Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase.

In smooth muscle and specific nonmuscle cells the phosphorylation of the 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 CaM phosphorylated by casein kinase II fails to activate bovine platelet MLCK (Sacks et al. (1992) Biochem. J. 283, 21-24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is stimulated by saturating [Ca2+] (K0.5 = 16 microM). CaM phosphorylation was inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependency identical to that for inhibition of myosin light chain phosphorylation. Native and MLCK-phosphorylated CaM were indistinguishable in activating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylate CaM in a Ca(2+)-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubstrate) domain and one at the catalytic site. CaM phosphorylation by MLCK occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first Ca(2+)-binding pocket. In summary, CaM phosphorylation by MLCK differs from CaM phosphorylation catalyzed by other kinases (i.e., the insulin receptor or casein kinase II) in both basic peptide and Ca2+ requirements as well as in the sites of phosphorylation. Further investigations of this model may provide insight into the mechanisms of MLCK activation and substrate recognition.