Analysis of insulin-receptor phosphorylation sites in intact cells by two-dimensional phosphopeptide mapping.
Insulin stimulates the autophosphorylation of the partially purified insulin receptor initially on tyrosine residues 1146, 1150 and 1151. This is followed by increased autophosphorylation of tyrosine residues 1316, 1322 and two further residues, possibly tyrosine residues 953 and 960 or 972 [Tavaré & Denton (1988) Biochem. J. 252, 607-615]. In the present paper we have used two cell lines transfected with insulin-receptor cDNA (CHO.T and NIH 3T3 HIR3.5 cells) to assess which tyrosine residues are phosphorylated on the insulin receptor within intact cells. We show that: (1) insulin causes a rapid increase in phosphorylation of tyrosine residues 1146, 1150 and 1151 in both cell types; tyrosine residues 1316 and 1322 are also phosphorylated, but apparently to a lesser extent in NIH 3T3 HIR3.5 cells; (2) the sites that may correspond to tyrosine residues 953 and 960 or 972 appear to be very poorly phosphorylated in both intact cell types; (3) insulin also promotes a substantial and rapid increase in the phosphorylation of serine and threonine residues on insulin receptors on CHO.T cells; this results in the appearance of two phosphopeptides not evident in the maps of the solubilized receptor preparations autophosphorylated in vitro.
Tavaré
J M
JM
Department of Biochemistry, University of Bristol Medical School, U.K.
O'Brien
R M
RM
Siddle
K
K
Denton
R M
RM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002460:Cell Line; D011232:Chemical Precipitation; D005260:Female; D006801:Humans; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism*
D000596:Amino Acids; D010710:Phosphates; D010748:Phosphopeptides; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin