c-Src and phosphatidylinositol 3-kinase are involved in NGF-dependent tyrosine phosphorylation of Shc in PC12 cells.
The adaptor protein Shc exists in three isoforms; p46, p52, and p66, and is a key regulator of a variety of biological processes. Our previous studies have shown that the tyrosine kinase c-Src phosphorylates Shc in a phosphatidylinositol (PtdIns) 4,5-bisphosphate-dependent manner. Here we demonstrate that PtdIns 3,4,5-trisphosphate stimulates phosphorylation of Shc by c-Src. The phosphorylation is blocked by a glutathione S-transferase fusion protein containing Shc phosphotyrosine binding (PTB) domain or a phosphotyrosine-containing Shc PTB domain-binding peptide. In rat pheochromocytoma cell line PC12, nerve growth factor (NGF) stimulates tyrosine phosphorylation of both Triton-soluble and -insoluble Shc which was maximal at 2-5 min after NGF treatment. We find that pretreatment of PC12 cells with the PtdIns 3-kinase inhibitor wortmannin or LY294002 results in almost half inhibition of the NGF-dependent tyrosine phosphorylation of only Triton-insoluble Shc. Similar inhibitory effect is observed with tyrosine kinase inhibitors genistein and PP1. Upon NGF stimulation, c-Src also becomes tyrosine-phosphorylated and accumulates in the Triton-insoluble fraction. The c-Src events are insensitive to wortmannin but sensitive to genistein. These results suggest that coordinate action of PtdIns 3-kinase and/or PtdIns 3,4,5-trisphosphate and c-Src can function as positive regulator in tyrosine phosphorylation of Shc in vitro and in vivo.
Sato
K
K
Laboratory of Molecular Biology, Kobe University, Japan. kksato@kobe-u.ac.jp
Otsuki
T
T
Kimoto
M
M
Kakumoto
M
M
Tokmakov
A A
AA
Watanabe
Y
Y
Fukami
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000081247:CSK Tyrosine-Protein Kinase; D009414:Nerve Growth Factors; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D019061:src-Family Kinases; C528035:Shc1 protein, rat