Erpel T, Alonso G, Roche S, Courtneidge S A
Differentiation Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69012 Heidelberg, Federal Republic of Germany.
J Biol Chem. 1996 Jul 12;271(28):16807-12. doi: 10.1074/jbc.271.28.16807.
The Src family of protein tyrosine kinases has been implicated in the response of cells to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). We recently described a microinjection approach that we used to demonstrate that kinase activity of Src family members is required for PDGF- and EGF-induced S-phase entry of fibroblasts. We have now used this approach to ask whether a functional SH3 domain of Src is required to transduce the mitogenic signal upon PDGF or EGF stimulation. Microinjection of plasmids encoding Src mutants lacking the SH3 domain (SrcDeltaSH3) or point-mutated within the ligand binding surface of the SH3 domain, but with intact kinase domains, inhibited the mitogenic effect of PDGF and EGF in fibroblasts. SrcDeltaSH3 could still associate with the PDGF receptor, suggesting that the inhibitory effect of the Src SH3 mutants was brought about by a failure of the PDGF receptor.SrcDeltaSH3 complex to relay the mitogenic signal further downstream. Chimeric molecules in which the Src SH3 domain was replaced with that of spectrin or Lck also blocked PDGF-induced DNA synthesis, whereas a chimera containing the Fyn SH3 domain did not. These data suggest that the Src or Fyn SH3 domain is required either for correct substrate selection or to recruit other proteins to the PDGF receptor.
蛋白酪氨酸激酶的Src家族与细胞对血小板衍生生长因子（PDGF）或表皮生长因子（EGF）的反应有关。我们最近描述了一种显微注射方法，用于证明Src家族成员的激酶活性是成纤维细胞在PDGF和EGF诱导下进入S期所必需的。我们现在使用这种方法来探究在PDGF或EGF刺激下，Src的功能性SH3结构域是否是转导有丝分裂信号所必需的。显微注射编码缺乏SH3结构域的Src突变体（SrcDeltaSH3）或在SH3结构域的配体结合表面发生点突变但激酶结构域完整的质粒，会抑制成纤维细胞中PDGF和EGF的促有丝分裂作用。SrcDeltaSH3仍然可以与PDGF受体结合，这表明Src SH3突变体的抑制作用是由于PDGF受体-SrcDeltaSH3复合物无法将有丝分裂信号进一步向下游传递所致。用血影蛋白或Lck的SH3结构域替换Src的SH3结构域的嵌合分子也会阻断PDGF诱导的DNA合成，而含有Fyn SH3结构域的嵌合体则不会。这些数据表明，Src或Fyn的SH3结构域对于正确选择底物或招募其他蛋白质至PDGF受体是必需的。
