Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain.
Kinetic analysis of autophosphorylation was done using a non-Michaelis-Menten kinetic model. This model describes autophosphorylation in terms of a fast reaction phase, a slow reaction phase, and a partition function for the two phases. Kinetic parameters determined by this new approach show that insulin stimulates autophosphorylation by promoting (1) a 10-fold increase in the rate constant for the fast phase of the reaction and (2) a 2-fold increase in the partition function favoring the fast phase. Insulin did not significantly affect the binding constant for ATP in this fast phase. Kinetic parameters obtained for the cytoplasmic kinase domain were similar to those obtained for the native insulin receptor in the absence of insulin. The insulin receptor has three subdomains encompassing its seven autophosphorylation sites. The juxtamembrane sites react primarily in the slow kinetic phase, favored by the absence of stimulation and low ATP concentrations. The carboxy-terminal and central autophosphorylation subdomains react primarily in the fast kinetic phase, favored by raising the ATP concentration and/or the presence of insulin. These observations demonstrate that: (1) both ATP and insulin regulate reaction in each autophosphorylation subdomain, (2) insulin stimulation occurs predominantly in the central and carboxy-terminal regions, and (3) autophosphorylation observed with the cytoplasmic kinase domain was similar to native insulin receptor in the absence of insulin. These findings are consistent with conclusions based on the kinetic analysis of autophosphorylation.
Kohanski
R A
RA
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D009036:Moths; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins
D007328:Insulin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011972:Receptor, Insulin