Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Adaptor Proteins, Vesicular Transport*
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Amino Acid Substitution
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Animals
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Brain / metabolism
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Carcinoma, Squamous Cell
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Cattle
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Cell Line, Transformed
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ErbB Receptors / metabolism
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Glutathione Transferase
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Humans
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Mice
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Mutagenesis, Site-Directed
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Oncogene Protein pp60(v-src) / metabolism
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Phosphatidylinositol 4,5-Diphosphate / metabolism*
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Phosphatidylinositol 4,5-Diphosphate / pharmacology*
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Protein Kinases / metabolism
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Proteins / chemistry*
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Proteins / metabolism*
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Proto-Oncogene Proteins pp60(c-src) / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Shc Signaling Adaptor Proteins
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Src Homology 2 Domain-Containing, Transforming Protein 1
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Tumor Cells, Cultured
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Tyrosine*
Substances
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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Phosphatidylinositol 4,5-Diphosphate
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Proteins
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Recombinant Fusion Proteins
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SHC1 protein, human
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Shc Signaling Adaptor Proteins
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Shc1 protein, mouse
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Src Homology 2 Domain-Containing, Transforming Protein 1
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Tyrosine
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Glutathione Transferase
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Protein Kinases
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ErbB Receptors
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Oncogene Protein pp60(v-src)
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Proto-Oncogene Proteins pp60(c-src)