Abstract
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.
Highlights
Among 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 [1]
Our results indicate the importance of the 8rc homology 2 (8H2) domain of'v-Crk in transducing epidermal growth factor (EGF) receptor signals from the activated EGF receptor to cause neurite outgrowth
Mutations within the SH2 Domain ofv-crk Negate o-Crh-dependent Neurite Outgrowth-Our previous studies have demonstrated that stable expression of the v-crk oncogene product in PC12 cells accelerated Nerve growth factor (NGF)-promoted neuritogenesis and caused EGF, which is mitogenic for native PC12 cells, to induce a differentiated phenotype [26]
Summary
Reagents-Mouse 2.5 S NGF and EGF were purchased from Bioproducts for Science, (Indianapolis, IN) and were used at 50 ng/ml. [y_3 2 PJATP (6000 Ci/mmol) was obtained from DuPont NEN (Boston, MA), and [3 2PJorthophosphate and enhanced chemiluminesence (ECL) were from Amersham Corp. Cell lysis for experiments involving immunoprecipitations were carried out in HNTG buffer (1% Triton X-lOO buffer containing 10% glycerol, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.5) containing phosphatase and protease inhibitors and incubated with 1 f.Lg of either anti-Gag (monoclonal antibody 3C2), or anti-EGF receptor polyclonal antibody for 3 h prior to incubation with rabbit anti-mouse passified Protein A or Protein A-Sepharose beads, respectively, for an additional 1 h. MAP Kinase Assay-Soluble cellular lysates prepared in RIPA buffer (pH 7.5, containing 10 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) plus phosphatase and protease inhibitors were adjusted to equal protein concentrations (approximately 1 mg/ml), precleared with Protein A-Sepharose, and incubated with combined anti-ERK1 and -ERK2 antisera (each used at 1:400 dilution) for 2 h at 4 "C.
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