Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations.
The activation of insulin-stimulated protein-serine/threonine kinases has been investigated in CHO cell lines transfected with cDNAs encoding either wild-type or mutant human insulin receptors. (1) Insulin treatment of CHO cells over-expressing wild-type insulin receptors resulted in the rapid and substantial (5-10-fold) activation of cytosolic protein kinases which phosphorylated myelin basic protein, Kemptide and two peptide substrates based on sites phosphorylated on ribosomal protein S6 in vivo. (2) Further fractionation of cytosolic extracts by MonoQ chromatography revealed two peaks of insulin-stimulated myelin basic protein kinase activity which were highly related to the previously described mitogen-activated protein (MAP) kinases ERK1 and ERK2. In addition, at least two major peaks of S6 kinase activity were resolved, which exhibited properties similar to the 70 kDa and 90 kDa S6 kinases described by others; the predominant effect of insulin was on the activity of the 90 kDa enzyme and was in excess of 10-fold. (3) MonoQ fractionation of extracts from parental CHO cells, or cells expressing kinase-deficient receptors, showed all insulin-stimulated peaks of activity to be almost completely absent. (4) Further studies demonstrated that substitution of tyrosine residues 1162 and 1163 (or 1162 alone) with phenylalanine led to a substantial reduction in the ability of insulin to stimulate these protein kinase activities when assayed in cytosolic extracts. In contrast, deletion of 69 amino acids from the C-terminus of the insulin receptor beta-subunit caused a leftward shift in the insulin dose-response curve of the MAP kinase activity, but apparently not in that of the 90 kDa S6 kinase activity.
Dickens
M
M
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Chin
J E
JE
Roth
R A
RA
Ellis
L
L
Denton
R M
RM
Tavaré
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin; D011994:Recombinant Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D007328:Insulin; D011994:Recombinant Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases