Insulin-induced activation of phosphoinositide 3-kinase in Fao cells.
Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85 alpha). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4-6% of the total PI3-kinase, and their specific activity was 11-14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.
Hayashi
T
T
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Japan.
Okamoto
M
M
Yoshimasa
Y
Y
Inoue
G
G
Yamada
K
K
Kono
S
S
Shigemoto
M
M
Suga
J
J
Kuzuya
H
H
Nakao
K
K
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental; D008715:Methionine / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051381:Rats; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D007328:Insulin; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D008715:Methionine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)