Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C- terminal myosin-binding domain. The kinase domain is the best characterized ; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction. H owever, it is not known whether the myosin binding domain modifies the acti n-myosin interaction. We designed MLCK cDNA to overexpress the Asp-777-;Glu -972 sequence in Escherichia coli, The purified Asp-777-Glu-972 fragment, a lthough devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (V-max = 736 +/- 0.44-fold, K-m = 1.06 +/- 0.20 mu M, n = 4), When the N-terminal 39 residues of the fragme nt were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity. We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (V-max = 3.03 +/- 0.22 fold, K-m = 6.93 +/- 1.61 mu M, n = 3), When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser- 815 peptides, the stimulatory activity was found in the latter. We confirme d that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides, Therefore, we suggest that phosphorylati on is not obligatory for smooth-muscle myosin not to be active.