Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.
Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.
Howard Hughes Medical Institute, Dallas, Texas.
J M
JM
Tavaré
B
B
Zhang
L
L
Ellis
R A
RA
Roth
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine; D014162:Transfection
D007328:Insulin; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D010748:Phosphopeptides; D013912:Threonine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin