Changes in insulin-receptor tyrosine, serine and threonine phosphorylation as a result of substitution of tyrosine-1162 with phenylalanine.
Previous studies, by ourselves and others, have shown that tyrosine residues 1158, 1162 and 1163 are very rapidly autophosphorylated on the human insulin receptor after insulin binding and that this is followed by the autophosphorylation of tyrosine residues 1328 and 1334. The autophosphorylation of these tyrosine residues, and their role in transmembrane signalling, were examined by using Chinese-hamster ovary cells transfected with either normal intact insulin receptors or receptors in which tyrosine residues 1162 or 1162/1163 were substituted with phenylalanine. These studies show the following. (1) Tyrosine-1158 could still be autophosphorylated when tyrosine-1162 and -1163 were substituted with phenylalanine. (2) Insulin-stimulated insulin-receptor tyrosine phosphorylation in intact cells was complete within 30 s and was accompanied, after a lag of 2-5 min, by a rise in serine and threonine phosphorylation the beta-subunit. (3) Replacement of tyrosine-1162 with phenylalanine blocked insulin-stimulated threonine phosphorylation of the insulin receptor in intact cells. (4) Insulin-stimulated serine phosphorylation of the beta-subunit was found in both intact cells and partially purified receptor preparations incubated with [gamma-32P]ATP and was still apparent after the replacement of tyrosine-1162 with phenylalanine. (5) Our data strongly suggest that insulin-stimulated insulin-receptor serine and threonine phosphorylations are initiated through two distinct pathways, with only the latter showing a strict dependence on autophosphorylation of tyrosine-1162.
Tavaré
J M
JM
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Dickens
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine; D013912:Threonine; D014162:Transfection; D014357:Trypsin; D014443:Tyrosine
D010748:Phosphopeptides; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin