Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Abstract

MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.Force regulation in smooth muscle is dependent on the activities of myosin light chain (MLC) 1 kinase and MLC phosphatase (3, 4). The activity of MLC kinase is regulated by Ca 2ϩ -calmodulin (3), whereas MLC phosphatase was originally thought to be constitutively active and unregulated (5). However, there is abundant evidence that the activity of MLC phosphatase can be both inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 5-7) or an increase in force at a constant [Ca 2ϩ ] and enhanced to produce Ca 2ϩ desensitization (8) or a decrease in force at a constant [Ca 2ϩ ]. NO is the classical agent to produce Ca 2ϩ desensitization (1, 9, 10). Recent evidence (11) suggests that NO produces vasodilatation by activating the soluble pool of guanylate cyclase, which in turn produces cGMP and leads to the activation of type I cGMP-dependent protein kinase (PKGI). PKGI mediates smooth muscle cell relaxation by several mechanisms. It has been demonstrated that PKGI acts on the maxi K ϩ channel to produce hyperpolarization of the smooth muscle (12), decreases Ca 2ϩ flux (13,14), and also activates MLC phosphatase (1, 15) to decrease the level of MLC 20 phosphorylation and to produce smooth muscle relaxation. In addition, PKGI-dependent pathways for vasodilatation may also include phosphorylation of telokin (16, 17) and HSP20 (18).MLC phosphatase is a holoenzyme consisting of a catalytic subunit (PP1c␦); a myosin-binding subunit (MBS), which is also referred to as the myosin-targeting subunit (MYPT1); and a 20-kDa subunit of unknown function (5). The activation of MLC phosphatase by PKGI is hypothesized to be due to a leucine zipper-leucine zipper (LZ-LZ) interaction of the N-terminal LZ of PKGI␣ and the C-terminal LZ of the MBS of MLC phosphatase (1, 2). The MBS has four major isoforms, which are produced by alternative RNA splicing of two different exons (5). Tissue-specific and developmentally regulated alternative splicing of a 123-bp central exon produces a 41-amino acid central insert (19). Alternative splicing of the 31-bp 3Ј-exon is responsible for the expression of LZ ϩ or LZ Ϫ MBS isoforms (5). Specifically, exclusion of the 3Ј-exon shifts the reading frame of the MBS transcript to encode a C-terminal LZ (2).We previously demonstrated that sensitivity to cGMP-mediated relaxation correlates with the relative expression of LZ ϩ / LZ Ϫ MBS isoforms (2), which is consistent with the activation of MLC phosphatase activity resulting from a LZ-LZ interaction of PKGI␣ with the MBS (1). In this study, we tested the hypothesis that cGMP-dependent activation of MLC phosphatase activity and smooth muscle vasodilatation are due to a LZ-LZ interaction of PKGI␣ and MBS by changing the expression of the MBS isoform, in isolation, and determining the effect on cGMP-mediated MLC 20 dephosphorylation in primary cultured smooth muscle cells (SMCs). MATERIALS AND METHODSCloning of the Chicken MBS of the MLC Phosphatase cDNA Fragment-...

MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGIα

“…Four MYPT1 fragments (Fig. 1A) were subcloned from the full-length chicken aorta MYPT1 cDNA (13,23,24). The production of four endogenous MYPT1 isoforms depends on the presence or absence of a CI and a downstream LZ (12).…”

“…Studies with glutathione S-transferase (GST)-fusion peptides have demonstrated that the NH 2 -terminal LZ of PKGI␣ (24) and COOH-terminal LZ of MYPT1 (23) are important for the PKGI␣-MYPT1 interaction. However, using full-length MYPT1, we have demonstrated that although the LZ domain is important for cGMP-mediated activation of MLC phosphatase, it may not mediate the binding of PKGI␣ to MYPT1 (13). Therefore, the MYPT1-PKGI␣ interaction may depend on the presence of an upstream domain.…”

“…NO diffuses into the smooth muscle cells and activates guanylate cyclase, thereby increasing the production of cGMP. This second messenger can then bind to and activate type I␣ cGMP-dependent protein kinase (PKGI␣), which interacts with multiple targets within the smooth muscle cell, including the L-type Ca 2ϩ channel (8), maxi-K ϩ channels (1), sarcoplasmic reticulum channels (20), telokin (4, 25), and MYPT1 (13,23,24), all of which have been demonstrated to produce smooth muscle relaxation.MYPT1 (Fig. 1A) contains two putative COOH-terminal coiled-coil (CC) domains at aa 715-746 and aa 929 -969, which have 60% and 81% probabilities of CC formation, respectively (16), and a third domain at aa 1013-1039, which is known as the leucine zipper (LZ) (12).…”

ROS-mediated downregulation of MYPT1 in smooth muscle cells: a potential mechanism for the aberrant contractility in atherosclerosis

“…9 In vitro and in vivo evidence showed that the LZ motif present in the C-terminal of MYPT1 is required for the activation of MP by type I PKG. [10][11][12] The sensitivity of smooth muscle to cGMP/PKG-mediated relaxation is proportional to the expression ratio of LZ þ / LZ À MYPT1. [10][11][12] In smooth muscle cells, the relationship between MP activity and the phosphorylation of MYPT1 has been extensively studied.…”

“…[10][11][12] The sensitivity of smooth muscle to cGMP/PKG-mediated relaxation is proportional to the expression ratio of LZ þ / LZ À MYPT1. [10][11][12] In smooth muscle cells, the relationship between MP activity and the phosphorylation of MYPT1 has been extensively studied. 13 However, changes in MYPT1 abundance have only been reported by a few studies.…”