Phosphorylation of the regulatory light chain of myosin catalyzed by myosin light-chain kinase (MLCK) is the key reaction in the regulation of actin–myosin interaction in smooth muscle. It is shown that this reaction is of an ordered type, whereby kinase first binds ATP and then the light chain, and following phosphate transfer, the phosphorylated light chain is released before ADP. The MLCK also phosphorylates itself, and this intramolecular autophosphorylation is Ca2+ and calmodulin (CaM) dependent. It has, however, no pronounced effect on the kinase activity or on its affinity for Ca2+ and CaM. With the aim of understanding the cooperativity of MLCK activation, the activity of the kinase was systematically measured as a function of different ligands involved. In these measurements the isolated light chain and intact filamentous myosin, as well as native actomyosin, were used as substrates. The activation of the kinase by Ca2+ was positively cooperative but only at relatively low CaM levels. The activation by CaM (at saturating Ca2+ levels) was also cooperative, even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between CaM and the kinase. This cooperativity was shown to result from time-dependent changes in the MLCK that take place during incubation with Ca2+ and CaM before addition of ATP in phosphorylation assays. As a result, activity of the kinase as a function of its concentration at constant CaM was biphasic: there was optimum activity at a ratio of 1:1 CaM to kinase and almost complete inhibition of the activity at a three- to six-fold excess of the kinase over CaM. The modification required 10–15 min preincubation (with Ca2+ and CaM) and could be explained by a dimerization of the kinase, demonstrated by the use of a zero-length cross-linker.Key words: kinetic mechanism, autophosphorylation, calcium and calmodulin activation, cooperativity, myosin light chain kinase, smooth muscle.
An antibody to phosphorylated myosin light chain: Application to myosin light chain kinase activity assay without radioisotope.
