Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor.
In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s).
Asamoah
K A
KA
Department of Biochemistry, School of Biological Sciences, Bassett Crescent East, University of Southampton, U.K.
Atkinson
P G
PG
Carter
W G
WG
Sale
G J
GJ
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D038362:Glycogen Synthase Kinase 3; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine / Q000032:analysis; D010920:Placenta / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D007328:Insulin; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D004676:Myelin Basic Protein; D010455:Peptides; D010710:Phosphates; D010768:Phosphoserine; D019000:Phosphotyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D008274:Magnesium