Studies of calcineurin-calmodulin interaction: probing the role of arginine residues using peptidylarginine deiminase.
We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis shows that only 3 of 34 arginines in calcineurin were deiminated; citrulline seems to be localized only in the calcineurin A (CaN A) subunit. Upon incubation with deiminase, the Mn2+/calmodulin-stimulated phosphatase activity decreases to 20-40% of the original activity within 1 h. However, the reduction in enzyme activity is fully protected by addition of calmodulin to the deimination reaction, and only 1.5 mol citrulline/mol calcineurin is found in this case. Removal of the calmodulin binding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested native calcineurin and also results in the loss of all citrulline residues. The calmodulin activation curve of the deiminated enzyme is significantly shifted; the calculated apparent Kact using native calmodulin is 15-fold higher than that of native calcineurin while the apparent Kact using a fluorescent derivative of calmodulin, dansyl-calmodulin, is 10-fold higher. However, the Vm of deiminated calcineurin is similar to that of native if highly elevated levels of calmodulin are used to activate the modified calcineurin. To determine directly if the binding of calmodulin to calcineurin is affected upon deimination, fluorescence titrations using dansyl-calmodulin were performed. The Kd of deiminated calcineurin determined from these titrations is 10-fold higher than that of unmodified calcineurin, indicating that calmodulin binding is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calmodulin binding site of the CaN A subunit.
Imparl
J M
JM
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Senshu
T
T
Graves
D J
DJ
D000818:Animals; D001120:Arginine; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019703:Calcineurin; D002147:Calmodulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002956:Citrulline / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D013997:Time Factors
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases