Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca²⁺. Pretreatment of cells with 1-[N-O-bis(5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca²⁺](i) but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca²⁺ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca²⁺ and potentiated myosin light chain phosphorylation. We propose that the Ca²⁺-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca²⁺ sensitivity of myosin light chain phosphorylation in smooth muscle.