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Abstract

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

MLCK‐independent phosphorylation of MLC20 and its regulation by MAP kinase pathway in human bladder smooth muscle cells

“…In addition, it is believed that Ca 2+ binds to calmodulin, and that Ca 2+ -calmodulin complex with catalytic subunit of MLCK activates enzyme to phosphorylate serine at position of 19 on MLC20 [Hartshorne, 1987]. However, Ca 2+ concentration does not always parallel the degree of MLC phosphorylation and contraction [Morgan and Morgan, 1984; Kanaide, 1999; Hirano et al, 2003]. When the extent of MLC phosphorylation or force of contraction induced by agonist stimulation is higher than that caused by a depolarization induced increase in the Ca 2+ concentration, this is called Ca 2+ -sensitization [Somlyo and Somlyo, 1994].…”

Characterization of the Zebrafish Homolog of Zipper Interacting Protein Kinase

“…MLC2 phosphorylation is required for numerous cellular processes including cellular morphogenesis, cell movement, smooth muscle contraction, cytokinesis, and tumor cell invasion [22,23]. MLC2 is phosphorylated, primarily at serine 19, but also threonine 18, by a number of protein kinases, including Myosin Light Chain Kinase (MLCK), Rho-Associated Protein Kinase (ROCK), MRCKs, Integrin-linked kinase (ILK), and Zipper-Interacting Protein Kinase (ZIPK) [24]. In smooth muscle MLC2 is most often regulated by calcium-dependent pathways that leads to MLCK activation or by the RhoA-ROCK pathway [25].…”

“…The dephosphorylation of both sites on MLC2 is mediated by a highly conserved Myosin Phosphatase (MP) complex consisting of a targeting subunit Mypt1 and the catalytic subunit Protein Phosphatase 1 Beta (PP1β) [26]. MLC2 kinases and phosphatases are in turn precisely regulated by reversible phosphorylation in response to a variety of signaling pathways [24]. Importantly, not only do ROCK and ZIPK phosphorylate MLC2, but they also phosphorylate and inhibit the myosin phosphatase [26].…”

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis

“…Recent evidence has documented that the Rho-induced activation of ROCK promoted actin stress fiber formation by directly phosphorylating MLC in some cells. 34 Based on this fact, we hypothesized that MLC2 phosphorylation might participate in the actin-mediated oocyte meiosis in conjunction with RhoA. Our recent work demonstrated that both RhoA and ROCK participated in oocyte cytokinesis and spindle migration by mediating actin organization during oocyte meiosis.…”