Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases.
Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.
Scimeca
J C
JC
INSERM U 145, Faculté de Médecine, Nice, France.
Ballotti
R
R
Filloux
C
C
Van Obberghen
E
E
D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013268:Stimulation, Chemical; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis; D014638:Vanadates / Q000494:pharmacology*
D007328:Insulin; D007527:Isoenzymes; D009842:Oligopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; C037642:kemptide; D004247:DNA; D011494:Protein Kinases; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases