Purification and characterization of an autophosphorylation-activated protein serine threonine kinase that phosphorylates and inactivates protein phosphatase 2A.
Protein phosphatase 2A2 is inactivated by phosphorylation following incubation with purified preparations of an autophosphorylation-activated protein kinase (Hong Guo and Zahi Damuni (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504). This protein kinase was purified about 250,000-fold from extracts of bovine kidney to apparent homogeneity. The purified preparations exhibited a single polypeptide of apparent M(r) approximately 36,000. Up to 1 mol of phosphoryl groups was incorporated per mol of the purified kinase following incubation with ATP. This autophosphorylation reaction (t1/2 approximately 0.5-1 min) was accompanied by a approximately 10-fold activation of the kinase. Autophosphorylation and activation were reversed by protein phosphatase 2A2 or the catalytic subunit of protein phosphatase 1. Phosphoamino acid analysis indicated that the kinase underwent autophosphorylation on threonines. The rate of autophosphorylation was independent of the concentration of the enzyme and a slope of 0.97 (gamma = 0.998) was obtained by van't Hoff's plot indicating that autoposphorylation was intramolecular. Relative to myelin basic protein, the enzyme exhibited about 8, 62, 130, 33, 5, and < 0.1% activity with histones H1, H2A, H2B, H3, and H4 and with glycogen synthase alpha, respectively. Heparin inhibited the activity of the enzyme half-maximally at about 20 micrograms/ml. The results indicate that this autophosphorylation-activated kinase is a new protein kinase.
Guo
H
H
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Reddy
S A
SA
Damuni
Z
Z
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2