Immunological detection of phosphotyrosine-containing proteins in rat livers after insulin injection.
Tyrosine-phosphorylated proteins in Triton X-100-solubilized fractions of rat livers were examined by immunoblotting with anti-phosphotyrosine antibodies. After 2 min of insulin injection via the portal vein into livers, three major bands of 170,000, 140,000, and 95,000 Mr were stimulated. Because the incubation of nitrocellulose membrane with anti-phosphotyrosine antibodies in the presence of 40 mM phosphotyrosine completely abolished these bands, the anti-phosphotyrosine antibodies appear to recognize the phosphotyrosine residues of these proteins. Insulin injection (2-2000 micrograms) very quickly stimulated the tyrosine phosphorylation of these proteins in a dose-dependent fashion. In contrast, insulinlike growth factor I or epidermal growth factor injection had little effect in stimulating the tyrosine phosphorylation of these proteins. Because anti-insulin-receptor antibodies immunoprecipitated a tyrosine-phosphorylated 95,000-Mr protein, this protein must be the beta-subunit of the insulin receptor; i.e., the beta-subunit of the insulin receptor and two other proteins were phosphorylated at tyrosine residues in vivo by insulin injection. These data suggest that the tyrosine phosphorylation and tyrosine kinase activity of the insulin receptor may have important roles in in vivo insulin action.
Institute of Diabetes Care and Research, Asahi Life Foundation, Chiyoda-Ku, Japan.
K
K
Tobe
O
O
Koshio
Y
Y
Tashiro-Hashimoto
F
F
Takaku
Y
Y
Akanuma
M
M
Kasuga
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000008:administration & dosage; D015151:Immunoblotting; D007328:Insulin / Q000008:administration & dosage* / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000008:administration & dosage; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011169:Portal Vein; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D007328:Insulin; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin