No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A switch from p130Cas/Crk to Gab1/Crk signaling correlates with anchorage independent growth and JNK activation in cells transformed by the Met receptor oncoprotein
Abstract
Cell transformation is associated with anchorage independent growth and morphological changes characterized by reduced adhesion and spreading. The molecular signals that control these events are poorly understood. The Met receptor tyrosine kinase is deregulated in human tumors and an oncogenic derivative of this receptor transforms cells. In this paper we demonstrate that fibroblasts transformed by the Met oncoprotein display decreased cell spreading consistent with the loss of actin stress fibers and vinculin staining focal adhesions. In contrast to control cells, focal adhesion kinase, p130Cas and paxillin are weakly or not detectably tyrosine phosphorylated in Met transformed cells. Moreover, although paxillin and p130Cas associate with the Crk adapter protein in control cells, they fail to associate with Crk in Met transformed cells, yet these cells are motile and capable of wound closure to the same extent as control cells. In Met transformed cells, Crk predominantly associates with the Cbl and Gab1docking proteins in a tyrosine phosphorylation dependent manner. The coupling of Gab1, but not Cbl, with Crk is retained in cells grown in suspension and enhances JNK activation. We propose that the loss of adhesion dependent signals required for cell cycle progression is compensated through Met induced Gab1/Crk signals.
... 2a). Following MET-dependent stimulation, the JNKs and p38s control a range of cellular processes as diverse as cell proliferation, differentiation, transformation and apoptosis [70][71][72][73][74] . ...
... However, blocking a single network node is sufficient to perturb cellular responses, indicating that the targeted inhibition of a specific signalling pathway cannot be compensated for by other, still active, regulatory tiers. This is in line with the well-established observation that obstruction of individual MET-dependent pathways adversely affects tumour growth, survival and migration in various cancer cell types and under different experimental settings 37,48,50,[56][57][58][70][71][72]76,139 . For example, the integrity of JNK-and p38-dependent signals is required for MET-stimulated proliferation and anchorage-independent growth in MET-transformed fibroblasts and melanoma cells [70][71][72] , and STAT3 signalling and NF-κb activity are necessary for MET-induced onset of leiomyosarcomas and for the survival of prostate cancer cells, respectively 48,76 . ...
... This is in line with the well-established observation that obstruction of individual MET-dependent pathways adversely affects tumour growth, survival and migration in various cancer cell types and under different experimental settings 37,48,50,[56][57][58][70][71][72]76,139 . For example, the integrity of JNK-and p38-dependent signals is required for MET-stimulated proliferation and anchorage-independent growth in MET-transformed fibroblasts and melanoma cells [70][71][72] , and STAT3 signalling and NF-κb activity are necessary for MET-induced onset of leiomyosarcomas and for the survival of prostate cancer cells, respectively 48,76 . ...
The MET tyrosine kinase receptor (also known as the HGF receptor) promotes tissue remodelling, which underlies developmental morphogenesis, wound repair, organ homeostasis and cancer metastasis, by integrating growth, survival and migration cues in response to environmental stimuli or cell-autonomous perturbations. The versatility of MET-mediated biological responses is sustained by qualitative and quantitative signal modulation. Qualitative mechanisms include the engagement of dedicated signal transducers and the subcellular compartmentalization of MET signalling pathways, whereas quantitative regulation involves MET partnering with adaptor amplifiers or being degraded through the shedding of its extracellular domain or through intracellular ubiquitylation. Controlled activation of MET signalling can be exploited in regenerative medicine, whereas MET inhibition might slow down tumour progression.
... After stimulation with HGF, Gab1 couples with the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and the majority of Met-dependent PI3 kinase activity is associated with Gab1 (Maroun et al., 1999a). In response to HGF, Gab1 also associates with phospholipase C (PLC)-1, the tyrosine phosphatase SHP-2, the adapter protein Crk (Gar- Guzman et al., 1999; Maroun et al., 1999a; Gual et al., 2000; Lamorte et al., 2000; Sakkab et al., 2000) and acts to recruit these signaling proteins to the Met receptor (Maroun et al., 1999a ). The Gab1-dependent recruitment of SHP-2 is required for sustained mitogen-activated protein kinase (MAPK) activity and epithelial morphogenesis downstream from the Met receptor (Maroun et al., 2000). ...
... Membranes were stripped and subsequently blotted with MAPK or JNK antibodies. L.S. Lock et al. al., 1999; Maroun et al., 1999a; Gual et al., 2000; Lamorte et al., 2000; Pratt et al., 2000; Sakkab et al., 2000; Crouin et al., 2001). SHP-2 was identified as a major binding protein for Gab2 (Gu et al., 1998), and by coimmunoprecipitation, both Gab1 and Gab2 associate with endogenous SHP-2 protein after stimulation of cells with HGF (Figure 2B ). ...
... Two potentially phosphorylated tyrosines (Y307 and Y373) are absent in Gab2. However, although these tyrosines contain a consensus binding site for Crk/PLC, Gab2 retains the ability to bind these signaling proteins (Figure 2B) (Gual et al., 2000; Lamorte et al., 2000; Sakkab et al., 2000;). Alternatively, tyrosines 307 and 373 in Gab1 may be required for the association with an unidentified protein that is critical for the morphogenic response. ...
The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis.
... c-Met was first identified as a member of the receptor tyrosine kinases family (RTKs) in 1987 [3] and has since attracted widespread interest because of its aberrant activation during the malignant progression of various cancers. Upon binding to its ligand, hepatocyte growth factor (HGF), c-Met triggers the induction of several downstream signalling cascades leading to a multitude of outcomes: increased survival, proliferation, anchorage-independent growth, enhanced motility, migration, scattering, metastasis and invasion [4][5][6][7][8][9][10]. ...
... Both of these steps result in translocation of ERKs to the nucleus to promote ETS/AP1-mediated transcriptional regulation of cell cycle modulators and adhesion proteins in order to control cell proliferation and motility [5][6][7][8][9]26]. c-Met can also activate the Jun amino-terminal kinases (JNKs) and p38 MAPKs via the same pathways, thereby regulating cytoskeleton-associated proteins that are important in cell migration and scattering [10,27]. Alternatively, PI3K was found to be directly activated by c-Met or indirectly by Ras-driven Akt/PKB signalling, which, in turn, leads to BCL2 antagonist of cell death (Bad) inactivation and to MDM2-mediated p53 degradation, promoting enhanced cell survival [28][29][30]. ...
The c-Met receptor, also known as the HGF receptor, is one of the most studied tyrosine kinase receptors, yet its biological functions and activation mechanisms are still not fully understood. c-Met has been implicated in embryonic development and organogenesis, in tissue remodelling homeostasis and repair and in cancer metastasis. These functions are indicative of the many cellular processes in which the receptor plays a role, including cell motility, scattering, survival and proliferation. In the context of malignancy, sustained activation of c-Met leads to a signalling cascade involving a multitude of kinases that initiate an invasive and metastatic program. Many proteins can affect the activation of c-Met, including a variety of other cell surface and membrane-spanning molecules or receptors. Some cell surface molecules share structural homology with the c-Met extracellular domain and can activate c-Met via clustering through this domain (e.g., plexins), whereas other receptor tyrosine kinases can enhance c-Met activation and signalling through intracellular signalling cascades (e.g., EGFR). In this review, we provide an overview of c-Met interactions and crosstalk with partner molecules and the functional consequences of these interactions on c-Met activation and downstream signalling, c-Met intracellular localization/recycling and c-Met degradation.
... Gab1 contains a pleckstrin homology (PH) domain, which tethers Gab1 to membranes through interactions with PtdIns(3,4,5)P 3 (Maroun et al., 1999) and is recruited to and phosphorylated on multiple tyrosine residues by an activated Met receptor (Birchmeier et al., 2003;Peschard et al., 2007). Upon phosphorylation, these residues serve as docking sites for numerous SH2-domain-containing adaptor and signaling proteins, including Crk, Nck, P85 subunit of PI3K, Shp2 and PLCc (Abella et al., 2010;Schaeper et al., 2000;Garcia-Guzman et al., 1999;Gual et al., 2000;Lamorte et al., 2000;Maroun et al., 1999;Maroun et al., 2000;Cunnick et al., 2001). Gab1 contains six proline rich motifs, two of which (proline-rich motifs four and five) are implicated in the constitutive association with the adaptor protein growth factor receptor-bound protein 2 (Grb2) through its C-terminal SH3 domain (Lock et al., 2002). ...
... Gab1 contains six proline rich motifs, two of which (proline-rich motifs four and five) are implicated in the constitutive association with the adaptor protein growth factor receptor-bound protein 2 (Grb2) through its C-terminal SH3 domain (Lock et al., 2002). Once recruited to a Met-Gab1 complex, these proteins trigger activation of multiple signaling cascades, including PI3K-Akt (Maroun et al., 1999), Ras-MAPK (Maroun et al., 2000;Schaeper et al., 2000), Rac and Rap1 (Lamorte et al., 2000) and Nck/N-WASP (Abella et al., 2010) that promote cell survival, actin cytoskeleton remodeling, as well as increased migration and invasion (Benvenuti and Comoglio, 2007;Birchmeier et al., 2003;Lai et al., 2009;Peschard and Park, 2007). ...
Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure-function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1-cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.
... Scale bars: 10m. tyrosine residues for phenylalanine (as in Gab1DCrk) is necessary to uncouple Crk from Gab1 (Lamorte et al., 2000) (Fig. 5A). To address the role for Crk, CrkII was either overexpressed or depleted through targeted siRNA in MDCK cells and dorsal ruffle formation assayed. ...
... This apparent heterogeneity in Nck binding, might, in part, reflect preferential tyrosine phosphorylation of Y407 by RTKs in vivo, whereas Y242 and Y307 are preferentially phosphorylated by activated Src (Chan et al., 2009). Moreover, Y242, Y307, Y407 represent only three of the six binding sites for Crk proteins on Gab1 (Lamorte et al., 2000) (Fig. 5A) and following overexpression, CrkII can compete with Nck for binding to Gab1 (Fig. 4E; supplementary materialFig. S4C,D), possibly restricting the preferential recruitment of Nck to Y407 (Fig. 4E). ...
The polarised distribution of signals downstream from receptor tyrosine kinases (RTKs) regulates fundamental cellular processes that control cell migration, growth and morphogenesis. It is poorly understood how RTKs are involved in the localised signalling and actin remodelling required for these processes. Here, we show that the Gab1 scaffold is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream from the Met, EGF and PDGF RTKs. Gab1 associates constitutively with the actin-nucleating factor N-WASP. Following RTK activation, Gab1 recruits Nck, an activator of N-WASP, into a signalling complex localised to dorsal ruffles. Formation of dorsal ruffles requires interaction between Gab1 and Nck, and also requires functional N-WASP. Epithelial cells expressing Gab1DeltaNck (Y407F) exhibit decreased Met-dependent Rac activation, fail to induce dorsal ruffles, and have impaired cell migration and epithelial remodelling. These data show that a Gab1-Nck signalling complex interacts with several RTKs to promote polarised actin remodelling and downstream biological responses.
... Gab1 is phosphorylated upon stimulation of cells with various growth factors (Holgado-Madruga et al., 1996; Weidner et al., 1996; Kallin et al., 2004), as well as upon activation of certain cytokines and antigen receptors (Takahashi-Tezuka et al., 1998; Nishida et al., 1999). Gab1 has six tyrosine residues (Y242, Y259, Y307, Y317, Y373 and Y406) in Tyr-X-X-Pro (YXXP) motifs, which serve as the binding sites for the SH2 domain of the adaptor protein Crk (Lamorte et al., 2000). However, Crk is not the only molecule bound to those residues in YXXP motifs in Gab1. ...
... In order to verify the result inFigure 2, four residues (Y242, Y259, Y317 and Y373) were mutated in one Gab1 construct. As these four residues are in YXXP motif and are known for the binding of the SH2 domain of Crk (Lamorte et al., 2000), the resulting Gab1 mutant was named as YF–Crk mutant (Figure 3a ). The level of Srcinduced phosphorylation of the YF–Crk mutant was 27% of that of the wt Gab1 in HEK293 cells (Figure 3b). ...
The docking protein Grb2-associated binder1 (Gab1) has a central role in the integration of the growth-factor signaling. In this study, we aimed to examine the significance of Src-mediated Gab1 phosphorylation in the hepatocyte growth factor (HGF) signaling. Using both mutagenesis and mass spectrometry approaches, Y242, Y259, Y317, Y373 and Y627 of Gab1 were identified to be phosphorylated by c-Src. It is interesting to note that the binding of the tyrosine phosphatase SHP2 to the Y627 antagonized the effect of c-Src on the phosphorylation of the other four tyrosine residues. Moreover, the tyrosine residues predominantly phosphorylated by c-Src were different from those predominantly phosphorylated by the HGF receptor. Gab1 overexpression potentiated both mitogenic and motogenic activities of HGF. However, a Gab1 mutant with substitutions of the Src phosphorylation sites (Y242, Y259, Y317 and Y373) failed to promote HGF-induced DNA synthesis, but retained its ability to facilitate HGF-induced chemotaxis. Taken together, our results not only suggest that the phosphorylation of Gab1 by c-Src is important for HGF-induced DNA synthesis, but also provide an example to illustrate how a docking protein (for example, Gab1) is differentially phosphorylated by c-Src and a receptor tyrosine kinase to emanate full spectrum of signals to the downstream.
... GAB1 is phosphorylated at tyrosine residues in response to a wide variety of stimuli including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), and insulin and stem cell factor (SCF); cytokines such as erythropoietin (EPO), interleukin-6 (IL-6), interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-3 (IL-3), and thrombopoietin (TPO); and various immune stimuli such as anti-IgM or F(ab')2 and lysophosphatidic acid (LPA) [11]. These phosphotyrosine residues are recognized by different proteins with SH2 domains, such as the SH2-containing protein tyrosine phosphatase-2 (SHP2) (Y627, Y659), p85 subunit of phosphoinositide 3-kinase (PI3K) (Y447, Y472, Y589), chicken tumor virus number 10 (CT10) regulator of kinase (CRK) (Y424, Y259, Y317), and others triggering the activation of different signaling pathways [4,14,18]. Furthermore, GAB1 is not only phosphorylated by receptor protein tyrosine kinases, but also by kinases of the SRC family [19,20]. An approximately 40% reduction in GAB1 phosphorylation levels was observed following HGF stimulation in cells derived from SRC null mice. ...
Simple Summary
GRB2-associated binder 1 (GAB1) is a docking protein pivotal in linking multiple stimuli to various intracellular signaling pathways. Embryonic development is disrupted in GAB1-deficient mice, and oncogenic mutations have been noted in cancer cases. In numerous types of cancer, high GAB1 expression levels correlate with a poor prognosis. Studies reveal that GAB1 significantly influences cellular transformation by changes in proliferation, evasion of apoptosis, metastasis, and angiogenesis—all fundamental processes in cancer development. Furthermore, GAB1 is implicated in the resistance/sensitivity to antitumor treatments, thus establishing its potential as an anticancer therapy target.
Abstract
GRB2-associated binder 1 (GAB1) is the inaugural member of the GAB/DOS family of pleckstrin homology (PH) domain-containing proteins. Upon receiving various stimuli, GAB1 transitions from the cytoplasm to the membrane where it is phosphorylated by a range of kinases. This event recruits SH2 domain-containing proteins like SHP2, PI3K’s p85 subunit, CRK, and others, thereby activating distinct signaling pathways, including MAPK, PI3K/AKT, and JNK. GAB1-deficient embryos succumb in utero, presenting with developmental abnormalities in the heart, placenta, liver, skin, limb, and diaphragm myocytes. Oncogenic mutations have been identified in the context of cancer. GAB1 expression levels are disrupted in various tumors, and elevated levels in patients often portend a worse prognosis in multiple cancer types. This review focuses on GAB1’s influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis—each of these processes being a cancer hallmark. GAB1 also modulates the resistance/sensitivity to antitumor therapies, making it a promising target for future anticancer strategies.
... MAPKs also activate the N-terminal Jun kinases (JNK) and p38MAPK. ese proteins, in turn, activate cell cycle regulators stimulating cell proliferation and functional changes in the cytoskeleton necessary for cell migration and invasion [36,37]. Y1356 phosphorylation of c-MET triggers PI3K phosphorylation, which in turn activate focal adhesion kinase (FAK) inducing cell mobility [38]. ...
Background:
c-MET is a receptor tyrosine kinase receptor (RTK) for the hepatocyte growth factor (HGF). The binding of HGF to c-MET regulates several cellular functions: differentiation, proliferation, epithelial cell motility, angiogenesis, and epithelial-mesenchymal transition (EMT). Moreover, it is known to be involved in carcinogenesis. Comprehension of HGF-c-MET signaling pathway might have important clinical consequences allowing to predict prognosis, response to treatment, and survival rates based on its expression and dysregulation. Discussion. c-MET represents a useful molecular target for novel engineered drugs. Several clinical trials are underway for various solid tumors and the development of new specific monoclonal antibodies depends on the recent knowledge about the definite c-MET role in each different malignance. Recent clinical trials based on c-MET molecular targets result in good safety profile and represent a promising therapeutic strategy for solid cancers, in monotherapy or in combination with other target drugs.
Conclusion:
The list of cell surface receptors crosslinking with the c-MET signaling is constantly growing, highlighting the importance of this pathway for personalized target therapy. Research on the combination of c-MET inhibitors with other drugs will hopefully lead to discovery of new effective treatment options.
... The activation of the MAPK cascade will sequentially activate different protein kinases whose terminal effectors include extracellular signal-regulated kinases (Erk1 and Erk2), Jun amino-terminal kinases (JNK1, JNK2 and JNK3) and p38. These downstream elements activate cell cycle regulators leading to cell proliferation and promote alterations in cytoskeletal functions that control cell migration and invasion (Rodrigues, Park et al. 1997;Lamorte, Kamikura et al. 2000). ...
... Six tyrosine residues in Gab1 (Y242, Y259, Y307, Y317, Y373 and Y406) are located in Tyr-X-X-Pro (YXXP) motifs, which serve as binding sites for the adaptor protein Crk [45] and for phospholipase Cγ (PLCγ) that binds to Y307, Y373 and Y406 respectively [46]. Interestingly, the two identified major oxidized cysteines Cys374 and Cys405 are located within or in direct proximity of an YXXP motif with non-phosphorylated Y373 and Y406 if the cysteines were oxidized. ...
Aim
NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.
In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins.
Results
TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1.
Innovation & conclusion
BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
... This signaling axis plays a pivotal role in both normal development and breast cancer progression including survival, proliferation, anchorage-independent growth, enhanced motility, migration, scattering, metastasis, and invasion. 4 The aggressive phenotype of breast cancers correlates with high levels of HGF and/or c-Met overexpression in 20-30% of breast cancers. 5 Therefore, aberrant HGF-dependent c-Met signaling activation driven by various mechanisms can lead to progression of breast cancer cells. ...
The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been correlated with the overexpression of the mesenchymal‐epithelial transition factor (c‐Met) receptor. Thus, the hepatocyte growth factor (HGF)/c‐Met signaling axis has gained considerable attention as a valid molecular target for breast cancer therapy.
This study reports for the first time the discovery of the 13¹‐oxophorbines pheophorbide A and protopheophorbide A along with chlorophyllide A from Ziziphus lotus, an edible typical Tunisian plant, as the potent antiproliferative compounds against the human breast cancer cells MDA‐MB‐231 and MCF‐7. Compared to other compounds, protopheophorbide A exerted the highest light‐independent antiproliferative effect against the metastatic TNBC MDA‐MB‐231 cells (IC50 = 6.5µM). In silico, this compound targeted the kinase domain of multiple c‐Met crystal structures. It potently inhibited the kinase domain phosphorylation of wild and mutant c‐Met in Z‐LYTE kinase assay. Protopheophorbide A inhibited HGF‐induced downstream c‐Met‐dependent cell proliferation, survival, adhesion and migration through RAF/MEK/ERK and PI3K/PTEN/AKT signaling pathways modulation, ROS generation and activation of JNK and p38 pathways. Interestingly, this compound impaired the ability of the MDA‐MB‐231 cells to adhere at different extracellular matrix proteins by reducing the HGF‐induced expression of integrins αv, β3, α2 and β1. Moreover, protopheophorbide A exhibited anti‐migratory properties (IC50 = 2.2µM) through impacting the expression levels of E‐cadherin, vimentin, β‐catenin, FAK, Brk, Rac, and Src proteins. Importantly, treatment with protopheophorbide A significantly inhibited the MDA‐MB‐231 tumor growth in vivo. Our results suggest that protopheophorbide A could be a novel c‐Met inhibitory lead with promise to control c‐Met/HGF‐dependent breast malignancies. This article is protected by copyright. All rights reserved
... Crk proteins have been well documented for overexpression in various cancers including PCa [10] [11] [12] [13] [14]. Crk II, a member of Crk adaptor proteins, has been reported to promote migration and invasion of breast cancer cells and induce anchorageindependent growth [15] [16] [17]. A study also showed that IGF-IR can interact with Crk II and induce downstream signaling through Crk II phosphorylation at tyrosine-221 in NIH-3T3 overexpressing IGF-IR cells [18]. ...
Crk (C10 regulator of kinase) adaptor proteins are highly expressed in many types of human cancers and often contribute to aggressive cancer phenotypes. Crk II, a member of CRK family, has been reported to regulate cell migration and metastasis in breast cancer cells. However, its role in other cancer types has not been reported. In this study, we investigated the molecular function of Crk II in prostate cancer (PCa) cells (CWR-22rv1) in vitro and using a mouse tumor model. Results showed that Crk II knockdown by shRNA-mediated silencing (Crk II-shRNA) in the PCa cells significantly inhibited both cancer cell migration and invasion in cell culture study. Crk II-shRNA cancer cells also significantly decreased colony formation in vitro, but had no significant reduction of tumor volume after 4 weeks of cancer cell xenografting in vivo when compared to the scramble control. Interestingly, Crk II-shRNA cancer cells showed a greatly reduced level of insulin-like growth factor 1 receptor (IGF-1R) and decreased signaling of the IGF-1R/PI3K/Akt axis upon IGF-1 ligand stimulation. A close interaction between Crk II and IGF-1R was demonstrated upon co-immunoprecipitation of IGF-1R with Crk II protein. Further, treatment of cells with either proteosomal degradation or protein synthesis inhibitor showed higher proportion of ubiquitin-associated IGF-1R and faster degradation of IGF-1R in Crk II-shRNA cells in comparison with that in the control cancer cells. Taken together, these data suggest that Crk II plays an important role in the regulation of IGF-1R protein stability and affects downstream of IGF-1R signaling pathways. Therefore, targeting Crk-II can block IGF-1R growth signaling and suppress cancer cell invasion and progression.
... STAT3 activation can be induced by the recruitment and activation of SRC to MET multisubstrate docking site or directly binding to MET [45], and may also be involved in anchorage-independent growth and formation of branch tubules [79]. The RAS-RAF-ERK1/2 pathway mediates proliferation and cell cycle progression, and together with PI3K/AKT pathway have been shown to be involved in spreading and cell motility [93][94][95][96][97]. MET is also involved in cell migration and invasion mediated by CRKL and PCL-γ; morphogenesis also requires STAT3 and PLC-γ pathways [79,88,[98][99][100][101][102][103][104][105][106]. Likewise, activation of RAS-RAC1/CDC42/RHO-PAK regulates cell adhesion and cytoskeletal proteins MET in non-small-cell lung carcinoma Review domain is involved in MET downregulation, as it binds proteins such as E3 ubiquitin ligase and CBL [107,108]. ...
Non-small-cell lung cancer (NSCLC) leads cancer-related deaths worldwide. Mutations in the kinase domain of the EGFR gene provide sensitivity to tyrosine kinase inhibitors (TKI) drugs. TKI show initial response rates over 75% in mutant EGFR-NSCLC patients, although most of these patients acquire resistance to EGFR inhibitors after therapy. EGFR-TKI resistance mechanisms include amplification in MET and its ligand, and also MET mutations. MET signaling dysregulation has been involved in tumor cell growth, survival, migration and invasion, angiogenesis and activation of several pathways, therefore representing an attractive target for anticancer drug development. In this review, we will discuss MET-related mechanisms of EGFR-TKI resistance in NSCLC, as well as the main drugs targeted to inhibit MET pathway.
... Although pY1313 had the ability to recruit most SH2 domains, it notably lacked the ability of pY1295, pY1307, and pY1365 to recruit the Shp2/PTPN11 SH2 domain. GAB1 had six predicted recruitment sites for SH2 domains from CRK and CRKL based on SH2 domain interaction prediction algorithms: pY242, pY259, pY307, pY373, and pY406 [41,42]. However, we identified no CRK SH2 domain interactions with these predicted recruitment sites (Figure 1). ...
Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high-throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several RTKs and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used this data to build a permutation- based logistic (PEBL) regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains.
... As well as ERK, c-Met is able to activate c-Jun N-terminal kinase (JNK) and p38 MAPKs. JNK and p38 by c-Met-dependent stimulation control a range of diverse cellular processes such as cell proliferation, differentiation, transformation and apoptosis ( Fig. 2A) (Rodrigues et al., 1997;Lamorte et al., 2000;Recio and Merlino, 2002). ...
A primary hurdle in developing anticancer therapeutics is to selectively target cancer cells while sparing normal tissues. Oncogenic protein kinases represent a class of biologically important targets for cancer intervention. Among them, c-Met is a receptor tyrosine kinase (RTK) that has low activity in normal tissues but is dysregulated in many tumor types. The c-Met is the prototype member of a subfamily of RTKs, which includes Ron, which is structurally distinct from other RTK families. It is the only known high-affinity receptor for hepatocyte growth factor, also known as scatter factor. HGF and c-Met are both required for normal mammalian development. In adults, both are widely expressed in a variety of tissues; however, their expression is normally very low and is involved mainly in tissue damage, repair and regeneration. The results of in vitro and in vivo experiments have shown that this receptor-growth factor pair is involved in multiple physiologic cellular responses, including cell proliferation, survival, differentiation, motility, and invasion. Here, as well as presenting the biological aspects of c-Met signaling regulation, we consider recent findings that have provided new knowledge at the molecular, cellular, and animal study. Also, we describe how the c-Met pathway is tuned by the functional cooperation between various signal transducers. We then discuss the progress in the development of agents that target the c-Met pathway, with an emphasis on small molecules of c-Met kinase inhibitors. Finally, we provide our perspective in terms of possible future trends and limitation in this field.
... Movement of cells into scratch wounds requires modulation of cell adhesion and changes in the cytoskeleton, e.g., membrane protrusion and generation of new sites of substrate adhesion at the front, as well as actin disassembly and cell detachment at the rear. Small GTPases and protein kinases play essential roles in actin dynamics and cell migration processes, and closure of scratch wounds in cultured cells depends on Rho and Rac as well as c-Jun N-terminal kinase (Ridley et al., 1995; Fenteany et al., 2000; Lamorte et al., 2000; Raftopoulou and Hall, 2004). Wound closure in vitro is not only achieved by activities restricted to cells in the front row, but also involves cells further away from the wound edge (Farooqui and Fenteany, 2005). ...
Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.
... This enhances cellular migration and contributes to the membrane recruitment of Gab1 in HGF-stimulated MDCK cells and the organisation of focal adhesion complexes [93]. Furthermore, other studies have shown that formation of the Gab1/Crk complex is a critical event in c-Met induced activation of the JNK pathway, an event downstream of Rac activation and a prerequisite for several of the aforementioned morphological changes and efficient cellular transformation [72,131,132] . Interestingly , a recent report has demonstrated that the p85 and Crk binding sites in Gab1 play a pivotal role in the c- Met mediated entry of the intracellular bacterium Listeria monocytogenes, implicating the Gab1/Crk complex in promotion of cytoskeletal rearrangements required for pathogen internalization [133]. ...
Since their discovery a little more than a decade ago, the docking proteins of the Gab/DOS family have emerged as important signalling elements in metazoans. Gab/DOS proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine and antigen receptors as well as cell adhesion molecules. They also contribute to signal diversification by channelling the information from activated receptors into signalling pathways with distinct biological functions. Recent approaches in protein biochemistry and systems biology have revealed that Gab proteins are subject to complex regulation by feed-forward and feedback phosphorylation events as well as protein-protein interactions. Thus, Gab/DOS docking proteins are at the centre of entire signalling subsystems and fulfil an important if not essential role in many physiological processes. Furthermore, aberrant signalling by Gab proteins has been increasingly linked to human diseases from various forms of neoplasia to Alzheimer's disease.
In this review, we provide a detailed overview of the structure, effector functions, regulation and evolution of the Gab/DOS family. We also summarize recent findings implicating Gab proteins, in particular the Gab2 isoform, in leukaemia, solid tumours and other human diseases.
... As alluded to above, utilization of different scaffolds could allow for localized signaling, by virtue of being targeted by specific kinases, or could control the selectivity of Crk signaling. A good illustration of this comes from a study by Lamorte and colleagues, showing that a switch from a p130Cas/Crk to a Gab1/Crk complex in Met oncogene transformed cells correlates with a change from a motile phenotype to a proliferation phenotype[113]. In a few cases, Crk can interact directly with tyrosine kinase receptors. For example, direct recruitment of Crk to the PDGFRα[28]or the VEGFR-3[29]can regulate immediate post-receptor signals from a subset of growth factors at the plasma membrane. ...
The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses.
... Both pathways mediate cell adhesion, motility and invasion151617. Cell migration and invasion are mediated by Ras, Crk, and c-src/FAK, and branching morphogenesis further requires the STAT3 and PLC-γ pathways1819202122 . Specifically, activation of Ras-Rac1/Cdc42- PAK and Gab1-Crk-C3G-Rap1 regulates cell adhesion and cytoskeletal proteins. ...
Dysregulation of mesenchymal-epithelial transition factor receptor tyrosine kinase pathway leads to cell proliferation, protection from apoptosis, angiogenesis, invasion, and metastasis. It can be dysregulated through overexpression, constitutive activation, gene amplification, ligand- dependent activation or mutation. New drugs targeting various mesenchymal-epithelial transition factor pathways are being investigated with promising results. © 2009 by the International Association for the Study of Lung Cancer.
... An example is Crk, an adaptor that together with p130Cas defines the molecular switch for tumor cell migration and metastasis [112,[137][138][139][140], which is critically involved in Rho-mediated cell-rounding and detachment of ephrin-A5-stimulated EphA3-expressing cells [67]. While a number of studies have confirmed its role in cell adhesion, focal complex assembly and migration [33,113,114,141], more recent findings suggest that EphB4-dependent Abl-Crk signalling also affects proliferation of transformed breast cancer cell lines [115]. ...
Eph receptors and their membrane-bound ephrin ligands are developmental cell guidance cues that direct cell migration and orchestrate patterning processes by modulating adhesive or repulsive cell properties. During the past two decades, an exponentially growing interest in their function has resulted in a considerably advanced understanding of the cellular and molecular principles of Eph function in normal and oncogenic development. Ephs not only accurately guide the path of migrating cells, but also facilitate contact and communication between neighbouring cell populations, in particular at epithelial/mesenchymal boundaries. Precise cell positioning not only relies on accurately-graded expression of individual Eph/ephrin pairs, but on the sum of interactions within particular expression domains and their modulation through crosstalk with a range of other signalling systems. There is little doubt that Eph and ephrins provide exciting new targets for anti-cancer therapies, but in appreciation of the complexity of their signals and biological functions it is perhaps not surprising that the development of Eph-specific therapeutics is only emerging.
... Both the CrkII and CrkL adapter proteins coordinate cell motility (Klemke et al., 1998; Uemura and Griffin, 1999; Cho and Klemke, 2000; Petit et al., 2000; Spencer et al., 2000), yet their involvement in the regulation of EM transitions has not been addressed. We and others have demonstrated that after activation of the Met receptor tyrosine kinase, CrkII and CrkL are recruited into Met-dependent signaling complexes (Garcia-Guzman et al., 1999 Lamorte et al., 2000; Sakkab et al., 2000). Both CrkII and CrkL are expressed in MDCK epithelial cells and in well-differentiated breast cancer cell lines that retain the ability to grow as colonies (see Figures 1 and 6 and our unpublished results). ...
Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells.
... We also examined the putative role of c-Met, the receptor for HGF, in the UV-induced Gab1-JNK pathway. Previous reports showed that Gab1 is involved in JNK activation and cell transformation by an oncogenic form of c-Met, Tpr/Met (9,24). In contrast to the results of with EGFR, UV irradiation induced tyrosine phosphorylation of c-Met in wild-type cells that was correlated to induction of JNK activity. ...
Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH2-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK
activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this
signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat
shock, and this defect was rescued by reintroduction of Gab1 into Gab1−/− cells. Consistently, Gab1−/− cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively
complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest
the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation.
In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.
... Tyrosines 242, 259, 307, 317, 373 and 407 are within YxxP motifs, predicted to bind the SH2 domains of Crk family proteins (Feller, 2001). Simultaneous mutation of all six of these residues to phenylalanine results in a total loss of CrkII or CrkL binding to the mutant Gab1 D Crk protein (Lamorte et al ., 2000;Sakkab et al., 2000). ...
The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.
CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk. Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G1-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth.
Receptor for activated C kinase 1 (RACK1) is a WD-domain repeating protein which involves in the mediation of various biological processes, including innate immune response. In the present study, a RACK1 (designed as EsRACK1) gene from Chinese mitten crab E. sinensis was cloned by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA sequence of EsRACK1 was of 1117 bp with an open reading frame (ORF) of 957 bp encoding a polypeptide of 318 amino acids containing seven WD repeats. EsRACK1 shared 62%-99% similarities with previously identified RACK1s in amino acid sequence, and it was clustered with the RACK1 from Pacifastacus leniusculus in the phylogenetic tree. The mRNA transcripts of EsRACK1 were constitutively expressed in various tissues with the highest expression level in hepatopancreas. The expression of EsRACK1 mRNA in hemocytes were significantly up-regulated post the stimulations with Vibrio anguillarum and Pichia pastoris. After exposure to CdCl2 and pentachlorophenol, the transcripts of EsRACK1 in hemocytes were up-regulated at the late phase from 12 h. When EsRACK1 was knocked down by dsRNA based RNAi, the total hemocyte counts, new-born hemocytes and phosphorylation of JNK were all significantly decreased. In addition, EsRACK1 transcription and phosphorylation of JNK were both decreased in hematopoietic tissue post Aeromonas hydrophila challenge. All the results suggested that EsRACK1 was involved in the innate immune response of the crab and participated in the production of new-born hemocytes through activation of JNK.
The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis.
The Crk adaptor proteins (CrkI, CrkII and CrkL) play an important role during cellular signalling by mediating the formation of protein-protein complexes and are involved in cellular migration, invasion, and adhesion. Targeting CrkI and CrkII in breast cancer cell lines by RNA interference demonstrated that loss of Crk expression corresponded with a significant decrease in cell migration and invasion. This implies that Crk adaptor proteins play an important role in integrating signals for migration and invasion of highly malignant cancer cell lines. As migration and invasion are important components of the metastatic cascade, future work includes stable knockdown of Crk in breast cancer cell lines and performing in vivo metastasis assays. Furthermore, mouse models over-expressing CrkI/II result in delayed ductal outgrowth. MMTV-CrkII mice display enhanced branching and leads to tumour development. This has important implications as we have shown elevated levels of Crk are observed in human breast cancer. This project may provide information, which could be used to develop effective treatments for breast cancer, as well as other cancer types.
The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.
Tumor progression of oral and other head and neck squamous cell carcinomas (HNSCCs) from dysplasia to metastasis involves
a series of pathologic phenotypic changes considered to be the hallmarks of cancer, and these have been associated with a
number of genetic, epigenetic, and molecular alterations (Fig. 10.1). Pathologic phenotypic changes precede metastasis and
include increased cell proliferation, survival, and horizontal spread, which require certain molecular changes that together
with later events contribute to the metastatic phenotype. These steps commonly include altered expression of molecules regulating
the cell cycle and death (e.g., p53), growth factor response (epidermal growth factor receptor, EGFR), protein synthesis and
metabolism (mammalian targets of rapamycin, mTOR), and cell immortality (telomerase). The subsequent steps of invasion and
metastasis involve penetration and breakdown of the extracellular matrix (ECM) comprising the basement membrane and interstitial
connective tissue; formation and invasion of a new stroma of host inflammatory and mesenchymal cells; neoangiogenesis and
lymphangiogenesis; and distant spread via these lymphatics and blood vessels to secondary regional and distant sites. The
molecular events accompanying the metastatic stage commonly include loss of expression or function of tumor suppressor genes
or increased expression or function of oncogenes including numerous growth factors, cytokines, cell adhesion molecules and
proteases, signal kinases, and nuclear transcription factors. Nuclear transcription factors appear to play a central role
in malignant transformation and metastasis, since direct overexpression, mutation or activation of these molecules, or components
of various upstream signaling pathways that modulate their function (Chaps. 8, 9, 11, 13) result in altered regulation of
expression of diverse gene programs that produce the phenotypic changes characteristic of cancer and metastasis (Chaps. 4,
5, 12–14).
Transitions between epithelial and mesenchymal cell phenotypes occur as part of normal organ development and wound healing,
and are also observed in cancer, fibrosis and other diseases. Although the latter processes involve dysregulated interconversion
of epithelial and mesenchymal cell phenotypes, the molecular mechanisms and sequences of cellular changes often closely resemble
events that occur as a part of normal development and tissue repair. While primarily epithelial to mesenchymal transition
(EMT) has been implicated in cancer and fibrosis, both EMT and mesenchymal to epithelial transition (MET) occur during organogenesis
and development.
The hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/c-MET) receptor tyrosine kinase (RTK) pathway plays a pleotropic role in cell proliferation, migration, invasion, angiogenesis and survival. Although it has critical physiological functions in embryonic development and tissue repair, this signaling cascade is frequently deregulated in a wide range of tumors. Aberrant HGF/c-MET signaling, driven by various mechanisms, including constitutive activation and over-expression, has multifunctional effects in oncogenesis and is implicated in the acquisition of an aggressive phenotype with metastatic potential. The central role of c-MET activity in cancer progression, as well as disparities between quiescent HGF/c-MET signaling in normal tissue and overexpression in tumor may provide a degree of tumor selectivity for therapeutic intervention, making HGF or c-MET inhibition an attractive proposition in oncology. This review focuses on the underlying oncogenic role of aberrant HGF/c-MET signaling in malignant progression, as well as recent preclinical and clinical data on the different strategies employed in inhibiting HGF/c-MET function.
HGF, the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, which are processes that play a crucial role in cell motility during normal development and malignant transformation. We and others have shown earlier that the adapter protein CrkII and its associated proteins positively regulate cell migratory events in response to both haptotactic and chemotactic stimuli, including HGF. Here, we demonstrate for the first time that phosphorylation of CrkII serves as a negative feedback loop to regulate motile responses upon Met stimulation. Thus, we found that the treatment of cells with HGF induces tyrosine phosphorylation of CrkII at Y221, which in turn results in inhibition of CrkII signaling via formation of an intramolecular pY221-SH2-domain interaction. Accordingly, expression of a mutant form of CrkII, CrkII-Y221F, which is resistant to phosphorylation at this negative regulatory site, enhanced Met-induced cell motility. Furthermore, we demonstrate here that the Met-induced CrkII phosphorylation depends on the Abl tyrosine kinase activity. As a corollary, we found that Abl inhibitors, such as the STI571 compound, significantly enhanced Met-induced cell motility, but failed to do so in cells that expressed the CrkII-Y221F mutant protein. Taken together, these results demonstrate that the Abl tyrosine kinase functions as a negative regulator of Met-induced cell migration, and that it does so by inducing CrkII phosphorylation at the site Y221.
The receptor tyrosine kinase MET has been studied of a large variety of human cancers, including lung and mesothelioma. The MET receptor and its ligand HGF (hepatocyte growth factor) play important roles in cell growth, survival and migration, and dysregulation of the HGF-MET pathway leads to oncogenic changes including tumor proliferation, angiogenesis and metastasis. In small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and malignant pleural mesothelioma (MPM), MET is dysregulated via overexpression, constitutive activation, gene amplification, ligand-dependent activation, mutation or epigenetic mechanisms. New drugs targeted against MET and HGF are currently being investigated in vitro and in vivo, with promising results. These drugs function at a variety of steps within the HGF-MET pathway, including MET expression at the RNA or protein level, the ligand-receptor interaction, and tyrosine kinase function. This paper will review the structure, function, mechanisms of tumorigenesis, and potential for therapeutic inhibition of the MET receptor in lung cancer and mesothelioma.
Since Cas was first identified as a highly phosphorylated 130 kilodalton protein that associated with the v-Src and v-Crk-oncoproteins, considerable effort has been made to determine its function. Its predicted role as a scaffolding molecule based on its domain structure has been largely confirmed. Through its ability to undergo rapid changes in phosphorylation, subcellular localization and association with heterologous proteins, Cas may spatially and temporally regulate the function of its binding partners. Numerous proteins have been identified that bind to Cas in vitro and/or in vivo, but in only a few cases is there an understanding of how Cas may function in these protein complexes. To date, Cas-Crk and Cas-Src complexes have been most frequently implicated in Cas function, particularly in regards to processes involving regulation of the actin cytoskeleton and proliferation. These and other Cas protein complexes contribute to the critical role of Cas in cell adhesion, migration, proliferation and survival of normal cycling cells. However, under conditions in which these processes are deregulated, Cas appears to play a role in oncogenic transformation and perhaps metastasis. Therefore, in its capacity as an adapter protein, Cas serves as a point of convergence for many distinct signaling inputs, ultimately contributing to the generation of specific cellular responses.
Following discovery of c-Cbl, a cellular form of the transforming retroviral protein v-Cbl, multiple Cbl-related proteins have been identified in vertebrate and invertebrate organisms. c-Cbl and its homologues are capable of interacting with numerous proteins involved in cell signaling, including various molecular adapters and protein tyrosine kinases. It appears that Cbl proteins play several functional roles, acting both as multivalent adapters and inhibitors of various protein tyrosine kinases. The latter function is linked, to a substantial extent, to the E3 ubiquitin-ligase activity of Cbl proteins. Experimental evidence for these functions, interrelations between them, and their biological significance are addressed in this review, with the main accent placed on the adapter functions of Cbl proteins.
Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. Selective formation of multi-protein complexes by the Crk and Crk-like (CRKL) proteins depends on specific motifs recognized by their SH2 and SH3 domains. In the case of the first SH3 domains [SH3(1)] a P-x-x-P-x-K motif is crucial for highly selective binding, while the SH2 domains prefer motifs which conform to the consensus pY-x-x-P. Crk family proteins are involved in the relocalization and activation of several different effector proteins which include guanine nucleotide releasing proteins like C3G, protein kinases of the Abl- and GCK-families and small GTPases like Rap1 and Rac. Crk-type proteins have been found not only in vertebrates but also in flies and nematodes. Major insight into the function of Crk within organisms came from the genetic model organism C. elegans, where the Crk-homologue CED-2 regulates cell engulfment and phagocytosis. Other biological outcomes of the Crk-activated signal transduction cascades include the modulation of cell adhesion, cell migration and immune cell responses. Crk family adaptors also appear to play a role in mediating the action of human oncogenes like the leukaemia-inducing Bcr-Abl protein. This review summarizes some key findings and highlights recent insights and open questions.
Mitogen-activated protein kinase and Phosphatidylinositol-3 kinase/Akt-mediated signaling pathways play a major role in controlling cell proliferation, differentiation and cell death. Phosphorylation and dephosphorylation of their specific Thr/Tyr residues is critical in determining their activity. We determined the expression pattern and activity of MAP kinases and Akt in Primitive Neuroectodermal Tumors (PNETs). The kinase activity of extracellular signal-regulated kinase (ERK) was higher in both primary tumors and cell lines, as evident from the increased phosphorylation of ERK1 and ERK2. We did not observe the activation of C-jun N-terminal kinase (JNK) or p38 MAPK The expression of Raf-1, a kinase acting upstream of ERK, was significantly increased in primary tumors compared to normal brain. The PI-3 kinase-activated phosphorylation of Akt was also higher in primary tumors. These results suggest that activation of the Raf-1/ERK module of the MAP kinase pathway play an important role in PNETs.
The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.
Gab proteins, including mammalian Gab1, Gab2, Gab3, Drosophila DOS and Caenorhabditis elegans Soc1, comprise a growing family of scaffolding/docking molecules involved in multiple signaling pathways mediated by receptor tyrosine kinases (RTKs) and non-RTK receptors. This paper reviews the structure/function relationships of Gab proteins and their biological roles during normal growth, differentiation and development programs. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.
Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.
C-Jun NH(2)-terminal kinase (JNK) has been implicated in numerous functions including stress responses, apoptosis,and transformation. The role in transformation is based largely on studies of isolated cell types with little indication of whether JNK plays a general role in a specific human tumor type or whether this occurs in vivo.
We examined 9 human prostate carcinoma cell lines in vitro and a representative line in vivo.
For all of the cell lines proliferation is highly correlated with serum-supported JNK activity (r(Pearson) = 0.91; P = 0.004), whereas no relationship was observed for 10 human breast cancer cell lines (r(Pearson) = -0.32). Treatment with characterized antisense oligonucleotides complementary to sequences common to either the JNK1 or JNK2 family of isoforms showed that, whereas antisense JNK1 inhibited growth by a maximum of 57%, antisense JNK2 inhibited proliferation up to 80%. Sense and scrambled control oligonucleotides had little effect (average 3.7 +/- 1.5%). Moreover, systemic treatment of mice bearing established xenografts of PC3 prostate carcinoma cells with antisense JNK1 and JNK2 led to inhibition tumor growth by 57% (P < 0.002) and 80% (P < 0.001), respectively. The difference is significant (P < 0.012). Combined antisense treatment led to a significant increase in frequency of tumor regression (P = 0.022).
These results indicate that JNK is required for growth of prostate carcinoma cells in vitro and in vivo, and additionally indicate that JNK2 plays a dominant role. The JNK pathway is a novel target in the treatment of prostate carcinoma.
Ras proteins function as molecular switches that cycle between an inactive GDP-bound state, and an active GTP-bound form that triggers different signaling pathways. Because Ras can integrate both proliferative and anti-apoptotic stimuli, GTP-locked Ras mutants play a critical role in the development of human tumors. Moreover, wild-type Ras relays the transforming potential of a number of molecules involved in tumor development, including protein tyrosine kinases. Consequently, the molecular intermediates that control Ras activation are potential targets of anti-tumoral pharmacology. Besides the canonical Shc/Grb2/Sos module classically involved in Ras activation, novel effectors have recently been shown to participate in this pathway, including the multivalent Grb2-associated docking protein Gab1, the protein tyrosine phosphatase SHP-2, and the phosphoinositide 3-kinase. Recent genetic advances have shown that these proteins are critically involved in cell proliferation and survival, further suggesting that they could be interesting targets for selective tumor therapy. Here we review recent progress in our understanding of the role of Gab1 and its partners in Ras activation, and other survival/proliferation pathways. Implications for the pharmacological manipulation of this pathway in the treatment of cancer will also be discussed.
Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.
Hepatocyte growth factor/scatter factor and its receptor, the tyrosine kinase Met, arose late in evolution and are unique to vertebrates. In spite of this, Met uses molecules such as Gab1 — homologues of which are present in Caenorhabditis elegans and Drosophila melanogaster — for downstream signalling. Pivotal roles for Met in development and cancer have been established: Met controls cell migration and growth in embryogenesis; it also controls growth, invasion and metastasis in cancer cells; and activating Met mutations predispose to human cancer.
The hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the Met protein tyrosine kinase, form a classic ligand-receptor system for epithelial-mesenchymal communications in the normal and cancerous prostate. This review illustrates the expression and activities of HGF/SF and Met during prostate development, homeostasis, and carcinogenesis. The participation of HGF/SF in the morphogenetic program of rodent prostate development, the role of Met in normal human prostate epithelium, and underlying mechanisms of deregulated Met expression in localized and metastatic prostate cancer are discussed. On the basis of the commonly observed overexpression of Met in metastatic prostate cancer, HGF/SF-Met-targeted imaging and therapeutic agents can now be applied toward diagnosis and treatment.
Crk adaptor proteins play an important role during cellular signaling by mediating the formation of protein complexes. Increased levels of Crk proteins are observed in several human cancers and overexpression of Crk in epithelial cell cultures promotes enhanced cell dispersal and invasion, implicating Crk as a regulator of invasive responses. To determine the requirement of Crk for invasive signals, we targeted the CRKI/II gene by RNA interference. Consistent knockdown of CrkI/II was observed with two small interfering RNA targeting sequences in all human cancer cell lines tested. CrkI/II knockdown resulted in a significant decrease in migration and invasion of multiple malignant breast and other human cancer cell lines (MDA-231, MDA-435s, H1299, KB, and HeLa). Moreover, CrkI/II knockdown decreased cell spreading on extracellular matrix and led to a decrease in actin stress fibers and the formation of mature focal adhesions. Using immunohistochemistry, we show elevated CrkI/II protein levels in patients with breast adenocarcinoma. Together, these studies identify Crk adaptor proteins as critical integrators of upstream signals for cell invasion and migration in human cancer cell lines and support a role for Crk in metastatic spread.
Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, imatinib, bevacizumab, and gefitinib inhibitors have illustrated the utility of targeting this protein class for treatment of selected cancers. A unique member of the RTK family, c-Met, also represents an intriguing target for cancer therapy that is yet to be explored in a clinical setting. The proto-oncogene, c-Met, encodes the high-affinity receptor for hepatocyte growth factor (HGF) or scatter factor (SF). c-Met and HGF are each required for normal mammalian development and have been shown to be particularly important in cell migration, morphogenic differentiation, and organization of three-dimensional tubular structures (e.g. renal tubular cells, gland formation, etc.) as well as cell growth and angiogenesis. Both c-Met and HGF have been shown to be deregulated in and to correlate with poor prognosis in a number of major human cancers. New data describing the constitutive phosphorylation of c-Met in a number of human tumors is presented here along with a variety of mechanisms by which c-Met can become activated, including mutation and gene amplification. In support of the clinical data implicating c-Met activation in the pathogenesis of human cancers, introduction of c-Met and HGF (or mutant c-Met) into cells conferred the properties of motility, invasiveness, and tumorgenicity to the transformed cells. Conversely, the inhibition of c-Met with a variety of receptor antagonists inhibited the motility, invasiveness, and tumorgenicity of human tumor cell lines. Consistent with this observation, small-molecule inhibitors of c-Met were developed that antagonized c-Met/HGF-dependent phenotypes and tumor growth in mouse models. This review will address the potential for development of c-Met inhibitors for treatment of human cancers with particular emphasis on recent findings with small-molecule inhibitors.
Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.
Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin–Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin–Crk complex in the collagen-induced cell motility.
Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the beta subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase. Covalent cross-linking of 125I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF.
We show in this report that two v-src substrate proteins, p130 and cortactin, become tyrosine-phosphorylated during integrin-mediated cell adhesion to extracellular matrix substrata and
upon cell attachment onto immobilized anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach
to polylysine or through antibodies against major histocompatibility complex. It also does not take place when adhesion-mediated
reorganization of the actin cytoskeleton is inhibited with cytochalasin D. Tyrosine phosphorylation of p130 and cortactin coincides with tyrosine phosphorylation of focal adhesion kinase during integrin-mediated cell adhesion but
is independent of cell adhesion in v-src-transformed cells. The tyrosine-phosphorylated sites in p130 and cortactin may serve as binding sites for proteins containing Src homology 2 domains, as is the case with two other integrin-regulated
docking proteins, focal adhesion kinase and paxillin. Thus, these results suggest that ligand binding of integrins regulates
the tyrosine phosphorylation state of multiple docking proteins. These proteins may mediate anchorage dependence of growth;
their misregulation in v-src-transformed and other tumorigenic cells may be responsible for the anchorage independence of such cells.
Constitutive activation of the Met tyrosine kinase results in transformation of cells of diverse origin. Recent studies have demonstrated a role for the c-Jun N-terminal kinase (JNK) in Met-induced transformation, but little is known about the molecular mechanisms that connect Met to JNK activation. Our studies show that activated Met associates with, and phosphorylates, the docking protein Gab1, which in turn binds to the src homology 2 (SH2)-domain of the adapter protein Crk and recruits Crk to the Met signaling complex. Formation of the Gab1 - Crk complex correlates with Met-induced JNK activation, and mutant forms of Met that fail to induce the complex formation also fail to activate JNK. Importantly, expression of a loss-of-function mutant of Crk severely impairs activation of the JNK pathway by Met. We also show here that Met controls the transcription of the matrix metalloproteinase-1 (MMP-1) gene in carcinoma cells and that this transcriptional regulation occurs in a Crk - JNK-dependent manner through an AP-1 element in the MMP-1 promoter. Taken together, our data implicate the Gab1 - Crk signaling complex in Met-induced JNK activation and suggest that the Gab1 - Crk complex formation may be an important event in regulating the tumorigenic phenotype of Met-transformed cells.
p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2-containing molecules.
The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.
Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.
The Tpr-Met oncoprotein, which is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement,
consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused
downstream from sequences encoded by the tpr gene. We have previously demonstrated that a single tyrosine residue in the carboxyl terminus, Tyr489, is highly phosphorylated and is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and for the association
of Tpr-Met with the Grb2 adaptor protein and phosphatidylinositol 3′-kinase. We show here that Tyr489 is also required for association of Tpr-Met with phospholipase Cγ and the tyrosine phosphatase, SHPTP2/Syp. To distinguish
which of these substrates are required for cell transformation by the Tpr-Met oncoprotein, we generated a novel Tpr-Met mutant
that selectively fails to associate with the Grb2 adaptor protein. Utilizing this mutant, together with additional Tpr-Met
mutants containing Tyr to Phe substitutions, we have demonstrated that transformation of Fr3T3 fibroblasts by the Tpr-Met
oncoprotein is dependent upon pathways downstream of Shc and Grb2 and that pathways downstream of phosphatidylinositol 3′-kinase,
phospholipase Cγ, and SHPTP2/Syp are insufficient for transformation.
The concept of anchorage-dependent growth and the close relationship between anchorage independence and tumorigenicity were first appreciated more than a quarter century ago ([9][1], [16][2], [23][3], [24][4]). Penman and his coworkers then showed that incubation of cells in the absence of
Hereditary papillary renal carcinoma (HPRC) is a recently recognized form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumours. The pattern of inheritance of HPRC is consistent with autosomal dominant transmission with reduced penetrance. HPRC is histologically and genetically distinct from two other causes of inherited renal carcinoma, von Hippel-Lindau disease (VHL) and the chromosome translocation (3;8). Malignant papillary renal carcinomas are characterized by trisomy of chromosomes 7, 16 and 17, and in men, by loss of the Y chromosome. Inherited and sporadic clear cell renal carcinomas are characterized by inactivation of both copies of the VHL gene by mutation, and/or by hypermethylation. We found that the HPRC gene was located at chromosome 7q31.1-34 in a 27-centimorgan (cM) interval between D7S496 and D7S1837. We identified missense mutations located in the tyrosine kinase domain of the MET gene in the germline of affected members of HPRC families and in a subset of sporadic papillary renal carcinomas. Three mutations in the MET gene are located in codons that are homologous to those in c-kit and RET, proto-oncogenes that are targets of naturally-occurring mutations. The results suggest that missense mutations located in the MET proto-oncogene lead to constitutive activation of the MET protein and papillary renal carcinomas.
The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr489, is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3' kinase, phospholipase Cgamma, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa Tpr-Met substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in Tpr-Met-mediated cell transformation.
The ability of cancer cells to proliferate in the absence of adhesion to extracellular matrix (ECM)1 proteins, termed anchorage independence of growth, correlates closely with tumorigenicity in animal models (14). This property of cancer cells presumably reflects the tendency of tumor cells to survive and grow in inappropriate locations in vivo. Such incorrect localization, as occurs in invasion and metastasis, is the characteristic that distinguishes malignant from benign tumors (31).
Great progress has been made in the last 20 years toward understanding how growth is controlled in normal cells and how oncogenes usurp these controls. Yet studies on how oncogenes (or loss of tumor suppressors) overcome the mechanisms that govern cellular location have lagged considerably. The finding that integrins transduce signals that influence intracellular growth regulatory pathways provided some insight into anchorage dependence. Available evidence indicates that integrin-dependent signals mediate the growth requirement for cell adhesion to ECM proteins.
Our understanding of integrin signaling has now reached a stage that connections to oncogenesis are becoming clear, enabling us to place a number of proto-oncogenes and oncogenes with respect to their adhesion dependence or independence. While many details of molecular mechanisms remain to be elucidated, sufficient information is now available to propose a general framework for how oncogenes lead to anchorage-independent growth.
The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 --> N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 --> H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive-metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.
The ABL, PDGFR, RET, and ALK tyrosine kinases have been clearly implicated in the etiology of human malignancies. However, in other tumors, distinguishing between tyrosine kinases as causative agents, as opposed to markers of disease progression or reflections of aberrant tumor cells, has proven difficult. In the absence of a genetic mutation in a tyrosine kinase, it has been difficult to confirm a role for various tyrosine kinases in most malignant human diseases. Given the central role of tyrosine kinases in the control of cellular growth and differentiation, however, it is likely that tyrosine kinases will be shown to participate in crucial aspects of tumorigenesis, some of which may provide fruitful avenues for therapeutic intervention.
Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.
Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains, is believed to participate in signal relay downstream of growth factor receptors. We show here that this phosphatase also plays an important role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblast cells lacking a functional Shp-2 were impaired in their ability to spread and migrate on fibronectin compared with wild-type cells. Furthermore, Shp-2 mutant cells displayed an increased number of focal adhesions and condensed F-actin aggregation at the cell periphery, properties reminiscent of focal adhesion kinase (FAK)-deficient cells. This is consistent with our previous observations in vivo that mice homozygous for the Shp-2 mutation died at midgestation with similar phenotype to FAK and fibronectin-deficient embryos, having severe defects in mesodermal patterning, particularly the truncation of posterior structures. Biochemical analysis demonstrated that FAK dephosphorylation was significantly reduced in Shp-2 mutant cells in suspension. Furthermore, regulated association of Src SH2 domain with FAK and paxillin during cell attachment and detachment on fibronectin was disrupted in Shp-2 mutant cells. This report defines a unique role of the Shp-2 tyrosine phosphatase in cell motility, which might guide the design of a new strategy for pharmaceutical interference of tumor metastasis.
c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. Recent studies have demonstrated that the Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. We found here that transient expression of Crk induces JNK activation, and this activation was dependent on both the SH2- and SH3-domains of Crk. Expression of p130(Cas) (Cas), a major binding protein for the Crk SH2-domain, also induced JNK activation, which was blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk was effectively blocked by a dominant-negative form of Rac, suggesting for a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, was efficiently blocked by dominant-negative mutants of Crk. A dominant-negative form of Cas in turn blocked the integrin-, but not epidermal growth factor - nor v-Src-mediated JNK activation. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation.
In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (-/-) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130(CAS), a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (-/-) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (-/-) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130(CAS) was also observed. In (-/-) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow-associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.
The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.
Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.
Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.
Epithelia and mesenchyme interact during various physiologic and pathologic processes. Scatter factor is a mesenchyme-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. Recent studies suggest that scatter factor and its receptor (c-met) mediate mesenchyme/epithelia signalling and even interconversion. In this mini-review, we will discuss how scatter factor and c-met may mediate interactions between mesenchyme and epithelia during embryogenesis, organ repair, and neoplasia.
A distinctive property of Hepatocyte Growth Factor (HGF) is its ability to induce differentiation of tubular structures from epithelial and endothelial cells (branching tubulogenesis). The HGF receptor directly activates PI3 kinase, Ras and STAT signalling pathways and phosphorylates the adaptator GRB2 Associated Binder-1 (Gab1). Gab1 is also phosphorylated in response to Epidermal Growth Factor (EGF) but is unable to induce tubule formation. Comparison of 32P-peptide maps of Gab1 from EGF- versus HGF-treated cells, demonstrates that the same sites are phosphorylated in vivo. However, while both EGF and HGF induce rapid tyrosine phosphorylation of Gab1 with a peak at 15 min, the phosphorylation persists for over 1 h, only in response to HGF. Nine tyrosines are phosphorylated by both receptors. Three of them (Y307, Y373, Y407) bind phospholipase C-γ (PLC-γ). Interestingly, the overexpression of a Gab1 mutant unable to bind PLC-γ (Gab1 Y307/373/407F) did not alter HGF-stimulated cell scattering, only partially reduced the growth stimulation but completely abolished HGF-mediated tubulogenesis. It is concluded that sustained recruitment of PLCγ to Gab1 plays an important role in branching tubulogenesis.
p130Cas (Cas), the protein encoded by the Crkas
gene (also known as Cas), is an adaptor molecule with a unique
structure that contains a Src homology (SH)-3 domain followed by multiple
YXXP motifs and a proline-rich region1. Cas was originally cloned
as a highly tyrosine-phosphorylated protein in cells transformed by v-Src
(refs 2,3) or v-Crk
(ref. 4) and has subsequently been implicated
in a variety of biological processes including cell adhesion5,
cell migration6, growth factor stimulation7,
8,
9,
cytokine receptor engagement10,
11 and bacterial infection12,
13. To determine its role in vivo, we generated mice lacking
Cas. Cas-deficient embryos died in utero showing marked systemic congestion
and growth retardation. Histologically, the heart was poorly developed and
blood vessels were prominently dilated. Electron microscopic analysis of the
heart revealed disorganization of myofibrils and disruption of Z-disks. In
addition, actin stress fiber formation was severely impaired in Cas-deficient
primary fibroblasts. Moreover, expression of activated Src in Cas-deficient
primary fibroblasts did not induce a fully transformed phenotype, possibly
owing to insufficient accumulation of actin cytoskeleton in podosomes. These
findings have defined Cas function in cardiovascular development, actin filament
assembly and Src-induced transformation.
Mitogen-activated protein kinases (MAPKs) are components of a three kinase regulatory cascade. There are multiple members of each component family of kinases in the MAPK module. Specificity of regulation is achieved by organization of MAPK modules, in part, by use of scaffolding and anchoring proteins. Scaffold proteins bring together specific kinases for selective activation, sequestration and localization of signaling complexes. The recent elucidation of scaffolding mechanisms for MAPK pathways has begun to solve the puzzle of how specificity in signaling can be achieved for each MAPK pathway in different cell types and in response to different stimuli. As new MAPK members are defined, determining their organization in kinase modules will be critical in understanding their select role in cellular regulation.
Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.
The met oncogene activated in vitro by treatment of a human osteogenic sarcoma (HOS) cell line with N-methyl-N'-nitronitrosoguanidine (MNNG) is related to the tyrosine kinase gene family. Probes from the met oncogene locus recognize two distinct transcripts of 9.0 kb and 10.0 kb which are independently expressed in a cell-type-specific fashion. While the met proto-oncogene locus expresses the 9.0 kb RNA and maps to human chromosome 7q21-31, the locus expressing the 10.0 kb RNA, (tpr; translocated promoter region) maps to human chromosome 1. Both MNNG-HOS cells and met NIH 3T3 transformants express a novel 5.0 kb RNA which represents a hybrid transcript with 5' sequences derived from tpr and 3' sequences from the met proto-oncogene. Treating HOS cells in vitro with MNNG, a known clastogenic carcinogen, resulted in fusion of two chromosomally disparate loci, met and tpr, generating the active met oncogene.
Signaling by tyrosine kinase receptors is mediated by selective interactions between individual Src homology 2 (SH2) domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. Here, we report the existence in the hepatocyte growth factor/scatter factor (HGF/SF) receptor of a multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV. Phosphorylation of this site mediates intermediate- to high-affinity interactions with multiple SH2-containing signal transducers, including phosphatidylinositol 3-kinase, phospholipase C gamma, pp60c-src, and the GRB-2-Sos complex. Mutation of the two tyrosines results in loss of biological function, as shown by abrogation of the transforming activity in the oncogenic counterpart of the receptor. The same bidentate motif is conserved in the evolutionarily related receptors Sea and Ron, suggesting that in all members of the HGF/SF receptor family, signal transduction is channeled through a multifunctional binding site.
We show in this report that two v-src substrate proteins, p130Cas and cortactin, become tyrosine-phosphorylated during integrin-mediated cell adhesion to extracellular matrix substrata and upon cell attachment onto immobilized anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine or through antibodies against major histocompatibility complex. It also does not take place when adhesion-mediated reorganization of the actin cytoskeleton is inhibited with cytochalasin D. Tyrosine phosphorylation of p130Cas and cortactin coincides with tyrosine phosphorylation of focal adhesion kinase during integrin-mediated cell adhesion but is independent of cell adhesion in v-src-transformed cells. The tyrosine-phosphorylated sites in p130Cas and cortactin may serve as binding sites for proteins containing Src homology 2 domains, as is the case with two other integrin-regulated docking proteins, focal adhesion kinase and paxillin. Thus, these results suggest that ligand binding of integrins regulates the tyrosine phosphorylation state of multiple docking proteins. These proteins may mediate anchorage dependence of growth; their misregulation in v-src-transformed and other tumorigenic cells may be responsible for the anchorage independence of such cells.
The Rho subfamily of GTPases is involved in control of cell morphology in mammals and yeast. The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA did not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation was also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation.
The receptor for hepatocyte growth factor/scatter factor (HGF/SF) was originally identified as an oncogene, Tpr-Met, which consists of the cytoplasmic tyrosine kinase domain of the HGF/SF receptor (Met) fused down-stream of sequences encoded by the tpr gene. As a consequence of this rearrangement the Tpr-Met fusion oncoprotein is localized to the cytoplasm and is a constitutively activated kinase. To identify signalling pathways important for Tpr-Met-mediated cell transformation we have generated tyrosine to phenylalanine mutants of Tpr-Met that are compromised in their ability to transform Fischer rat 3T3 (Fr3T3) cells in culture. We show that a single tyrosine residue in the carboxy terminus of Tpr-Met (residue 489) is essential for efficient transformation of Fr3T3 cells by this oncoprotein. Mutation of tyrosine 489 to phenylalanine does not affect the exogenous kinase activity of the Tpr-Met oncoprotein toward casein, but it impairs the ability of the mutant protein to bind to and activate phosphatidylinositol 3 kinase in vivo and completely abolishes the in vivo association with the Grb2 adaptor protein as well as the association and/or phosphorylation of an unknown protein of 110 kDa. These data are consistent with a single tyrosine residue in the Tpr-Met oncoprotein being essential for the activation of several signalling pathways which lead to the transformation of Fr3T3 fibroblasts.
THE cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphory-lation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro
. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK asso-ciation and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduc-tion pathway.
Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement
that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor
kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic
activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif
within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene.
By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated
by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this
mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement
is discussed.
The Met receptor tyrosine kinase is the receptor for hepatocyte growth factor/scatter factor. HGF/SF is a multifunctional cytokine that can stimulate proliferation, motility, and morphogenesis in epithelial and endothelial cells. Oncogenic activation of the Met receptor occurs through a genomic rearrangement that generates a hybrid protein in which tpr sequences are directly fused amino terminal to the met receptor kinase domain. The resultant Tpr-Met hybrid protein possesses tyrosine kinase activity, is constitutively phosphorylated on tyrosine residues in vivo, and transforms fibroblasts in culture. We have identified two tyrosine residues within the catalytic domain of the Tpr-Met oncoprotein (Y365, Y366) and Met receptor (Y1234, Y1235) that are phosphorylated and essential for both the catalytic and biological activity of the oncoprotein and receptor. However, a detailed analysis of phosphorylation in these proteins has not been undertaken. In order to determine the sites of tyrosine phosphorylation in the Tpr-Met oncoprotein, in vitro mutagenesis, phosphopeptide mapping, and dephosphorylation protection assays were performed. Here we identify that a single tyrosine (Y489) in the carboxy terminus of the Tpr-Met oncoprotein is highly phosphorylated and is essential for biological activity. In contrast, additional tyrosines (Y482, Y498) located in the carboxy terminus are not phosphorylated at detectable levels and are not essential for the biological activity of the oncoprotein.
The specification of the R7 photoreceptor cell in the developing eye of Drosophila is dependent upon activation of the Sevenless (SEV) receptor tyrosine kinase. By screening for mutations that suppress signaling via a constitutively activated SEV protein, we have identified a novel gene, daughter of sevenless (dos). DOS is required not only for signal transduction via SEV but also in other receptor tyrosine kinase signaling pathways throughout development. The presence of an amino-terminally located pleckstrin homology domain and many potential tyrosine phosphorylation sites suggests that DOS functions as an adaptor protein able to interact with multiple signaling molecules. Our genetic analysis demonstrates that DOS functions upstream of Ras1 and defines a signaling pathway that is independent of direct binding of the DRK SH2/SH3 adaptor protein to the SEV receptor tyrosine kinase.
Cellular interactions with the extracellular matrix proteins play important roles in a variety of biological processes. Recent studies suggest that integrin-mediated cell-matrix interaction can transduce biochemical signals across the plasma membrane to regulate cellular functions such as proliferation, differentiation and migration. These studies have implicated a critical role of focal adhesion kinase (FAK) in integrin-mediated signal transduction pathways. We report here that overexpression of FAK in CHO cells increased their migration on fibronectin. A mutation of the major autophosphorylation site Y397 in FAK abolished its ability to stimulate cell migration, while phosphorylation of Y397 in a kinase-defective FAK by endogenous FAK led to increased migration. We also find that the wild-type and the kinase-defective FAK were associated with Src and Fyn in CHO cells whereas the F397 mutant was not. These results directly demonstrate a functional role for FAK in integrin signaling leading to cell migration. They also provide evidence for the functional significance of FAK/Src complex formation in vivo.
v-Crk, identified as an oncogene product of the CT10 retrovirus, became the first example of an adaptor protein. It consists mostly of the Src homology 2 (SH2) and Src homology 3 (SH3) domains. Two of the three major proteins bound to Crk SH2 have been identified as paxillin and p130Cas. Both paxillin and p130Cas are phosphorylated upon stimulation by integrin, suggesting that Crk transduces signals from integrin. The cloning of the complementary DNA of two major proteins bound to Crk SH3 was recently completed. Both cDNAs encoded novel proteins: C3G, a guanine nucleotide exchange protein for Rap1, and DOCK180, an SH3-containing protein of unknown function. The SH3 domain of Crk also binds to Sos, Abl, and Eps15. The variety of the proteins bound to Crk SH3 implies that Crk provides a set of effector proteins that are triggered together. Alternatively, other domains of the SH3-binding proteins enable Crk to specifically activate each of the SH3-binding proteins according to the particular form of stimulation.
Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase implicated in controlling cellular responses to the engagement of cell-surface integrins, including cell spreading and migration, survival and proliferation. Aberrant FAK signaling may contribute to the process of cell transformation by certain oncoproteins, including v-Src. Progress toward elucidating the events leading to FAK activation following integrin-mediated cell adhesion, as well as events downstream of FAK, has come through the identification of FAK phosphorylation sites and interacting proteins. A signaling partnership is formed between FAK and Src-family kinases, leading to tyrosine phosphorylation of FAK and associated 'docking' proteins Cas and paxillin. Subsequent recruitment of proteins containing Src homology 2 domains, including Grb2 and c-Crk, to the complex is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-MAP kinase pathway.
Crk, which belongs to the adaptor family of proteins composed of Src homology 2 (SH2) and SH3 domains, has a putative role in signaling. However, the downstream events of Crk signaling remain unclear. In this study, we found that Jun kinase (JNK) is moderately activated by v-Crk in both NIH 3T3 cells and chicken embryo fibroblasts. Transient expression of v-Crk, c-Crk-I, or c-Crk-II activated JNK1 in human embryo kidney cells, 293T. Coexpression of a guanine nucleotide exchange protein C3G, which specifically binds to Crk's SH3 domain, further enhanced the JNK activity as well as growth rate and anchorage-independent growth of v-Crk NIH 3T3 cells. Furthermore, overexpression of a dominant-negative form of C3G lacking the guanine nucleotide exchange domain abolished both the JNK activity and the colony forming potential of v-Crk NIH 3T3 cells. The requirement for JNK activation in v-Crk induced transformation was demonstrated by the suppression of colony forming activity of v-Crk NIH 3T3 cells when a dominant-negative form of JNK kinase, Sek1/MKK4 is expressed in these cells. These data strongly suggest the existence of a novel signaling cascade involving an adaptor protein v-Crk, which transmits signals through C3G toward JNK activation.
The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.
The c-cbl protooncogene was first identified as the cellular homologue of a viral oncogene v-cbl that induces pre-B lymphomas and myeloid leukemias in mice. Until recently, the biochemical basis for Cbl's transforming potential and its physiological role remained unclear. However, a convergence of biochemical studies in mammalian cells and genetic studies in C. elegans and Drosophila has now identified Cbl as a negative regulator of tyrosine kinase signaling. The N-terminal transforming region of Cbl (Cbl-N) and an adjacent RING finger domain are the elements most conserved during evolution. The Cbl-N region has now been shown to contain a novel phosphotyrosine-binding (PTB) domain that directly interacts with autophosphorylated tyrosine kinases via a D(N/D)XpY motif. A critical role of the PTB domain in Cbl function is demonstrated by the localization of a loss-of-function mutation in C. elegans Cbl homologue SLI-1 within this region. The corresponding mutation in human Cbl inactivates the PTB domain function and abrogates Cbl-mediated regulation of tyrosine kinase function. Recent studies have also identified a novel signaling pathway initiated by the interaction of mammalian Cbl proteins with the SH2 domains of Crk adaptor molecules, which results in Cbl's linkage with C3G, a guanine nucleotide exchange protein for Rap1 family of small G-proteins. Presently, Rap1 is thought to antagonize Ras function, although Rap1-specific targets have emerged recently. Thus, recent advances have firmly placed the little known protooncoprotein Cbl on the center stage of tyrosine kinase-mediated signal transduction.
The integrin family of transmembrane receptors have long been recognized for their structural roles in linking extracellular matrix proteins with the cellular actin cytoskeleton to regulate cell shape, cell migration and tissue architecture. Lately, it has become clear that integrin receptors can initiate intracellular signals that synergize with those from growth-factor receptor protein-tyrosine kinases in modulating cell growth. This review describes our current knowledge of integrin-stimulated tyrosine phosphorylation events and downstream signalling pathways, with emphasis on the recent progress made in understanding the molecular pathways linking the integrin receptors with mitogen-activated protein (MAP) kinase cascades.
Several components in cytokine signaling remain unidentified. We report the cloning and initial characterization of one such component, p97, a widely expressed scaffolding protein distantly related to Drosophila DOS and mammalian Gab1. Upon cytokine, growth factor, or antigen receptor stimulation, p97 becomes tyrosyl phosphorylated and associates with several SH2 domain-containing proteins, including SHP2. Expression of p97 mutants unable to bind SHP2 blocks cytokine-induced c-fos promoter activation, inhibiting Elk1-mediated and STAT5-mediated transactivation. Surprisingly, such mutants do not inhibit MAPK activation. Our results identify p97 as an important regulator of receptor signaling that controls a novel pathway to immediate-early gene activation and suggest multiple functions for SHP2 in cytokine receptor signaling.
Clustering of integrins into focal adhesions and focal complexes is regulated by the actin cytoskeleton. In turn, actin dynamics are governed by Rho family GTPases. Integrin-mediated adhesion activates these GTPases, triggering assembly of filopodia, lamellipodia and stress fibers. In the past few years, signaling pathways have begun to be identified that promote focal adhesion disassembly and integrin dispersal. Many of these pathways result in decreased myosin-mediated cell contractility.
The adhesive extracellular matrix protein fibronectin and its integrin receptors play important roles at several stages of tumor development. Tumor cells are generally less adhesive than normal cells and deposit less extracellular matrix. The loosened matrix adhesion that results may contribute to the ability of tumor cells to leave their original position in the tissue. Normal cells, when detached, stop growing and undergo anoikis (apoptosis caused by loss of adhesion). Integrin-activated pathways mediated by focal adhesion kinase (FAK) and the adapter protein She seem to be particularly important in anchorage dependence; many oncoproteins are capable of shunting these pathways. Malignant cells circumvent anchorage dependence with the help of oncoproteins. Once invading tumor cells have gained access to the circulation, adhesion to the endothelia and other tissue components facilitates the establishment of tumor colonies at distant sites. Specific tissue affinities may underlie the tendency of some tumors to metastasize preferentially to certain tissues. Interfering with tumor cell attachment with integrin-binding peptides has been shown to be an effective antimetastatic strategy in animal experiments. Tumor angiogenesis is yet another aspect of malignancy wherein extracellular matrices and integrins are important. Angiogenic endothelial cells in tumor vessels depend on the alpha v family of integrins for survival. Inhibiting angiogenesis with compounds that block the activity of alpha v integrins, and targeting drugs into tumors through these integrins, show promise as new anticancer strategies.
Signaling pathways mediated by adhesive molecules are tightly associated with cytoskeletal organization and cell growth regulation. Focal adhesion kinase (FAK) plays a prominent role in the adhesion signaling pathway through its tyrosine kinase activity and protein-protein interaction with other signaling molecules, including src, paxillin, and p130CAS, and other proteins. We explored the roles of these signaling molecules in the transformation of B104-1-1 cells, an NIH/3T3-derived cell line transformed by activated rat p185neu. The cytoskeletal organization of the p185neu-transformed cells was disrupted, and their morphology was dramatically altered. FAK, paxillin, and p130CAS appeared to be tyrosine phosphorylated in both NIH/3T3 and B104-1-1. However, the phosphorylation levels of paxillin and p130CAS were lower in B104-1-1 cells than in NIH/3T3 cells. Surprisingly, the association between FAK and paxillin was enhanced in B104-1-1 cells, suggesting reorganization of protein-protein interaction modulated by protein phosphorylation. Our results showed that even though cellular transformation by src and neu has similar consequences, such as focal adhesion disassembly and increased metastasis potential, the molecular events underlying the signaling pathways can be dramatically different.
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130
Cas
, were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKβ/RAFTK/CadTK.
FAK localizes to sites of transmembrane integrin receptor clustering and facilitates intracellular signaling events. FAK-null (FAK-) fibroblasts exhibit a rounded morphology, defects in cell migration, and an elevated number of cell-substratum contact sites. Here we show that stable re-expression of epitope-tagged FAK reversed the morphological defects of the FAK- cells through the dynamic regulation of actin structures and focal contact sites in fibronectin (FN) stimulated cells. FAK re-expressing fibroblasts (clones DA2 and DP3) exhibit a characteristic fibrillar shape and display indistinguishable FN receptor-stimulated migration properties compared to normal fibroblasts. Expression of various FAK mutants in the FAK- cells showed that FAK kinase activity, the Tyr-397/SH2 domain binding site, and the first proline-rich SH3 binding region in the FAK C-terminal domain were individually needed to promote full FAK-mediated FAK- cell migration to FN whereas direct paxillin binding to FAK was not required. Expression of the FAK Phe-397 mutant did not promote FAK- cell migration and overexpression of p50(csk) in DA2 cells inhibited migration to FN suggesting that Src-family PTKs play important roles in FAK-mediated motility events. Expression of the FAK C-terminal domain, FRNK, promoted FAK dephosphorylation at Tyr-397 and potently blocked FAK-mediated cell migration. This dominant-negative effect of FRNK was reversed by a point mutation (Leu-1034 to Ser) which prevented FRNK localization to focal contact sites. Our results show that FAK functions as a key regulator of fibronectin receptor stimulated cell migration events through the recruitment of both SH2 and SH3 domain-containing signaling proteins to sites of integrin receptor clustering.
Part of the cellular response to toxins, physical stresses and inflammatory cytokines occurs by signalling via the stress-activated protein kinase (SAPK) and p38 reactivating kinase pathways. This results in modification of cellular gene expression. These stress-responsive kinase pathways are structurally similar, but functionally distinct, from the archetypal mitogen-activated protein kinases (MAPKs or ERKs). The ERK pathway is a hierarchical cascade originating at the cell membrane with receptors for mitogens or growth factors, which recruit, via adapter proteins and exchange factors, the small guanosine triphosphatase (GTPase) Ras (see fig. 1). Ras activates raf, a serine threonine kinase, which activates MEK (MAPK/ERK kinase). MEK, in turn, phosphorylates and activates ERK1 and ERK2, which translocate to the nucleus and transactivate transcription factors, changing gene expression to promote growth, differentiation or mitosis. By transducing signals through a cascade of kinases, several options for control are introduced for amplifying and/or modifying the output signal. The SAPK and p38 pathways are also hierarchically arranged, but less is known about the upstream components and the downstream effects of stimulation of these pathways. Among the processes modulated by stress-responsive pathways are apoptosis, transformation, development, immune activation, inflammation and adaptation to environmental changes. This review outlines the upstream componentry of these pathways that interact with a variety of agonists to modify the activity of SAPK and p38, and explores the downstream functions of this activation.