CrkL mediates Ras-dependent activation of the Raf/ERK pathway through the guanine nucleotide exchange factor C3G in hematopoietic cells stimulated with erythropoietin or interleukin-3.
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CrkL is an SH2 and SH3 domain-containing adaptor protein implicated in pathogenesis of chronic myelogenous leukemia. Here, we demonstrate that overexpression of CrkL enhances the erythropoietin (Epo)- or interleukin (IL)-3-induced activation of Elk-1 and the c-fos gene promoter activity in 32D/EpoR-Wt cells. Moreover, the Epo-induced activation of ERK1 and ERK2 was augmented and prolonged in cells inducibly overexpressing CrkL. A moderate increase in Epo-induced activation of JNK was also observed in cells overexpressing CrkL. Overexpression of C3G enhanced the Elk-1 activation synergistically with CrkL, while a C3G mutant lacking the guanine nucleotide exchange domain showed an inhibitory effect. Studies using a dominant negative Ha-Ras mutant demonstrated that the Elk-1 and ERK2 activation enhanced by CrkL and C3G was dependent on Ras. Consistent with this, the Epo-induced activation of Ras was augmented in cells inducibly overexpressing CrkL. Most importantly, a CrkL mutant defective in the SH2 or N-terminal SH3 domain showed an inhibitory effect on the Epo-induced activation of ERK2. These data indicate that the CrkL-C3G complex plays a role in Epo- or IL-3-induced, Ras-dependent activation of the Raf/ERK pathway leading to the activation of Elk-1 and the c-fos gene transcription. (+info)
The adapter protein Crkl links Cbl to C3G after integrin ligation and enhances cell migration.
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Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through beta(1) integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1alpha, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 +/- 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1alpha was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl DeltaSH2, after integrin cross-linking. Full-length Crkl, but not CrklDeltaSH2, coprecipitated with a single major tyrosine phosphoprotein with an M(r) of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G. (+info)
IFN-gamma activates the C3G/Rap1 signaling pathway.
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IFN-gamma transduces signals by activating the IFN-gamma receptor-associated Jak-1 and Jak-2 kinases and by inducing tyrosine phosphorylation and activation of the Stat-1 transcriptional activator. We report that IFN-gamma activates a distinct signaling cascade involving the c-cbl protooncogene product, CrkL adapter, and small G protein Rap1. During treatment of NB-4 human cells with IFN-gamma, c-cbl protooncogene product is rapidly phosphorylated on tyrosine and provides a docking site for the src homology 2 domain of CrkL, which also undergoes IFN-gamma-dependent tyrosine phosphorylation. CrkL then regulates activation of the guanine exchange factor C3G, with which it interacts constitutively via its N terminus src homology 3 domain. This results in the IFN-gamma-dependent activation of Rap1, a protein known to exhibit tumor suppressor activity and mediate growth inhibitory responses. In a similar manner, Rap1 is also activated in response to treatment of cells with type I IFNs (IFN-alpha, IFN-beta), which also engage CrkL in their signaling pathways. On the other hand, IFN-gamma does not induce formation of nuclear CrkL-Stat5 DNA-binding complexes, which are induced by IFN-alpha and IFN-beta, indicating that pathways downstream of CrkL are differentially regulated by different IFN subtypes. Taken altogether, our data demonstrate that, in addition to activating the Stat pathway, IFN-gamma activates a distinct signaling cascade that may play an important role in the generation of its growth inhibitory effects on target cells. (+info)
Calmodulin-independent coordination of Ras and extracellular signal-regulated kinase activation by Ras-GRF2.
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Ras-GRF2 (GRF2) is a widely expressed, calcium-activated regulator of the small-type GTPases Ras and Rac. It is a multidomain protein composed of several recognizable sequence motifs in the following order (NH(2) to COOH): pleckstrin homology (PH), coiled-coil, ilimaquinone (IQ), Dbl homology (DH), PH, REM (Ras exchanger motif), PEST/destruction box, Cdc25. The DH and Cdc25 domains possess guanine nucleotide exchange factor (GEF) activity and interact with Rac and Ras, respectively. The REM-Cdc25 region was found to be sufficient for maximal activation of Ras in vitro and in vivo caused Ras and extracellular signal-regulated kinase (ERK) activation independent of calcium signals, suggesting that, at least when expressed ectopically, it contains all of the determinants required to access and activate Ras signaling. Additional mutational analysis of GRF2 indicated that the carboxyl PH domain imparts a modest inhibitory effect on Ras GEF activity and probably normally participates in intermolecular interactions. A variant of GRF2 missing the Cdc25 domain did not activate Ras and functions as an inhibitor of wild-type GRF2, presumably by competing for interactions with molecules other than calmodulin, Ras, and ligands of the PH domain. The binding of calmodulin was found to require several amino-terminal domains of GRF2 in addition to the IQ sequence, and no correlation between calmodulin binding by GRF2 and its ability to directly activate Ras and indirectly stimulate the mitogen-activated protein (MAP) kinase ERK in response to calcium was found. The precise role of the GRF2-calmodulin association, therefore, remains to be determined. A GRF2 mutant missing the IQ sequence was competent for Ras activation but failed to couple this to stimulation of the ERK pathway. This demonstrates that Ras-GTP formation is not sufficient for MAP kinase signaling. We conclude that in addition to directly activating Ras, GRF2, and likely other GEFs, promote the assembly of a protein network able to couple the GTPase with particular effectors. (+info)
Crk activation of JNK via C3G and R-Ras.
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v-crk is an oncogene identified originally in CT10 chicken tumor virus. C3G, a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, is postulated to transduce the oncogenic signal of v-Crk to c-Jun kinase (JNK). We have found that R-Ras, but not Rap1, mediates JNK activation by v-Crk in 293T and NIH 3T3 cells. Constitutively activated R-Ras, R-Ras(Val-38), but not Rap1(Val-12), activated JNK, as did the constitutively active H-Ras(Val-12) or Rac1(Val-12). v-Crk activation of JNK was inhibited by a dominant-negative mutant of R-Ras, R-Ras(Asn-43). JNK activation by R-Ras(Val-38) was inhibited by a dominant-negative mutant of mixed lineage kinase 3. Among six GEFs for Ras-family G proteins, mSos1, Ras-GRF, C3G, CalDAG-GEFI, Ras-GRP/CalDAG-GEFII, and Epac/cAMP-GEFI, GEFs for either H-Ras or R-Ras activated JNK and c-Jun-dependent transcription. CalDAG-GEFI and Epac/cAMP-GEFI, both of which are GEFs specific for Rap1, did not activate JNK or c-Jun-dependent transcription. These results demonstrate that R-Ras, but not Rap1, is the downstream effector of C3G to stimulate JNK. Finally, we found that expression of the dominant-negative R-Ras mutant induced flat reversion of NIH 3T3 cells transformed by v-Crk, suggesting that R-Ras-dependent JNK activation is critical for the transformation by v-Crk. (+info)
The association of CRKII with C3G can be regulated by integrins and defines a novel means to regulate the mitogen-activated protein kinases.
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In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1. (+info)
The Ras-mitogen-activated protein kinase pathway is critical for the activation of matrix metalloproteinase secretion and the invasiveness in v-crk-transformed 3Y1.
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To search for the intracellular signaling pathway critical for the secretion of matrix metalloproteinases (MMP), we studied the effects of dominant negative Ras (S17N Ras) and dominant negative MEK1 (MEK1AA) expression in v-crk-transformed 3Y1. Expression of either S17N Ras or MEK1AA dramatically suppressed the augmented secretion of MMP-2 and MMP-9 in v-crk-transfected 3Y1. Similarly, a Ras farnesyltransferase inhibitor, manumycin A, and a MEK1 inhibitor, U0126, suppressed MMP secretion in a dose-dependent manner, whereas a PI3 kinase inhibitor, wortmannin, could not. In addition, the suppression of MMP secretion by S17N Ras showed good correlation with the inhibition of in vitro invasiveness of the cells. In contrast, expression of dominant negative C3G did not suppress MMP secretion, although it substantially blocked the c-Jun N-terminal kinase activation. Taken together, the Ras-MEK1 pathway, but not the C3G-JNK pathway, seems to play a key role in the activation of MMP secretion and, hence, the invasiveness of v-crk-transformed cells. (+info)
Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion.
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The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion. (+info)