Predominance of tyrosine phosphorylation of insulin receptors during the initial response of intact cells to insulin.
Anti-phosphotyrosine antibody and anti-insulin receptor antibody were used to study insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor in [32P]orthophosphate-labeled Fao hepatoma cells. Without insulin, the receptor contained both phosphoserine and phosphothreonine and could be immunoprecipitated with anti-receptor antibody but not with the anti-phosphotyrosine antibody. After incubation of these cells with insulin, both antibodies immunoprecipitated the phosphorylated receptor. The beta-subunit of the receptor precipitated with anti-phosphotyrosine antibody from cells stimulated with insulin (100 nM) for 1 min contained predominantly phosphotyrosine, whereas, after 10 min with insulin, the amounts of phosphotyrosine and phosphoserine were nearly equal. These results suggest that insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the beta-subunit. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibody followed by precipitation of the remaining proteins with anti-receptor antibody suggests that insulin receptors which contain phosphoserine in the basal state are tyrosine phosphorylated more slowly than the dephosphorylated receptors or not at all after the addition of insulin. The beta-subunit of the insulin receptor was the major phosphorylated protein precipitated by the anti-phosphotyrosine antibody from insulin-stimulated Fao cells. These results confirm our notion that insulin initially stimulated tyrosine autophosphorylation and subsequently serine phosphorylation of the insulin receptor in intact cells and suggests that this sequence of reactions occurs faster on receptors that are dephosphorylated before the incubation with insulin.
Pang
D T
DT
Sharma
B R
BR
Shafer
J A
JA
White
M F
MF
Kahn
C R
CR
D000818:Animals; D000906:Antibodies; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin