Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease.
A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.
Tallant
E A
EA
Department of Pharmacology, University of Alabama, Birmingham 35294.
Brumley
L M
LM
Wallace
R W
RW
D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000097:blood*; D002417:Cattle; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D011480:Protease Inhibitors; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002154:Calpain