Sato K, Yamamoto H, Otsuki T, Aoto M, Tokmakov A A, Hayashi F, Fukami Y
Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Nada, Japan.
FEBS Lett. 1997 Jun 30;410(2-3):136-40. doi: 10.1016/s0014-5793(97)00539-5.
The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST-Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584-592], effect of PtdIns(4,5)P2 on the phosphorylation of GST-Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST-Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. Km for GST-Shc in the presence of 1 microM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST-Shc was inhibited by a GST-fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.
衔接蛋白Shc被制备成谷胱甘肽S-转移酶融合蛋白（GST-Shc），并用作c-Src的体外底物。由于已表明Shc的磷酸酪氨酸结合结构域可结合磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P2）[周等人（1995年），《自然》378卷，584 - 592页]，因此研究了PtdIns(4,5)P2对c-Src介导的GST-Shc磷酸化的影响。通过c-Src的自身磷酸化和对外源底物Cdc2肽的磷酸化判断，PtdIns(4,5)P2刺激了GST-Shc的磷酸化，而对c-Src活性没有任何影响。另一方面，磷脂酰丝氨酸、磷脂酸、磷脂酰肌醇和磷脂酰肌醇4-磷酸可刺激c-Src活性本身，但磷脂酰胆碱无此作用。在存在1 microM PtdIns(4,5)P2的情况下，GST-Shc的Km计算为90 nM。含有Shc磷酸酪氨酸结合结构域的GST融合蛋白可抑制GST-Shc的PtdIns(4,5)P2依赖性磷酸化。这些结果表明，PtdIns(4,5)P2可通过与Shc结合，作为c-Src介导的Shc磷酸化的调节剂。
