Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system.
The cytoplasmic domain of the human insulin receptor (HIR) from the baculovirus expression system (BIRK) is a soluble, constitutively activated protein-tyrosine kinase. In a cell-free system, BIRK is phosphorylated on serine and threonine residues by protein kinase C (PKC) purified from rat brain. Two-dimensional tryptic phosphopeptide mapping of PKC-phosphorylated BIRK identified one phosphothreonine and three phosphoserine peptides, which were also in tryptic digests of insulin receptors from insulin- or PMA-treated Chinese hamster ovary (CHO) cells transfected with the HIR. After Lys-C proteolysis of PKC-phosphorylated BIRK, radioactive phosphopeptides were purified on a C8 reverse-phase high pressure liquid chromatography column. Amino acid sequence analysis identified a phosphothreonine peptide corresponding to amino acids 1331-1340 of the HIR. This peptide contains only one threonine, amino acid 1336, which is identified as a site for PKC phosphorylation in BIRK. CHO cells transfected with the wild type (CHO/HIR) or a mutant human insulin receptor (CHO/HIRT1336N), in which threonine 1336 was substituted with asparagine, were 32P labeled and then stimulated with insulin or phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide analysis of the HIR revealed that phosphorylation of phosphothreonine peptide T, shown to be in PKC-phosphorylated BIRK, was increased by insulin or PMA. However, the corresponding peptide was not in the mutant receptor. Therefore, the present study directly identifies threonine 1336 in the HIR as a phosphorylation site for insulin and PMA. These data also show that BIRK can be used as a model for the study of the regulation of the insulin receptor kinase.
Ahn
J
J
Memorial Sloan-Kettering Cancer Center, New York, New York.
Donner
D B
DB
Rosen
O M
OM
D000818:Animals; D016367:Baculoviridae; D016466:CHO Cells; D002460:Cell Line; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D009036:Moths; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism
D007328:Insulin; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate