A novel substrate for insulin-sensitive serine/threonine kinase in intact cells.
We have studied insulin-stimulated threonine phosphorylation of cellular proteins by immunoblotting and immunoprecipitation using antiphosphothreonine antibody (anti-P-Thr). A 50-kilodalton protein (p50) was found to be greatly phosphorylated on threonine residues upon insulin stimulation in intact rat hepatoma cells (Fao) and Chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR). Insulin induced threonine phosphorylation of this protein in a dose-dependent manner, with an ED50 of 3-6 x 10(-9) M. The 50-kilodalton phosphoprotein (pp50) was detectable 20 min after exposure of the cells to insulin, and phosphorylation reached a maximum after 90 min. Immunoprecipitation of pp50 with anti-P-Thr required extraction of the cellular proteins with sodium dodecyl sulfate and dithiothreitol, and subcellular fractionation of the cells revealed that pp50 is present in the membrane fraction, implying that pp50 is a protein integrated into the membrane component in the cells. Tryptic phosphopeptide mapping of the pp50 was distinct from that of the insulin receptor beta-subunit. Phosphoamino acid analysis of the pp50 demonstrated that insulin increased phosphorylation, mainly of threonine and moderately of serine, whereas pp50 did not contain phosphotyrosine. Cycloheximide, a protein synthesis inhibitor, did not affect the insulin-induced appearance of pp50 in the cells. pp50 was not detectable in A431 cells and KB cells stimulated by epidermal growth factor. These data suggest that p50 is a novel endogenous substrate for insulin-sensitive serine/threonine kinase in intact cells.
Kono
S
S
Department of Medicine, Kyoto University Faculty of Medicine, Sakyo-ku, Japan.
Kuzuya
H
H
Yamada
K
K
Yoshimasa
Y
Y
Okamoto
M
M
Inoue
G
G
Hayashi
T
T
Suga
J
J
Imura
H
H
Nakao
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D016466:CHO Cells; D002294:Carcinoma, Squamous Cell / Q000737:chemistry* / Q000201:enzymology / Q000473:pathology*; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000032:analysis; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000737:chemistry* / Q000201:enzymology / Q000473:pathology*; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism*; D008970:Molecular Weight; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011972:Receptor, Insulin / Q000032:analysis; D013912:Threonine / Q000276:immunology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007328:Insulin; D008565:Membrane Proteins; D013912:Threonine; D004815:Epidermal Growth Factor; D003513:Cycloheximide; D066246:ErbB Receptors; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases