Agonist-induced Force Enhancement

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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-...

Section: Preparation Of Chicken Smoothmentioning

confidence: 99%

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

Huang

Fisher

Brozovich

2004

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“…The additional minor sites of phosphorylation were identified by determining the cycles in which 32 P was released when 32 P-labeled fractions of the protein digests were subjected to sequential Edman degradation under conditions that optimized recovery of 32 P. Solid phase Edman sequencing of peak I and CRP analysis of MYPT1 (Fig. 5) revealed that Thr 675 was the only residue that could yield the release of 32 P in the eighth cycle following digestion with endoproteinase Lys-C and in the fourth cycle following digestion with endoproteinase Arg-C. From a similar analysis of peak II, we have identified Ser 849 , which corresponds to Ser 854 in Rat3 MBS and Ser 808 in chicken M130, as a minor phosphorylation site.…”

Section: Fig 2 Recombinant Mypt1 Kinase Induces Camentioning

confidence: 99%

“…Indeed, they have suggested that MYPT1 phosphorylation by native MYPT1 kinase may be physiologically irrelevant because in their hands MYPT1 is a poor substrate for recombinant ZIPK. It is possible, as suggested by Richards et al (32), that ZIPK (and native MYPT1 kinase) only phosphorylates the M133 isoform of MYPT1 (i.e. Thr 695 site) and not the shorter, spliced-out M130 isoform (i.e.…”

mentioning

confidence: 97%

“…The phosphopeptide samples were immobilized to Immobilon membrane (Millipore) following the manufacturer's instructions. The phosphorylated residues within phosphopeptides were located by determining the cycles in which 32 P was released when the samples were subjected to sequential Edman degradation with a vapor phase amino acid sequencer (Applied Biosystems Procise 494) under conditions that optimize recovery of 32 P (22). The CRP analysis program (23) was used to assign a phosphorylation site to the 32 P released in a specific cycle.…”

mentioning

confidence: 99%

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Smooth Muscle Myosin Phosphatase-associated Kinase Induces Ca2+ Sensitization via Myosin Phosphatase Inhibition

Borman¹,

MacDonald²,

Murányi³

et al. 2002

102

Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in ␤-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr 654 (M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1M phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.

“…This aspect of smooth muscle phenotypic diversity has not been examined. We have selected the MYPT1 subunit of smooth muscle myosin phosphatase as a model gene to address this question for several reasons: 1) MYPT1 isoforms are thought to determine smooth muscle relaxant properties and tissue-specific relaxant responses to signaling pathways (15,16). 2) Isoforms of MYPT1 are generated by cassette-type alternative splicing of single exons in the central and 3Ј-end of the pre-mRNA.…”

mentioning

confidence: 99%

Splicing of a Myosin Phosphatase Targeting Subunit 1 Alternative Exon Is Regulated by Intronic Cis-elements and a Novel Bipartite Exonic Enhancer/Silencer Element

Dirksen¹,

Mohamed²,

Fisher³

2003

Isoforms of the smooth muscle myosin phosphatase targeting subunit 1 (MYPT1) are generated by cassettetype alternative splicing of exons. Tissue-specific expression of these isoforms is thought to determine smooth muscle-relaxant properties and unique responses to signaling pathways. We used mini-gene deletion/mutation constructs to identify cis regulators of splicing of the chicken MYPT1 central alternative exon. Comparisons of alternative exon splicing were made between smooth muscle cells of the fast-phasic contractile phenotype (gizzard), in which the central alternative exon is skipped, and slow tonic contractile phenotype (aorta), in which the alternative exon is included. We demonstrate that splicing of the alternative exon requires a cis-enhancer complex in the vicinity of the alternative exon 5-splice site. This complex consists of two UCUU motifs in an intronic U-rich sequence (putative PTB (polypyrimidine tract binding) or T cell inhibitor of apoptosis-1 binding sites), an intronic 67-nucleotide enhancer that has similarities with the cardiac Troponin T MSE3 enhancer, and a potentially novel exonic splicing enhancer. The exonic enhancer contains the palindromic sequence UCCUACAUCCU present in many other transcripts where alternative splicing of exons occurs, suggesting that it may be more broadly active. The exonic enhancer is adjacent to a potentially novel exonic silencer element that contains a 13-nucleotide imperfect palindromic sequence. This silencer, in conjunction with a distal intronic silencer, is proposed to mediate the silencing of splicing of the MYPT1 central alternative exon in the fast phasic smooth muscle phenotype.Smooth muscle tissues show considerable phenotypic and functional diversity. Much of the phenotypic diversity in smooth muscle tissues is generated by the alternative splicing of exons from single gene transcripts. As many as 50% of vertebrate genes are estimated to have alternative splicing of exons as a mechanism by which multiple protein isoforms with different or modified functions are generated from a single gene (1-3). The process of splice site selection and exon definition for exons that are constitutively spliced has been well described (4 -7). Binding of U1 small nuclear ribonucleoprotein particle (snRNP) 1 to the consensus 5Ј-splice site sequence and binding of splicing factor 1 (SF1) and U2 snRNP auxiliary factor (U2AF) to the consensus 3Ј-splice site sequences (branchpoint and polypyrimidine tract, respectively) commits the exon to the splicing pathway (7,8). A number of factors are known to contribute to the "weakening" of the splicing reaction so that splicing of an exon may be regulated. These factors include exon and intron size, RNA secondary structure, and the extent to which the 5Ј-splice site, branchpoint, and polypyrimidine tract sequences match the consensus sequences for U1 snRNP, SF1, and U2AF binding, respectively (9 -11).A number of cis-elements that regulate the splicing of alternative exons have been identified (reviewed in Refs. 10, 12-14). W...