Autophosphorylation and autoactivation of spleen protein tyrosine kinase.
Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration.
Kong
S K
SK
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Chohan
I S
IS
Wang
J H
JH
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*
D010748:Phosphopeptides; D000804:Angiotensin II; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D000469:Alkaline Phosphatase; D008274:Magnesium