Cyclic GMP-Dependent Stimulation Reverses G-Protein-Coupled Inhibition of Smooth Muscle Myosin Light Chain Phosphatase

Wu, Xuqiong; Somlyo, Avril V.; Somlyo, Andrew P.

doi:10.1006/bbrc.1996.0460

Cited by 146 publications

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“…The Ca 2ϩ sensitivity of WT aortic SM was unaffected by the deletion of telokin, likely reflecting its low endogenous telokin content. Taken together, these results, and those of our previous studies (2,3,23), support the hypothesis that telokin directly or indirectly increases myosin phosphatase activity and, thereby, contributes to the Ca 2ϩ desensitization of force and cyclic nucleotide-induced relaxation in telokin-rich SMs, such as in the gut, bladder, portal vein, resistance vessels, and trachealis.…”

Section: Discussionsupporting

confidence: 87%

“…Depletion of endogenous telokin from permeabilized SM correlates with a loss of 8-Br-cGMP-induced relaxation, which is rescued by the addition of recombinant telokin and further enhanced by PKG (2,3,10). Site-specific mutation of Ser-13 to an acidic, Asp residue to mimic phosphorylation, but not to a nonphosphorylatable Ala, enhances the Ca 2ϩ -desensitizing effect on force.…”

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confidence: 96%

“…Ca 2ϩ sensitization due to inhibition of MLCP activity is mediated by an agonist G protein-coupled, Ca 2ϩ -independent process that activates RhoA͞Rho-kinase, phosphorylates the myosin regulatory subunit of MLCP (MYPT1), and leads to increased force (1). On the other hand, cyclic nucleotideactivated kinases, in addition to decreasing cytosolic Ca 2ϩ , make a significant contribution to dephosphorylation of RLC 20 and relaxation through Ca 2ϩ -desensitization processes (2)(3)(4)(5) and can reverse G protein-coupled Ca 2ϩ sensitization (2,3). Interaction between the leucine zipper motifs of protein kinase G and MYPT1 leads to direct stimulation of MLCP (6).…”

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confidence: 99%

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Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation

Khromov¹,

Wang²,

Choudhury³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cyclic nucleotides can relax smooth muscle without a change in [Ca 2؉ ]i, a phenomenon termed Ca 2؉ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth musclespecific protein and target for cyclic nucleotide-induced Ca 2؉ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an Ϸ30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca 2؉ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca 2؉ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally Ϸ5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca 2؉ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 M 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues. S mooth muscle (SM) myosin II ATPase activity and associated contraction is activated by actin only when Ser-19 of the myosin regulatory light chain (RLC 20 ) is phosphorylated. The extent of RLC 20 phosphorylation is a reflection of the balance between Ca 2ϩ calmodulin-dependent myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP) activities. Although the major mechanism for initiating contraction is the rise in [Ca 2ϩ ] i , force can be further increased or decreased through signaling pathways that modulate MLCK and͞or MLCP activities giving rise to processes termed Ca 2ϩ sensitization or -desensitization (reviewed in ref. 1). Ca 2ϩ sensitization due to inhibition of MLCP activity is mediated by an agonist G protein-coupled, Ca 2ϩ -independent process that activates RhoA͞Rho-kinase, phosphorylates the myosin regulatory subunit of MLCP (MYPT1), and leads to increased force (1). On the other hand, cyclic nucleotideactivated kinases, in addition to decreasing cytosolic Ca 2ϩ , make a significant contribution to dephosphorylation of RLC 20 and relaxation through Ca 2ϩ -desensitization processes (2-5) and can reverse G protein-coupled Ca 2ϩ sensitization (2, 3). Interaction between the leucine zipper motifs of protein kinase G and MYPT1 leads to direct stimulation of MLCP (6). A role for this interaction in Ca 2ϩ desensitization is supported by the observation that chicken gizzard MYPT1 lacking the leucine zipper motifs is resistant to 8-BrcGMP-induced depho...

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Section: Discussionsupporting

confidence: 87%

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confidence: 96%

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confidence: 99%

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Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation

Khromov¹,

Wang²,

Choudhury³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Both vasoconstrictor signaling pathways, which lead to inhibition of the phosphatase and cell contraction (reviewed in Ref. 10), and vasodilator signaling pathways, which lead to cell relaxation via activation of myosin phosphatase have been recently defined (11)(12)(13)(14)(15).…”

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confidence: 99%

Myosin Phosphatase-Rho Interacting Protein

Surks

Richards

Mendelsohn

2003

Journal of Biological Chemistry

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Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116 RIP3 , is expressed in vascular smooth muscle, and is localized to actin myofilaments. Blood vessel tone is regulated by the contractile state of vascular smooth muscle cells in the blood vessel wall. Diseases characterized by abnormal vascular smooth muscle cell contraction include hypertension, blood vessel spasm, and atherosclerosis (1-5). Smooth muscle contraction is tightly coupled to myosin light chain phosphorylation (6), which in turn is regulated by the relative activities of myosin light chain kinase and myosin phosphatase. Myosin light chain kinase is activated by intracellular calcium and phosphorylates myosin light chains, leading to cell contraction (7,8). Myosin phosphatase dephosphorylates myosin light chains, leading to smooth muscle cell relaxation (9). Myosin phosphatase activity, once thought to be constitutive, is now known to be highly regulated. Both vasoconstrictor signaling pathways, which lead to inhibition of the phosphatase and cell contraction (reviewed in Ref. 10), and vasodilator signaling pathways, which lead to cell relaxation via activation of myosin phosphatase have been recently defined (11-15).Myosin phosphatase is a heterotrimer consisting of a PP1 catalytic subunit, a 130-kDa myosin binding subunit (MBS) 1 and a 20-kDa subunit of unknown function (9, 16 -18). The MBS is a regulatory subunit that targets PP1 to its substrate, myosin light chain (9), and has multiple protein interaction domains, including ankyrin repeats at its amino terminus, and a leucine zipper domain at its carboxyl terminus. MBS binds PP1 and myosin light chain at its amino terminus and the M20 subunit and cGMP-dependent protein kinase 1␣ (cGK) at its carboxyl terminus (Ref. 11, reviewed in Ref. 19). The MBS-cGK interaction is necessary for NO/cGMP-mediated activation of myosin phosphatase (11).In vascular smooth muscle, G-protein-coupled receptor agonists cause contraction in part by inhibition of myosin phosphatase activity (20). Several downstream signaling pathways that inhibit myosin phosphatase activity have been discovered re...

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“…5 In contrast, relaxation of smooth muscle results from nitric oxide (NO)-stimulated activation of MP, which is mediated by cGMP and cGMP-dependent protein kinase (PKG). 6,7 Several MYPT1 isoforms identified to date are products of a gene with alternative splicing of a central and a 3 0 exons. No distinct functions are known for the central insert isoforms.…”

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confidence: 99%

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

Cheng

Tsai

et al. 2013

Laboratory Investigation

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Reactive oxygen species (ROS) mediates the aberrant contractility in hypertension. Abnormal contractility occurs in atherosclerotic vessels but changes in proteins that regulate contractility remain poorly understood. Myosin phosphatase (MP) activity, which regulates smooth muscle relaxation, is regulated by the phosphorylation of its regulatory subunit, MP targeting subunit 1 (MYPT1). In the present study, we examined the roles of ROS in MP subunit expression both in cultured human aortic smooth muscle cells (HASMCs) and during atherosclerosis progression in apolipoprotein E-knockout (apoE-KO) mice. Furthermore, the effect of decreased MYPT1 on actin cytoskeleton and cell migration activity was assessed in HASMCs. Short hairpin RNA-mediated knockdown of MYPT1 increased stress fibers and attenuated platelet-derived growth factor-induced cell migration in HASMCs. Superoxide anion-inducing agent LY83583 downregulated MYPT1 mRNA and protein levels, but did not affect the phosphorylation of MYPT1 and catalytic subunit of MP, PP1d. The LY83583-induced decrease in MYPT1 was abolished by co-treating with superoxide dismutase or by inhibiting NADPH oxidase with diphenyleneiodonium. Treatment of peroxynitrite, but not hydrogen peroxide (H 2 O 2 ), downregulated MYPT1 protein expression and induced MYPT1 phosphorylation without affecting mRNA levels. Co-treatment with a proteasome inhibitor, MG-132, eliminated peroxynitrite-induced MYPT1 downregulation. In apoE-KO mice, MYPT1 protein, but not mRNA, levels were markedly decreased in 16-week-and 24-week-old mice. Oral estrogen treatment, which was previously shown to decrease aortic ROS levels, upregulated aortic MYPT1 expression. Moreover, reduction in MYPT1 expression correlated with increased aortic sensitivity toward vasoconstrictors. These results suggested that during atherosclerosis progression oxidative stress mediates the downregulation of MYPT1, which may inhibit smooth muscle cell migration and contribute to the aberrant contractility.Laboratory Investigation (2013) 93, 422-433; doi:10.1038/labinvest.2013 published online 18 February 2013 KEYWORDS: atherosclerosis; contractility; myosin phosphatase; reactive oxygen species; smooth muscle At the molecular level, phosphorylation of the 20-kDa myosin light chains (MLC 20 ) is a key determinant for smooth muscle contraction. MLC 20 phosphorylation levels are determined by the activity ratio between myosin light chain kinase (MLCK) and myosin phosphatase (MP). 1 Although MLCK activation depends on cytoplasmic calcium concentration, MP activity is subject to modulation by various signaling molecules. 2 MP is a heterotrimer comprised of a 37-to 38-kDa catalytic subunit (PP1d), a 110-to 130-kDa regulatory subunit (MP targeting subunit 1; MYPT1), and a 20-kDa subunit of unknown function. The phosphorylation of MYPT1 by protein kinases is a major regulatory mechanism for MP that results in either the inhibition or enhancement of MP activity. 3 Small GTPase RhoA and its effecter, Rho-kinase, inhibit MP activity through ...

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Cyclic GMP-Dependent Stimulation Reverses G-Protein-Coupled Inhibition of Smooth Muscle Myosin Light Chain Phosphatase

Cited by 146 publications

References 0 publications

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation

Myosin Phosphatase-Rho Interacting Protein

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

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Cyclic GMP-Dependent Stimulation Reverses G-Protein-Coupled Inhibition of Smooth Muscle Myosin Light Chain Phosphatase

Cited by 146 publications

References 0 publications

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca 2+ and decreased cGMP-induced relaxation

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca 2+ and decreased cGMP-induced relaxation

Myosin Phosphatase-Rho Interacting Protein

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

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Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca ²⁺ and decreased cGMP-induced relaxation