Abstract
The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.
Highlights
The human PDGF receptor binding site for Src has been identified as Tyr(P)-579/Tyr(P)-581 located in the juxtamembrane region of the activated receptor dimer [9]
Volvement of the Src SH3 domain in PDGF and epidermal growth factor (EGF) kinase-inactive Src and Fyn mutants act as dominant negative receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling
The dominant negative effect of both these mutants on PDGF signaling was shown to be SH2-dependent. Since both tyrosines are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling
Summary
In Vitro Mutagenesis and Subcloning—A 1.8-kilobase mouse neuronal c-Src cDNA [10] was cloned into the BamHI site of M13mp. This deleted construct, equivalent to wild type mouse c-Src, was used as a template for further mutagenesis. The SH2-inactivating mutation, R177K, was made in the wt c-Src cDNA single-stranded template using the mutagenic oligonucleotide TCCTCGTGAAGGAGAGTGA. Wild type and mutant murine c-Src retrovirus expression constructs were used to generate 2 retrovirus-producing cell lines [16] by calcium-phosphate transfection [17] followed by G418 selection (Geneticin, Life Technologies, Inc., number 1811– 031). Six-cm dishes of SrcϪ cells were infected at 20 –30% confluence with the 10-ml 2 supernatants for 24 h followed by selection with 600 g/ml G418 in DMEM plus 10% FCS. The bound mAb 327 was detected using a biotinylated horse anti-mouse IgG (Vector Labs, number BA2000) at a 1:300 dilution of the 1.5 mg/ml stock or 5 g/ml final concentration followed by addition of fluorescein-labeled avidin (Vector Labs, A-2011) at 10 g/ml. A standard deviation was calculated for each set of results for a given c-Src cell line and is presented in Fig. 3 and Fig. 4
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