Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Abstract

Ca 2؉ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by ''mixed-peptide'' Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca 2؉ sensitizing kinase cascade.

Novel ZIP kinase isoform lacks leucine zipper

“…Recently, it was found that ZIP kinase phosphorylates the regulatory light chain (MLC 20 ) of smooth muscle myosin [5,6]. This finding raised a possibility that ZIP kinase plays a role in cell contractile activity since the function of smooth muscle and non-muscle myosin is regulated by the phosphorylation of its MLC 20 [7][8][9].…”

“…On the other hand, it is known that MLC 20 is also phosphorylated at Thr18 in addition to Ser19 [10,11], which stabilizes the formation of myosin filaments and the myosin phosphorylated at both sites tends to form large thick filaments [12,13].…”

“…While the physiological significance of Thr18 phosphorylation is obscure, Thr18 phosphorylation occurs in vivo and it has been reported that di-phosphorylation of MLC 20 at Thr 18 and Ser19 occurs in smooth muscles induced by external stimuli [14,15] and in nonmuscle cells in conjunction with the cellular shape change and exocytosis [16][17][18]. However, physiological relevance and regulation of di-phosphorylation of MLC 20 has been puzzling because MLCK, which has been thought to be the predominant kinase responsible for MLC 20 phosphorylation, phosphorylates the Thr18 at a much slower rate than that for Ser19 of MLC 20 [10].…”

“…However, physiological relevance and regulation of di-phosphorylation of MLC 20 has been puzzling because MLCK, which has been thought to be the predominant kinase responsible for MLC 20 phosphorylation, phosphorylates the Thr18 at a much slower rate than that for Ser19 of MLC 20 [10]. Interestingly, it was found that ZIP kinase phosphorylates Ser19 and Thr18 of MLC 20 at the same rate constant, suggesting that this kinase is responsible for the production of di-phosphorylated myosin in smooth muscle and non-muscle cells [6].…”

“…However, physiological relevance and regulation of di-phosphorylation of MLC 20 has been puzzling because MLCK, which has been thought to be the predominant kinase responsible for MLC 20 phosphorylation, phosphorylates the Thr18 at a much slower rate than that for Ser19 of MLC 20 [10]. Interestingly, it was found that ZIP kinase phosphorylates Ser19 and Thr18 of MLC 20 at the same rate constant, suggesting that this kinase is responsible for the production of di-phosphorylated myosin in smooth muscle and non-muscle cells [6]. Actually, it was found that ZIP kinase is primarily responsible for MLC 20 phosphorylation in motile mammalian cells [18], although a recent study suggested that integrin linked kinase is rather responsible for MLC 20 phosphorylation in the Triton-X100 permeabilized rat caudal artery fiber using a protein kinase inhibitor, AV25, as a probe [19].…”