Jm. Imparl et al., STUDIES OF CALCINEURIN-CALMODULIN INTERACTION - PROBING THE ROLE OF ARGININE RESIDUES USING PEPTIDYLARGININE DEIMINASE, Archives of biochemistry and biophysics, 318(2), 1995, pp. 370-377
We have used an enzyme, peptidylarginine deiminase, to convert certain
arginyl groups in calcineurin to citrulline. Amino acid analysis show
s that only 3 of 34 arginines in calcineurin were deiminated; citrulli
ne seems to be localized only in the calcineurin A (CaN A) subunit, Up
on incubation with deiminase, the Mn2+/calmodulin-stimulated phosphata
se activity decreases to 20-40% of the original activity within 1 h. H
owever, the reduction in enzyme activity is fully protected by additio
n of calmodulin to the deimination reaction, and only 1.5 mol citrulli
ne/mol calcineurin is found in this case. Removal of the calmodulin bi
nding domain of the deiminated CaN A by limited proteolysis results in
the reactivation of the phosphatase to the same level as digested nat
ive calcineurin and also results in the loss of all citrulline residue
s. The calmodulin activation curve of the deiminated enzyme is signifi
cantly shifted; the calculated apparent K-act using native calmodulin
is 15-fold higher than that of native calcineurin while the apparent K
-act using a fluorescent derivative of calmdulin, dansyl-calmodulin, i
s 10-fold higher. However, the V-m of deiminated calcineurin is simila
r to that of native if highly elevated levels of calmodulin are used t
o activate the modified calcineurin. To determine directly if the bind
ing of calmodulin to calcineurin is affected upon deimination, fluores
cence titrations using dansyl-calmodulin were performed. The K-d of de
iminated calcineurin determined from these titrations is 10-fold highe
r than that of unmodified calcineurin, indicating that calmodulin bind
ing is indeed affected. These data indicate that at least one arginine
is important for calmodulin binding and is likely located at the calm
odulin binding site of the CaN A subunit. (C) 1995 Academic Press, Inc
.