Characterization of a 60-kilodalton substrate of the insulin receptor kinase.
A 60-kDa tyrosine-phosphorylated protein has been observed after insulin treatment of cells in immunoprecipitations of the GTPase-activating protein of Ras (called GAP) as well as the phosphatidylinositol 3-kinase. In the present studies, these two 60-kDa proteins have been shown to differ by limited proteolytic digestions as well as by immunoprecipitation with a monoclonal antibody. This monoclonal antibody was also utilized to show that the 60-kDa GAP-associated protein was rapidly phosphorylated in intact cells after insulin stimulation and to associate with GAP only after insulin treatment of the cells. In addition, the 60-kDa protein was found to be phosphorylated in vitro by the insulin receptor. Finally, the 60-kDa protein immunoprecipitated by this antibody was found not to react with a polyclonal antibody directed against a 62-kDa tyrosine-phosphorylated GAP-associated protein previously observed in src-transformed cells. These studies indicate that insulin stimulates the tyrosine phosphorylation of at least two distinct 60-kDa proteins, one that becomes associated with GAP and appears to be a direct substrate of the insulin receptor kinase and another that associates with the phosphatidylinositol 3-kinase.
Hosomi
Y
Y
Hyogo Institute of Clinical Research, Akashi, Japan.
Shii
K
K
Ogawa
W
W
Matsuba
H
H
Yoshida
M
M
Okada
Y
Y
Yokono
K
K
Kasuga
M
M
Baba
S
S
Roth
R A
RA
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D019000:Phosphotyrosine; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D007328:Insulin; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin