Sato K, Gotoh N, Otsuki T, Kakumoto M, Aoto M, Tokmakov A A, Shibuya M, Fukami Y
Laboratory of Molecular Biology, Kobe University, Japan.
Biochem Biophys Res Commun. 1997 Nov 17;240(2):399-404. doi: 10.1006/bbrc.1997.7667.
In the previous study (Sato K.-I. et al. (1997) FEBS Lett. 410, 136-140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F). We found that Y317F Shc but not Y239/240F or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns(4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2.
在先前的研究中（佐藤K.-I.等人，(1997)《欧洲生物化学学会联合会快报》410, 136 - 140），我们表明c-Src对Shc蛋白的磷酸化作用依赖于磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P2）与Shc的PTB结构域的结合。在本研究中，我们证明，与c-Src不同，v-Src和表皮生长因子（EGF）受体能够以不依赖PtdIns(4,5)P2的方式且在不同的磷酸化位点对Shc进行磷酸化。为了确定Shc中的磷酸化位点，我们使用了酪氨酸残基（Y）317和/或239及240被苯丙氨酸残基（F）取代的突变型Shc蛋白。我们发现Y317F Shc可被c-Src磷酸化，而Y239/240F或Y239/240/317F Shc则不能。该反应依赖于PtdIns(4,5)P2，并被添加的Shc的PTB结构域所抑制。另一方面，v-Src和EGF受体能够以不依赖PtdIns(4,5)P2的方式使Y317F和Y239/240F Shc磷酸化，但不能使Y239/240/317F Shc磷酸化。这些结果突出了c-Src与v-Src或EGF受体之间的差异，并表明只有当Shc的PTB结构域与PtdIns(4,5)P2结合时，c-Src才能主要在Shc的Tyr239/240位点进行磷酸化。
