PHOSPHORYLATION OF CALMODULIN IN THE FIRST CALCIUM-BINDING POCKET BY MYOSIN LIGHT-CHAIN KINASE

In smooth muscle and specific nonmuscle cells the phosphorylation of t he regulatory myosin light chains by myosin light chain kinase (MLCK) is an obligatory step in actin-induced activation of myosin ATPase and subsequent contractile events, We have previously demonstrated that C aM phosphorylated by casein kinase II fails to activate bovine platele t MLCK (Sacks ed al. (1992) Biochem. J. 283, 21-24), While myosin ligh t chains are perceived as the only known substrate for MLCK phosphoryl ation activity, we now show that MLCK phosphorylates CaM. This phospho rylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is sti mulated by saturating [Ca2+] (K-0.5 = 16 mu M). CaM phosphorylation wa s inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependen cy identical to that for inhibition of myosin light chain phosphorylat ion. Native and MLCK-phosphorylated CaM were indistinguishable in acti vating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylat e CaM in a Ca2+-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubst rate) domain and one at the catalytic site, CaM phosphorylation by MLC K occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first C a2+-binding pocket, In summary, CaM phosphorylation by MLCK differs fr om CaM phosphorylation catalyzed by other kinases (i.e., the insulin r eceptor or casein kinase II) in both basic peptide and Ca2+ requiremen ts as well as in the sites of phosphorylation, Further investigations of this model may provide insight into the mechanisms of MLCK activati on and substrate recognition. (C) 1996 Academic Press, Inc.