Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myos in light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosph orylation. Inhibition of SMPP-1M is modulated through phosphorylation of th e myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membr ane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar- associated. This raises a conundrum about the mechanism of interaction betw een these enzymes. We present ZIP-like kinase, identified by "mixed-peptide " Edman sequencing after affinity purification, as the previously unidentif ied SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosph orylation of ZIP-like kinase was associated with an increase in kinase acti vity during carbachol stimulation, suggesting that the enzyme may be a term inal member of a Ca2+ sensitizing kinase cascade.