggable. KRAS mutations frequently occur in lung, colorectal, and pancreatic cancers, the three most deadly cancers in the United States. Studies within the last decade demonstrated that the covalent modification of KRAS C12, which accounts for about 10% of all RAS mutations, led to the discovery of an adjacent pocket (called the switch II pocket) that accommodated a portion of the drug. This led to the development of sotorasib as a second-line treatment of KRASG12C mutant non-small cell lung cancer. Considerable effort also has been expended to develop MAP kinase and PI3-kinase pathway inhibitors as indirect RAS antagonists.
The extracellular signal-regulated kinase (ERK) MAP kinase is utilized downstream of Ras>Raf>MEK signaling to control activation of a wide array of targets. Activation of ERK is elevated in Ras-driven tumors and RASopathies, and is thus a target for pharmacological inhibition. Regulatory mechanisms of ERK activation have been studied extensively in vitro and in cultured cells but little in living animals. We tagged the C. elegans ERK-encoding gene, mpk-1. MPK-1 is ubiquitously expressed with elevated expression in certain contexts. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During patterning of vulval precursor cells, MPK-1 is necessary and sufficient for the central cell, P6.p, to assume 1˚ fate. Yet MPK-1 translocates to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into regulation of MPK-1 activation within a complex intercellular signaling network.
Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.
Small molecules targeting aberrant RAF activity, like Vemurafenib (PLX4032), are highly effective against cancers harboring the V600E BRAF mutation, and are now approved for clinical use against metastatic melanoma. However, in tissues showing elevated RAS activity and in RAS-mutant tumors, these inhibitors stimulate RAF dimerization, resulting in inhibitor resistance and downstream paradoxical ERK activation. To understand the global signaling response of cancer cells to RAF inhibitors, we profiled the temporal changes of the phosphoproteome of two colon cancer cell lines (Colo205 and HCT116) that respond differently to Vemurafenib. Comprehensive data mining and filtering identified a total of 37910 phosphorylation sites, 660 of which were dynamically modulated upon treatment with Vemurafenib. We established that 83% of these dynamic phosphorylation sites were modulated in accordance with the phospho-ERK profile of the two cell lines. Accordingly, kinase-substrate prediction algorithms linked most of these dynamic sites to direct ERK1/2-mediated phosphorylation, supporting a low off-target rate for Vemurafenib. Functional classification of target proteins indicated the enrichment of known (nuclear pore, transcription factors, RAS-RTK signaling) and novel (Rho GTPases signaling, actin cytoskeleton) ERK-controlled functions. Our phosphoproteomic data combined with experimental validation established novel dynamic connections between ERK signaling and the transcriptional regulators TEAD3 (Hippo pathway), MKL1 and MKL2 (Rho-SRF pathway). We also confirm that an ERK docking site found in MKL1 is directly antagonized by overlapping actin binding, defining a novel mechanism of actin-modulated phosphorylation. Altogether, time-resolved phosphoproteomics further documented Vemurafenib selectivity and identified novel ERK downstream substrates.
Functional enrichment analysis is a key step in interpreting gene lists discovered in diverse high-throughput experiments.
g:Profiler studies flat and ranked gene lists and finds statistically significant Gene Ontology terms, pathways and other
gene function related terms. Translation of hundreds of gene identifiers is another core feature of g:Profiler. Since its
first publication in 2007, our web server has become a popular tool of choice among basic and translational researchers. Timeliness
is a major advantage of g:Profiler as genome and pathway information is synchronized with the Ensembl database in quarterly
updates. g:Profiler supports 213 species including mammals and other vertebrates, plants, insects and fungi. The 2016 update
of g:Profiler introduces several novel features. We have added further functional datasets to interpret gene lists, including
transcription factor binding site predictions, Mendelian disease annotations, information about protein expression and complexes
and gene mappings of human genetic polymorphisms. Besides the interactive web interface, g:Profiler can be accessed in computational
pipelines using our R package, Python interface and BioJS component. g:Profiler is freely available at http://biit.cs.ut.ee/gprofiler/.
Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation invitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.
Cancer is often considered a genetic disease. However, much of the enormous plasticity of cancer cells to evolve different phenotypes, to adapt to challenging microenvironments and to withstand therapeutic assaults is encoded by the structure and spatiotemporal dynamics of signal transduction networks. In this Review, we discuss recent concepts concerning how the rich signalling dynamics afforded by these networks are regulated and how they impinge on cancer cell proliferation, survival, invasiveness and drug resistance. Understanding this dynamic circuitry by mathematical modelling could pave the way to new therapeutic approaches and personalized treatments.
Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community.
Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus.
PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.
The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype.
Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4,784 proteins. This included 1,317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. While the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.
A major constituent of the nuclear basket region of the NPC, nucleoporin Tpr plays a multi-dimensional role in regulating important processes. We have previously established that Tpr is phosphorylated in both, MAP kinase dependent and independent manner, and found that Tpr acts as both a substrate and as a scaffold for ERK2. Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as the minor ERK independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that Protein Kinase A phosphorylates the S2094 residue, and the site is hyperphosphorylated during mitosis. Further, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during the telophase. Abrogation of S2059 phosphorylation abolishes its interaction with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, and results in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study open fresh avenues for the better understanding of Tpr functions.
The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.
Cancer often arises when normal cellular growth goes awry due to defects in critical signal transduction pathways. A growing number of inhibitors that target specific components of these pathways are in clinical use, but the success of these agents has been limited by the resistance to inhibitor therapy that ultimately develops. Studies have now shown that cancer cells respond to chronic drug treatment by adapting their signaling circuitry, taking advantage of pathway redundancy and routes of feedback and cross-talk to maintain their function. This review focuses on the compensatory signaling mechanisms highlighted by the use of targeted inhibitors in cancer therapy.
Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1,30,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase-substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase-substrate interactions in PhosphoSitePlus®.
The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 2 (ERK2) is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2, a form of ERK2 engineered to use an analog of adenosine 5'-triphosphate (ATP), to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, mRNA processing, and regulation of the activity of the Rho family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E twenty-six family of transcriptional regulators), was extensively phosphorylated on sites within canonical and noncanonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.
High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.
Cancer is now appreciated as not only a highly heterogenous pathology with respect to cell type and tissue origin but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes such as death, proliferation, differentiation and migration. Thus, the activities of molecular networks that execute metabolic or cytoskeletal processes, or regulate these by signal transduction, are altered in a complex manner by diverse genetic mutations in concert with the environmental context. A major challenge therefore is how to develop actionable understanding of this multivariate dysregulation, with respect both to how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments. While high-throughput experimental platform technologies ranging from genomic sequencing to transcriptomic, proteomic and metabolomic profiling are now commonly used for molecular-level characterization of tumor cells and surrounding tissues, the resulting data sets defy straightforward intuitive interpretation with respect to potential therapeutic targets or the effects of perturbation. In this review article, we will discuss how significant advances can be obtained by applying computational modeling approaches to elucidate the pathways most critically involved in tumor formation and progression, impact of particular mutations on pathway operation, consequences of altered cell behavior in tissue environments and effects of molecular therapeutics.
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development.
Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected
is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work,
we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction.
ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote
ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr
resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation.
Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2
in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing
role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.
Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins.
Phospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins.
Phospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.
Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.
Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.
MAPK (mitogen-activated protein kinase) signalling pathways contribute to the regulation of diverse responses, including normal and pathological aspects of cell growth, division, differentiation and death. Their ubiquity and versatility raise the issue of how they achieve specific coupling of signal with cellular response. How do the kinases in the cascade distinguish their correct substrates from the vast excess of incorrect substrates? Furthermore, how do different signals elicit distinct responses when they are transmitted by the same components? This short review highlights several mechanisms that can promote specificity in MAPK signalling, including tethering interactions between MAPKs and their substrates and regulators mediated by docking sites, feedback loops and cross-pathway regulatory circuits, and the selective activation of scaffold proteins.
Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny leading to the development of pharmacologic inhibitors for the treatment of cancer. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAPK/ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Finally, Ras is a key downstream effector of the epidermal growth factor receptor (EGFR), which is mutationally activated and/or overexpressed in a wide variety of human cancers. ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Thus, the EGFR-Ras-Raf-MEK-ERK signaling network has been the subject of intense research and pharmaceutical scrutiny to identify novel target-based approaches for cancer treatment. In this review, we summarize the current status of the different approaches and targets that are under evaluation and development for the therapeutic intervention of this key signaling pathway in human disease.
The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data we highlight some downstream phosphoproteins that are frequently altered in cancer. This article is protected by copyright. All rights reserved.
Overexpression of stathmin (STMN1) is closely linked to tumor metastases and poor prognosis in endometrial carcinoma (EC). However, the underlying mechanism is little known. In the present study, we investigated the expression of STMN1 in EC. Subsequently, we assessed the role of STMN1 in EC cell proliferation and migration. Our data show that STMN1 is upregulated in EC, and elevated expression of STMN1 is correlated positively with tumor stage and lymph node metastasis. In vitro, forced expression of STMN1 promoted cell invasion and migration. In contrast, knockdown of STMN1 inhibited cell aggressive behaviors. Moreover, the expression and the activity changes of matrix metalloproteinases (MMP)-2/9 were observed in EC cells after the cells being silenced or overexpression of STMN1. In conclusion, STMN1 is an oncogene and it enhances the growth and invasion of EC possibly by mediating the secretion and activation of MMP2 and MMP9 protein.
Cell signaling pathways control cells' responses to their environment through an intricate network of proteins and small molecules partitioned by intracellular structures, such as the cytoskeleton and nucleus. Our understanding of these pathways has been revised recently with the advent of more advanced experimental techniques; no longer are signaling pathways viewed as linear cascades of information flowing from membrane-bound receptors to the nucleus. Instead, such pathways must be understood in the context of networks, and studying such networks requires an integration of computational and experimental approaches. This understanding is becoming more important in designing novel therapies for diseases such as cancer. Using the MAPK (mitogen-activated protein kinase) and PI3K (class I phosphoinositide-3' kinase) pathways as case studies of cellular signaling, we give an overview of these pathways and their functions. We then describe, using a number of case studies, how computational modeling has aided in understanding these pathways' deregulation in cancer, and how such understanding can be used to optimally tailor current therapies or help design new therapies against cancer. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 17 is July 11, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAP kinase able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized, however how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identified thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that co-inhibition of the pleiotropic acidophilic kinase CK2 and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These findings highlight a role for CK2 blockade in potentiating the anti-proliferative effects of BRAF and MEK inhibition in BRAF cancers.
THE normal cellular homologue of the acutely transforming oncogene v-ra/is c-raf-l, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli1. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases ErkI and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K)2-7. Here we report that MAPK-K as well as Erkl and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells3 can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with Mr 50,000 wh