Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.     

Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors

B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.     

Guanine-nucleotide exchange protein C3G activates JNK1 by a ras-independent mechanism. JNK1 activation inhibited by kinase negative forms of MLK3 and DLK mixed lineage kinases

Recently we have reported that the adaptor protein Crk transmits signals to c-Jun kinase (JNK) through C3G, a guanine-nucleotide exchange protein for the Ras family of small G proteins. Transient expression of C3G in 293T cells induced JNK1 activation without a significant effect on extracellular signal-related kinase 1 (ERK1), whereas mSos1 activated equally both JNK1 and ERK1. Coexpression of the dominant negative form of Ras-N17 did not suppress C3G-induced JNK1 activation but reduced the activity of JNK1 induced by mSos1, suggesting that Ras is not required for JNK activation by C3G. Ras-independent activation of JNK was supported by the finding that C3G-induced JNK activation was not inhibited by the dominant negative forms of Rac or Pak, which are components of the signaling pathway from Ras leading to JNK activation. In contrast, C3G-induced JNK1 activation was strongly inhibited by coexpression of the kinase negative forms of the mixed lineage kinase (MLK) family of proteins, MLK3 and dual leucine zipper kinase (DLK). In addition, MLK3-induced JNK1 activation was found to be suppressed by the kinase negative form of DLK, which bound to MLK3. These results suggest that C3G activates JNK1 through a pathway involving the MLK family of proteins.     

C3G is tyrosine-phosphorylated after integrin-mediated cell adhesion in normal but not in Bcr/Abl expressing cells

The SH2-SH3 adaptor protein Crkl has been implicated in the signal transduction pathways of several membrane-bound receptors. Tyrosine phosphorylation of proteins associated with such signalling complexes can generate binding sites for the Crkl SH2-domain and can recruit proteins constitutively bound to Crkl via the Crkl SH3 domain into such complexes. In the current study we show that Crkl, but only a minor amount of the related Crk, form constitutive complexes in vivo with guanine nucleotide exchange factor C3G in 3T3 fibroblasts. Adhesion of both normal and transformed cells to fibronectin or other extracellular matrix proteins consistently induces the tyrosine-phosphorylation of C3G. Adhesion-induced tyrosine phosphorylation of C3G is dependent on an intact cytoskeleton and peaks at 5-10 min after attachment. In contrast, 3T3 cells stably transfected with Bcr/Abl P210 show a prominent reduction in the amount of C3G complexed to Crkl and do not exhibit tyrosine-phosphorylation of C3G upon spreading and attachment. These data establish that integrin-mediated cell adhesion results in Crkl-mediated tyrosine phosphorylation of C3G, a pathway which can be disrupted by Bcr/Abl.     

Cellular proteins binding to the first Src homology 3 (SH3) domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.     

Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.     

Transformation activity of Cdc42 requires a region unique to Rho-related proteins

The Rho subfamily GTP-binding protein Cdc42 mediates actin cytoskeletal rearrangements and cell cycle progression and is essential for Ras transformation. Expression of a Cdc42 mutant (Cdc42(F28L)) that undergoes spontaneous activation (guanine nucleotide exchange) results in transformation of NIH3T3 fibroblasts. In this report, we show that deletion of residues 120-139 from Cdc42(F28L), which comprise an insert region unique to Rho subfamily proteins but is missing in other GTP-binding proteins, yields a Cdc42 molecule that still undergoes spontaneous GTP-GDP exchange and stimulates both actin cytoskeletal changes and the activation of the cellular targets p21-activated kinase and the c-Jun kinase (JNK1). However, this Cdc42 mutant is unable to transform cells. These findings indicate that the Rho subfamily insert region is dispensable for many of the known signaling pathways initiated by activated Cdc42 but is essential for its regulation of cell growth.     

Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)

We have demonstrated previously that growth hormone (GH) activates focal adhesion kinase (FAK), and this activation results in the tyrosine phosphorylation of two FAK substrates, namely paxillin and tensin. We now show here in Chinese hamster ovary cells stably transfected with rat GH receptor cDNA that human (h)GH induces the formation of a large multiprotein signaling complex centered around another FAK-associated protein, p130(Cas) and the adaptor protein CrkII. hGH stimulates the tyrosine phosphorylation of both p130(Cas) and CrkII, their association, and the association of multiple other tyrosine-phosphorylated proteins to the complex. Both the c-Src and c-Fyn tyrosine kinases are tyrosine phosphorylated and activated by cellular hGH stimulation and form part of the multiprotein signaling complex as does tensin, paxillin, IRS-1, the p85 subunit of phosphatidylinositol 3-kinase, C3G, SHC, Grb-2, and Sos-1. c-Cbl and Nck are also tyrosine-phosphorylated by cellular stimulation with hGH and associate with the p130(Cas)-CrkII complex. c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is activated in response to hGH in accordance with the formation of the abovementioned signaling complex, and hGH stimulated JNK/SAPK activity is increased in CrkII overexpressing NIH3T3 cells compared with vector transfected NIH3T3 cells. The formation of such a large multiprotein signaling complex by GH, with the resultant activation of multiple downstream effector molecules, may be central to many of the pleiotropic effects of GH.     

Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation

Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.     

Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crk and CrkL leads to synergistic activation of c-Jun N-terminal kinase

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.     

Activation of Rap1, antagonist to ras, by Crk-C3G

Rap1 was identified as gene whose overexpression suppressed transformation by ras. Rap1 belongs to the Ras family. The amino acid sequences of Rap1 and Ras show 55% identity to each other. Due to this high sequence similarity, Rap1 binds to effector molecules of Ras, however, Rap1 does not activate them. Thus, Rap1 functions are antagonistic to Ras in the cells. C3G was identified as a Crk SH3-binding guanine nucleotide exchange factor. Biochemical and cell biological analyses revealed that C3G is a Rap1 activator. Since it has been considered that Crk transduces signals from tyrosine kinases, this finding suggests that the activity of Rap1 is also under the control of tyrosine kinases. Overexpression of C3G in ras-transformed cells caused the morphology of the cells to revert to that of normal cells. Moreover, a mutant cell line that was resistant to EGF-dependent transformation was isolated. In the cell line a mutation was found in crk gene that was the cause of the resistance. These findings suggest that Crk-C3G-Rap1 pathway may function as an anti-transformation machinery.     

Identification of Tyr-762 in the platelet-derived growth factor alpha-receptor as the binding site for Crk proteins

Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) alpha-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF alpha-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor. In contrast, the Y762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF alpha-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous position to Tyr-762 in the alpha-receptor, binds RasGAP. RasGAP is not bound to the alpha-receptor. Thus, this region in the kinase inserts of the two receptors may be important for the divergency in signaling from the two PDGF receptors.     

The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway

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.     

Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G

p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.     

Networks of interaction of p120cbl and p130cas with Crk and Grb2 adaptor proteins

P120cbl, the product of the c-cbl proto-oncogene, has previously been shown to become tyrosine phosphorylated following EGF stimulation of cells, and to bind constitutively to the SH3 domain of the adaptor protein Grb2. Here we show that another adaptor protein, Crk, binds through its SH2 domain to tyrosine phosphorylated p120cbl. In addition, Crk becomes phosphorylated on tyrosine and serine following EGF treatment of PC12 and other cell lines. In unstimulated cells, while Grb2 is not bound to any tyrosine phosphoprotein, Crk is bound via its SH2 domain to tyrosine phosphorylated p130cas, the Crk-associated v-Src substrate. Following EGF treatment, Crk dissociates from p130cas, possibly due to a higher affinity of Crk SH2 for p120cbl compared with p130cas. Interaction between Grb2 and p120cbl increases threefold following EGF treatment of cells; in vitro, this induction of Grb2 association with unphosphorylated p120cbl can be mimicked by the addition of tyrosine phosphorylated Shc, suggesting a transfer of information between the SH2 and SH3 domains of Grb2. These data indicate that adaptor proteins can exchange binding partners in response to stimuli, and that different adaptor proteins can bind to the same partners by different mechanisms.     

The tyrosine phosphatase PTP1C associates with Vav, Grb2, and mSos1 in hematopoietic cells

The association of the murine motheaten phenotype of severe hemopoietic dysregulation with loss of PTP1C tyrosine phosphatase activity indicates a critical role for this SH2 domain-containing phosphotyrosine phosphatase in the regulation of hemopoietic cell growth and differentiation. To explore the molecular basis for PTP1C effects on hematopoiesis, we have investigated the possibility that this enzyme interacts with the product of the Vav proto-oncogene, a putative guanine nucleotide exchange factor expressed exclusively in hemopoietic cells. Our data indicate that PTP1C physically associates with Vav in murine spleen cells and in EL4 T lymphoma and P815 mastocytoma cells, and that this interaction is increased following mitogenic stimulation and the induction of both PTP1C and Vav tyrosine phosphorylation. The results also reveal tyrosine phosphatase activity to be present in Vav immunoprecipitates from stimulated splenic and P815 cells and suggest that a major portion of total cellular PTP1C catalytic activity is associated with Vav. As Vav-associated tyrosine phosphatase activity was not detected in PTP1C-deficient motheaten splenic cells, it appears that PTP1C accounts for most, if not all, Vav-coprecipitable tyrosine phosphatase activity in normal cells. The data also demonstrate the capacity of the Vav SH2 domain alone to bind to PTP1C in activated P815 cells, but suggest a role for the two Vav SH3 domains in enhancing this interaction. In addition, the results reveal PTP1C association with two other molecules implicated in Ras activation, the Grb2 adaptor protein and mSos1, a GTP/GDP exchanger for Ras. PTP1C therefore has the capacity to bind and potentially modulate various signaling effectors involved in activation of Ras or Ras-related proteins, and, accordingly, regulation of Ras activation represents a possible mechanism whereby PTP1C influences hemopoietic cellular responses.     

Activation of G1 progression, JNK mitogen-activated protein kinase, and actin filament assembly by the exchange factor FGD1

Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.     

Crk protein binds to PDGF receptor and insulin receptor substrate-1 with different modulating effects on PDGF- and insulin-dependent signaling pathways

We have studied the involvement of murine c-Crk, an SH2/SH3 containing adaptor protein, in signaling pathways stimulated by different receptor tyrosine kinases. We show here that c-Crk is associated with components of insulin- and PDGF-dependent signaling pathways. Insulin treatment of murine myoblast cells induces the formation of stable complex of endogenous c-Crk with insulin receptor substrate-1 (IRS-1) mediated via the SH2 domain of Crk. The ligand dependent physical association of c-Crk with IRS-1 is direct. However IRS-1 is also co-precipitated with c-Crk from quiescent L6 cells. The association of IRS-1 with c-Crk in quiescent cells is probably not direct since Far Western blot analysis did not reveal the binding of neither SH2 domain nor amino-terminal SH3 domain of c-Crk to IRS-1 from unstimulated cells. We also show that PDGF treatment of murine myoblast cells induces association of c-Crk with the PDGF receptor and tyrosine phosphorylation of c-Crk. Overexpression of c-Crk enhanced insulin- but not PDGF-induced activation of MAP kinases when compared to parental cell lines. Thus, the formation of the direct IRS-1/Crk complex appears to be crucial for Crk-mediated insulin-induced activation of MAP kinase, whereas Crk is probably involved in other PDGF-induced responses. These data provide support to the hypothesis that insulin and PDGF employ different mechanisms for activation of MAP kinase cascade.