Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src.
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.
Sato
K
K
Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Nada, Japan. ksato@inherit.biosig.kobe-u.ac.jp
Yamamoto
H
H
Otsuki
T
T
Aoto
M
M
Tokmakov
A A
AA
Hayashi
F
F
Fukami
Y
Y
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002417:Cattle; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D019269:Phosphatidylinositol 4,5-Diphosphate; D011506:Proteins; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016392:Proto-Oncogene Proteins pp60(c-src)