v-Crk Modulation of Growth Factor-induced PC12 Cell Differentiation Involves the Src Homology 2 Domain of v-Crk and Sustained Activation of the Ras/Mitogen-activated Protein Kinase Pathway

Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H.(1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation. Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H.(1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation. INTRODUCTIONAmong the molecular determinants that generate and maintain diverse cellular morphologies and functions, peptide growth factors, via their cognate receptors, are known to elicit a wide range of biological responses that include proliferation and survival as well as differentiation(1Cantley L. Auger K. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). Collective efforts to understand the mechanisms of growth factor actions have revealed that many growth factor receptors possess intrinsic tyrosine kinase activity (2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar, 3Fantl W.J. Johnson D. Williams L.T. Annu. Rev. Biochem. 1993; 62: 453-481Crossref PubMed Scopus (927) Google Scholar) and become phosphorylated in the intracellular domain following ligand binding. A general paradigm for the activation and signaling of receptor-type tyrosine kinases involves the recruitment of cytoplasmic molecules that contain Src homology (SH) 1The abbreviations used are: SHSrc homologyEGFepidermal growth factorNGFnerve growth factorMAPmitogen-activated protein. 2 domains (for review, see (4Pazin M.J. Williams L.T. Trends Biochem. Sci. 1992; 17: 374Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 5Pawson T. Gish G. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 6Margolis B. Cell Growth 3: 73-80PubMed Google Scholar, 7Mayer B.J. Baltimore D. Trends Cell Biol. 1993; 3: 8-13Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 8Birge R.B. Hanafusa H. Science. 1993; 262: PubMed Scopus Google A of the signal as and with receptor tyrosine L. Auger K. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). cellular responses to growth factor are and in the signaling mechanisms that growth factor a are pheochromocytoma PC12 is a model for the of neuronal L. A. Sci. S. A. PubMed Scopus Google Scholar, L. A. Cell. 3: Google Scholar). PC12 cells to the NGF (for review, see and by many of the of as well as the of to cells and their Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). that the and of MAP kinase activation may with the of in PC12 1991; Full Text PDF PubMed Scopus Google Scholar, S. H. C. Biochem. J. 1992; PubMed Scopus Google Scholar, S. H. Cell. Full Text PDF PubMed Scopus Google Scholar). is by that show that of signaling molecules that MAP kinase as the NGF receptor D. B. D. L. Science. 1991; PubMed Scopus Google Schlessinger J. Biol. Full Text Full Text PDF PubMed Scopus Google A. D. T. T. PubMed Scopus Google growth factor Johnson Mol. Cell. Biol. 1992; 3: Scopus Google J. G. G. R. A. S. J. Schlessinger J. T. 1992; PubMed Scopus Google S. H. Cell. Full Text PDF PubMed Scopus Google or H. G. S. Sci. S. A. 1993; PubMed Scopus Google Scholar) in or neurite in PC12 cells. the that of the EGF receptor in PC12 cells results in neurite S. K. H. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that tyrosine kinase growth factor receptors are and that the strength of a tyrosine kinase signal may as a molecular for the responses of PC12 cells toward EGF and or is a protein that is from the of with the SH2 and the domains of the adaptor protein and growth activity in B. Hanafusa H. PubMed Scopus Google Scholar, B.J. Hanafusa H. Science. 1990; PubMed Scopus Google Scholar). However, stably expressed in PC12 v-Crk neurite and expression of neuronal following EGF R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). To the of neurite by we the responses of v-Crk or v-Crk mutants with to their ability to the EGF receptor, MAP and neurite outgrowth. results the of the SH2 domain of v-Crk in EGF receptor from the EGF receptor to cause neurite outgrowth. However, sustained activation of the MAP kinase pathway to a in cells v-Crk or the v-Crk SH2 mutants, that sustained MAP kinase activation, by itself, may be not sufficient to cause neurite in the v-CrkPC12 NGF and EGF from for and used was from and and enhanced from was from from Mol. Cell. Biol. PubMed Scopus Google and the have D. J. PubMed Scopus Google Scholar). include and from for SOS and and to from and to EGF receptor from of SH2 PC12 Cell SH2 mutants and by and B. Hanafusa H. J. 1990; 64: PubMed Google Scholar). was to and was to These the of the is in known SH2 protein tyrosine and is within the B. Hanafusa H. J. 1990; 64: PubMed Google Scholar). the is in SH2 domains and is the of the A the of the SH2 was from on of the or mutants to the in of and for protein expression by with PC12 cells and v-CrkPC12 and as R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). To the of neurite cells in in the or of growth two in as Ras Expression the Ras expression we a with and the of the D. Mol. Cell. Biol. 1991; PubMed Scopus Google Scholar) by and of Ras of and of was with of and used for was used as a to in J. Mol. Google Scholar, PubMed Scopus Google Scholar). in for of was for an was and with for to the cells in and of cells was by the cells in as J. Mol. Google Scholar, PubMed Scopus Google and was as R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). a and of protein by with by Full Text PDF PubMed Scopus Google which the to on a activity was by of the by of and from from the of to the the protein of used to the of was that of J. S. 1991; Scholar). or v-CrkPC12 cells on and in for to with NGF or EGF for to with and of guanine of was for in and to with the as Ras or a of to was the following which the on as with within the SH2 of have that expression of the in PC12 cells and which is for PC12 to a R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). To the of the v-Crk SH2 domain in neurite outgrowth, we have stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells and These have by to with the of the G. D. J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). v-Crk mutants have to be in expressed as B. Hanafusa H. J. 1990; 64: PubMed Google Scholar). a the comparable in the SH2 domain binding to EGF receptor and T. J. Biol. 1992; Full Text PDF PubMed Google PC12 stably the SH2 two the v-Crk(R273N) and the v-Crk(H294R) for contain of protein comparable with in the v-Crk PC12 v-CrkPC12 that we have R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). of expression of the protein that of by responses neurite to growth in PC12 cells and PC12 cells or v-Crk 2 and the of growth factors, the cells the PC12 and PC12 cells following NGF with v-CrkPC12 cells an of NGF-induced neurite R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). However, the of neurite following NGF is in cells v-Crk(R273N) or as with the PC12 that the v-Crk act as dominant negative for the NGF signaling pathway 2 and and of the v-CrkPC12 and PC12 neurite two the of the v-Crk(R273N)PC12 cells have that a from the However, prolonged NGF induces neurite in v-Crk(R273N)PC12 and that sufficient cells have the to neurite EGF of v-Crk(R273N)PC12 or cells to 2 not a the v-CrkPC12 cells are to in the of factor of v-Crk(R273N) and v-Crk(H294R) mutants results not the SH2 domain of v-Crk is to an in PC12 neurite in PC12 cells v-Crk or v-Crk(R273N) or PC12 cells with NGF or EGF for to two in as neurite PC12 cells; in A the of of and v-Crk(R273N) cells following with PC12 cells and and PC12 cells v-Crk(R273N) and or v-Crk protein and in the or of NGF for is are in tyrosine kinase signaling in to EGF or PC12 cells are in not binding to cellular PC12 or or v-Crk(R273N) cells in the of EGF or NGF for as or cells and with to and with or of the tyrosine-phosphorylated are on the and the of molecular are on the of SOS is by an v-Crk(R273N) and v-Crk(H294R) are not tyrosine phosphorylated following NGF or EGF of cellular was by and with in of tyrosine phosphorylated v-Crk is on the and v-Crk SH2 the of v-Crk with the EGF investigate v-Crk SH2 mutants not the as v-Crk protein in EGF-dependent neurite outgrowth, we the ability of the v-Crk protein to cellular known to with v-Crk in show that v-Crk mutants to and tyrosine-phosphorylated and in PC12 cells and in EGF stimulation, v-Crk stably to the tyrosine-phosphorylated EGF was in the v-Crk(R273N) and in both v-Crk(R273N) and v-Crk(H294R) to become tyrosine-phosphorylated following EGF or NGF mutants are not for a tyrosine kinase. To point mutants within the SH2 domain of v-Crk the binding activity of the v-Crk we the binding of v-Crk to the the Ras guanine nucleotide exchange which to to the domain of in a R. M.J. G. J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, B. Hanafusa H. J. PubMed Scopus Google Scholar). A be by in both v-Crk and v-Crk(R273N) cells. the of EGF stimulation, SOS was as from to ligand stimulation, a was tyrosine kinase activity is with v-Crk or with and to an in kinase the of EGF stimulation, a protein was the tyrosine-phosphorylated in v-Crk with tyrosine of a protein. However, and not in the or v-Crk(R273N) kinase and in from the of tyrosine was to the for the of a in cells not v-Crk(R273N)PC12 cells and the tyrosine-phosphorylated receptor was not in from cells not was a tyrosine-phosphorylated to To that the EGF receptor, we the EGF receptor from of cells in which v-Crk was by the with in the v-CrkPC12 cells not v-Crk(R273N)PC12 cells and with the EGF is to that an EGF receptor was from the of v-Crk(R273N)PC12 cells with receptor the that within the EGF receptor kinase was the cells v-Crk(R273N) mutants not as v-Crk with to neurite kinase activity is with v-Crk in PC12 cells. or v-Crk(R273N) as in that the complexes in kinase and with of and for and the was and for 2 in to with an of or v-Crk(R273N) cells as in that the in A was with an receptor and with of EGF on Ras in v-CrkPC12 PC12 cellular differentiation Ras activation A. D. T. T. PubMed Scopus Google Scholar, S. PubMed Scopus Google and v-Crk expression in PC12 cells growth neurite outgrowth, we to be with a in the kinetics of the Ras PC12 Ras is by the of Ras in the is by and to within of NGF results in sustained activation of Ras to the the of for However, of the v-CrkPC12 cells with NGF or EGF induces a sustained of that of the was in the These results that v-Crk Ras activation that kinetics following NGF is to that the of to growth factor are in the and v-Crk PC12 with of the Ras in the induces sustained activation following EGF or NGF in PC12 cells. PC12 cells of and with for in cells with NGF or EGF for to was with and the and data are as the to the and are the of and v-Crk for a to v-Crk can in the of growth factor receptor activation, was expressed in or PC12 cells by with the and the of for with oncogenic is to for and D. Mol. Cell. Biol. 1991; PubMed Scopus Google expression of oncogenic Ras can neurite in PC12 A. D. T. T. PubMed Scopus Google Scholar). in in expression of to neurite in PC12 in v-Crk PC12 cells growth factor of the cells with the that was not in v-Crk PC12 cells results that v-Crk is to growth factor of v-Crk and to for growth factor receptor tyrosine PC12 cellular differentiation. However, expression of in or v-Crk(R273N)PC12 cells neurite with comparable of the cells neurite that of the actions of v-Crk on growth factor signaling of Ras activation expression of the Ras protein can the dominant negative of the v-Crk SH2 on NGF-induced neurite in or PC12 cells stably v-Crk or with or with of the with of as a the cells in with and with to in a EGF and of MAP in v-CrkPC12 and v-Crk(R273N) that of EGF and NGF on PC12 differentiation are by in the and of MAP kinase 1991; Full Text PDF PubMed Scopus Google Scholar, S. H. C. Biochem. J. 1992; PubMed Scopus Google Scholar). to the of v-Crk on growth differentiation, we the of tyrosine of MAP kinases in growth v-CrkPC12 and v-Crk(R273N)PC12 cells with S. K. H. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Schlessinger J. Biol. Full Text Full Text PDF PubMed Scopus Google tyrosine of MAP kinase in PC12 cells was EGF that NGF stimulation, that tyrosine was in the NGF-treated cells in the v-CrkPC12 MAP kinase was sustained to PC12 cells EGF stimulation, and the of tyrosine in NGF-treated PC12 cells in and EGF- or NGF-treated v-Crk(R273N)PC12 cells a in the of MAP kinase to PC12 cells kinetics of MAP kinase tyrosine by v-Crk and in to EGF or NGF of PC12 or v-CrkPC12 cells with EGF or NGF for to 2 and of cellular protein and with a and the tyrosine in and v-Crk(R273N)PC12 cells. the of the and by the with and of tyrosine-phosphorylated and is by are of prolonged tyrosine of and MAP kinases a prolonged activation, the activity of MAP kinase was by an PC12 NGF results in a sustained activation of MAP kinase EGF PC12 cells EGF causes a MAP kinase activation that is of that in NGF-treated PC12 cells and NGF-treated v-CrkPC12 cells kinetics of MAP kinase to PC12 cells v-Crk(R273N)PC12 cells a sustained of MAP activity following EGF that was from that of the v-Crk cells and NGF of v-Crk(R273N)PC12 cells in a and prolonged MAP kinase activation with PC12 cells v-Crk's ability to growth neurite may with to the of tyrosine and activity of MAP kinase with PC12 the that the of MAP kinase activation, and the on neurite can be kinetics of MAP kinase activity by v-Crk and v-Crk(R273N) in to EGF or NGF of PC12 cells. of PC12 v-CrkPC12 or cells for to 2 with EGF and or NGF and of cellular with kinase for MAP kinase activity and protein as a activity was by of protein from the the of two on data that the of MAP kinase in PC12 cells of growth factor have that PC12 cells the to EGF by a neuronal that is of NGF-treated PC12 R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). Here we that v-Crk can stably to both the EGF receptor and the guanine nucleotide exchange protein to in a sustained activation of Ras and MAP kinase following EGF sustained MAP kinase activity may be to elicit EGF-dependent neurite in PC12 cells the that the SH2 mutants of v-Crk MAP kinase activity not cause neurite that sustained activation, by itself, may not be sufficient to switch the fate of PC12 cells from proliferation toward neuronal differentiation growth factor that v-Crk the signal from EGF receptor to Ras in to neurite is by we have of Ras or MAP kinase activation in PC12 cells in the of a ligand both Ras and MAP kinase prolonged following EGF to PC12 cells. in the SH2 domain of binding of v-Crk to the EGF receptor and to neurite outgrowth. and with not we neurite in the or PC12 that for a tyrosine kinase signal in the v-CrkPC12 cells. These data point to the of a signal and of an adaptor in the cellular to a signal as neurite that v-Crk can act in a or analogous manner to in an signal to Ras in PC12 cells. the SH2 domain of both and can tyrosine-phosphorylated EGF receptor, and the domains of both and can R. M.J. G. J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, B. Hanafusa H. J. PubMed Scopus Google Scholar, R. J. T. D. 1993; PubMed Scopus Google Scholar, S. Schlessinger J. D. 1993; PubMed Scopus Google Scholar, S. B. L. A. R. 1993; PubMed Scopus Google Scholar). the stably with an guanine nucleotide exchange protein as revealed in Ras S. T. S. S. K. T. T. K. Sci. S. A. PubMed Scopus Google Ras signaling pathway is for both NGF-induced differentiation and in PC12 A. D. T. T. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). Expression of a dominant negative in PC12 cells Mol. Cell. Biol. PubMed Scopus Google Scholar). of NGF-induced neurite in PC12 S. PubMed Scopus Google Scholar). However, as and consistent with results of 1991; Full Text PDF PubMed Scopus Google Scholar, K. T. K. S. S. Google the activation of Ras to the following EGF and NGF of PC12 cells are a activation by and to by was sustained for following NGF in Ras activity is to a in to be as a PC12 the of Ras and MAP kinase sustained that the following EGF or NGF and both growth neurite that v-Crk SH2 point mutants their ability to with the tyrosine phosphorylated EGF receptor their ability to the kinetics of growth MAP kinase activation that the domain may be for activation of the kinase pathway in is not by the that v-Crk(R273N) to by the that a on the SOS EGF a on SOS or to following to the in to receptor J. Biol. Full Text PDF PubMed Google Scholar). by A. D. Schlessinger J. Cell. Full Text PDF PubMed Scopus Google Scholar) that of to by is sufficient to Ras and MAP kinase. the of in v-Crk to v-Crk to the B. Hanafusa H. J. 1990; 64: PubMed Google Scholar). by v-Crk(R273N) may SOS or to the which can the activation of Ras and MAP kinase ligand in is that MAP kinase activation growth factor stimulation, and may a recruitment of signaling a to the of the However, results suggest that the and domains not be to sustained MAP kinase activation, binding of v-Crk to the EGF receptor is for neurite results of the v-Crk SH2 mutants the of the of MAP kinase is for in PC12 cells. to signaling molecules to MAP kinase activation can the for PC12 cells of the receptor, and of and Schlessinger J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. D. T. T. PubMed Scopus Google Scholar, J. G. G. R. A. S. J. Schlessinger J. T. 1992; PubMed Scopus Google Scholar, G. S. J. Cell Biol. 1991; PubMed Scopus Google Scholar, G. S. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, C. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). the that expression of of MAP kinase activation and neurite suggest that an activation for MAP kinase may be a point for PC12 S. H. Cell. Full Text PDF PubMed Scopus Google Scholar). S. K. H. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) have by the EGF receptor in PC12 that a activation of MAP kinase may the for the responses to EGF and results are consistent with the that sustained MAP kinase activity can be for v-Crk's ability to cause EGF to be that of the SH2 mutants are of MAP kinase. the that cells v-Crk(R273N) or mutants a dominant negative on NGF-induced neurite the that mutants may a signaling neurite or growth However, the that of cells mutants cause neurite that the of for neurite are not in expression of v-Crk in PC12 cells the kinetics of NGF-induced neurite R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google and results that may via the ability of v-Crk to the NGF-induced activation of the kinase we have that the domain can tyrosine-phosphorylated in of cells R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google we have to of v-Crk with or kinase activity from cellular for is may be to in binding of a protein However, v-Crk is an for in and tyrosine phosphorylated within following NGF in R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). is that to to the EGF receptor, not the K. T. K. S. S. Google Scholar, T. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, A. K. A. Schlessinger J. A. J. PubMed Scopus Google suggest that to the the pathway EGF receptor to T. W.J. B. J. Biol. Full Text PDF PubMed Google Scholar). and S. S. T. K. S. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar) have that the SH2 domain of the tyrosine phosphorylated and may suggest that v-Crk with receptor via with However, results suggest that the of v-Crk with the EGF and receptors, are not the in PC12 fate both growth differentiation is PC12 the EGF receptor with that of v-Crk to the EGF receptor, and suggest that v-Crk can act to the that SH2 mutants v-Crk's ability to of that pathways may be we have a between kinase activation and PC12 differentiation by within v-Crk and be to the of tyrosine kinase signaling pathways to kinase activation and in v-Crk growth factor neurite outgrowth. INTRODUCTIONAmong the molecular determinants that generate and maintain diverse cellular morphologies and functions, peptide growth factors, via their cognate receptors, are known to elicit a wide range of biological responses that include proliferation and survival as well as differentiation(1Cantley L. Auger K. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). Collective efforts to understand the mechanisms of growth factor actions have revealed that many growth factor receptors possess intrinsic tyrosine kinase activity (2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar, 3Fantl W.J. Johnson D. Williams L.T. Annu. Rev. Biochem. 1993; 62: 453-481Crossref PubMed Scopus (927) Google Scholar) and become phosphorylated in the intracellular domain following ligand binding. A general paradigm for the activation and signaling of receptor-type tyrosine kinases involves the recruitment of cytoplasmic molecules that contain Src homology (SH) 1The abbreviations used are: SHSrc homologyEGFepidermal growth factorNGFnerve growth factorMAPmitogen-activated protein. 2 domains (for review, see (4Pazin M.J. Williams L.T. Trends Biochem. Sci. 1992; 17: 374Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 5Pawson T. Gish G. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 6Margolis B. Cell Growth 3: 73-80PubMed Google Scholar, 7Mayer B.J. Baltimore D. Trends Cell Biol. 1993; 3: 8-13Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 8Birge R.B. Hanafusa H. Science. 1993; 262: PubMed Scopus Google A of the signal as and with receptor tyrosine L. Auger K. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). cellular responses to growth factor are and in the signaling mechanisms that growth factor a are pheochromocytoma PC12 is a model for the of neuronal L. A. Sci. S. A. PubMed Scopus Google Scholar, L. A. Cell. 3: Google Scholar). PC12 cells to the NGF (for review, see and by many of the of as well as the of to cells and their Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). that the and of MAP kinase activation may with the of in PC12 1991; Full Text PDF PubMed Scopus Google Scholar, S. H. C. Biochem. J. 1992; PubMed Scopus Google Scholar, S. H. Cell. Full Text PDF PubMed Scopus Google Scholar). is by that show that of signaling molecules that MAP kinase as the NGF receptor D. B. D. L. Science. 1991; PubMed Scopus Google Schlessinger J. Biol. Full Text Full Text PDF PubMed Scopus Google A. D. T. T. PubMed Scopus Google growth factor Johnson Mol. Cell. Biol. 1992; 3: Scopus Google J. G. G. R. A. S. J. Schlessinger J. T. 1992; PubMed Scopus Google S. H. Cell. Full Text PDF PubMed Scopus Google or H. G. S. Sci. S. A. 1993; PubMed Scopus Google Scholar) in or neurite in PC12 cells. the that of the EGF receptor in PC12 cells results in neurite S. K. H. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that tyrosine kinase growth factor receptors are and that the strength of a tyrosine kinase signal may as a molecular for the responses of PC12 cells toward EGF and or is a protein that is from the of with the SH2 and the domains of the adaptor protein and growth activity in B. Hanafusa H. PubMed Scopus Google Scholar, B.J. Hanafusa H. Science. 1990; PubMed Scopus Google Scholar). However, stably expressed in PC12 v-Crk neurite and expression of neuronal following EGF R.B. R. D. R. Hanafusa H. Mol. Cell. Biol. PubMed Scopus Google Scholar). To the of neurite by we the responses of v-Crk or v-Crk mutants with to their ability to the EGF receptor, MAP and neurite outgrowth. results the of the SH2 domain of v-Crk in EGF receptor from the EGF receptor to cause neurite outgrowth. However, sustained activation of the MAP kinase pathway to a in cells v-Crk or the v-Crk SH2 mutants, that sustained MAP kinase activation, by itself, may be not sufficient to cause neurite in the v-CrkPC12