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Avril V. Somlyo - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of telokin mediated disinhibition of myosin light chain Phosphatase and camp cgmp induced relaxation of gastrointestinal smooth muscle
Journal of Biological Chemistry, 2012Co-Authors: Alexander S Khromov, Mykhaylo V Artamonov, Ko Momotani, John D Shannon, Avril V. SomlyoAbstract:Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca2+-activated contraction and Ca2+ sensitization of force induced by a GST-MYPT1(654–880) fragment inhibiting myosin light chain Phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain Phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.
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Molecular Mechanism of Telokin-mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-induced Relaxation of Gastrointestinal Smooth Muscle
The Journal of biological chemistry, 2012Co-Authors: Alexander S Khromov, Mykhaylo V Artamonov, Ko Momotani, Masumi Eto, John D Shannon, Li Jin, Avril V. SomlyoAbstract:Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain Phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain Phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.
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the camp responsive rap1 guanine nucleotide exchange factor epac induces smooth muscle relaxation by down regulation of rhoa activity
Journal of Biological Chemistry, 2011Co-Authors: Bartosz J Zieba, Mykhaylo V Artamonov, Ko Momotani, Andra S Stevenson, Alexander S Khromov, Aaron S Franke, Ronald L Neppl, Ruoya Ho, Magdalena Chrzanowskawodnicka, Avril V. SomlyoAbstract:Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain Phosphatase activity, an increase in myosin light chain (RLC20) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP (“007”), significantly reduced agonist-induced contractile force, RLC20, and myosin light chain Phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI2 analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca2+ desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.
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thromboxane a2 induced bi directional regulation of cerebral arterial tone
Journal of Biological Chemistry, 2009Co-Authors: Ronald L Neppl, Ko Momotani, Lubomir T. Lubomirov, Gabriele Pfitzer, Masumi Eto, Avril V. SomlyoAbstract:Abstract Myosin light chain Phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain Phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin Phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces “buffered” vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.
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Cyclic GMP-Dependent Stimulation Reverses G-Protein-Coupled Inhibition of Smooth Muscle Myosin Light Chain Phosphatase
Biochemical and biophysical research communications, 1996Co-Authors: Avril V. Somlyo, Avril V. SomlyoAbstract:8-bromo-cyclic guanosine monophosphate (8-br-cGMP), at constant Ca2+, accelerated the dephosphorylation of the regulatory myosin regulatory light chain and relaxation of permeabilized rabbit ileum smooth muscle. These effects were independent of myosin light chain kinase activity. Similarly, 8-br-cGMP relaxed GTPgammaS-induced force and inhibited the GTPgammaS-induced increase in myosin light chain phosphorylation at constant [Ca2+]. We conclude that cyclic GMP-dependent kinase, activated by 8-br-cGMP, increases smooth muscle myosin light chain Phosphatase activity and counteracts the inhibition of the latter enzyme by G-protein(s).
Michael P. Walsh - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Smooth Muscle Myosin Light Chain Phosphatase by Multisite Phosphorylation of the Myosin Targeting Subunit, MYPT1.
Cardiovascular & hematological disorders drug targets, 2018Co-Authors: Justin A. Macdonald, Michael P. WalshAbstract:BACKGROUND Smooth muscle contraction is triggered primarily by activation of Ca2+/calmodulin-dependent myosin light chain kinase leading to phosphorylation of the regulatory light chains of myosin II. Numerous contractile stimuli also induce inhibition of myosin light chain Phosphatase thereby prolonging the contractile response. The Phosphatase is a trimeric enzyme containing a catalytic subunit, a regulatory, myosin-binding subunit (MYPT1) and a third subunit of uncertain function. MYPT1 is phosphorylated at multiple sites by several kinases, which regulate Phosphatase activity, protein-protein interactions and subcellular localization. The best-characterized phosphorylation events involve phosphorylation by Rho-associated coiled-coil kinase (ROCK) at T697 and T855, which inhibits Phosphatase activity, and phosphorylation by cAMP- or cGMPdependent protein kinases (PKA and PKG, respectively) at S696, T697, S854 and T855, which has no effect on Phosphatase activity. Furthermore, phosphorylation by ROCK at T697 and T855 prevents phosphorylation by PKA or PKG at the neighboring serine residues. Some 30 phosphorylation sites have been identified in MYPT1 with many more suggested by large-scale phosphoproteomic studies. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation of MYPT1 functions in part because of their involvement in pathological processes. For example, dysfunctional MYPT1 phosphorylation has been implicated in the pathogenesis of several vascular disorders, including type 2 diabetes. CONCLUSION Much effort is now being devoted to the development of novel therapeutics targeting MYPT1 and specific kinases involved in the phosphorylation of MYPT1.
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Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain Phosphatase by Phos-tag SDS-PAGE
The FASEB Journal, 2016Co-Authors: Michael P. Walsh, Justin A. Macdonald, Cindy SutherlandAbstract:Phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain Phosphatase plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylatio...
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Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain Phosphatase by Phos-tag SDS-PAGE
American journal of physiology. Cell physiology, 2016Co-Authors: Cindy Sutherland, Justin A. Macdonald, Michael P. WalshAbstract:Phosphorylation of the myosin-targeting subunit 1 of myosin light chain Phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant Phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant Phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain Phosphatase activity and smooth muscle contraction.
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Abnormal myosin Phosphatase targeting subunit 1 phosphorylation and actin polymerization contribute to impaired myogenic regulation of cerebral arterial diameter in the type 2 diabetic Goto-Kakizaki rat.
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2015Co-Authors: Khaled S. Abd-elrahman, Michael P. Walsh, Olaia Colinas, Emma J. Walsh, Hai-lei Zhu, Christine Campbell, William C ColeAbstract:The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain Phosphatase by phosphorylating its targeting subunit myosin Phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain Phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8-10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin Phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18-20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.
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Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi, 2013Co-Authors: Mitsuo Mita, Michael P. Walsh, Cindy Sutherland, Hitoshi Tanaka, Hayato Yanagihara, Jun-ichi Nakagawa, Shigeru Hishinuma, Masaru ShojiAbstract:Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine Phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain Phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain Phosphatase, RhoA, Rho-associated kinase, tyrosine kinase
Masumi Eto - One of the best experts on this subject based on the ideXlab platform.
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Reconstituted human myosin light chain Phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, MYPT1.
Biochemistry, 2014Co-Authors: Mukta D. Khasnis, Akiko Nakatomi, Kristyn Gumpper, Masumi EtoAbstract:The myosin light chain Phosphatase (MLCP) is a cytoskeleton-associated protein Phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation. The recombinant MLCP partially purified from mammalian cell lysates retained characteristics of the native enzyme, such that it was fully active without Mn2+ and sensitive to PP1 inhibitor compounds. Selective thio-phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 thio-phosphorylation did not alter the Phosphatase activity. Interference with the docking of phospho-Thr696 at the active site weakened the inhibition, suggesting selective auto...
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Molecular Mechanism of Telokin-mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-induced Relaxation of Gastrointestinal Smooth Muscle
The Journal of biological chemistry, 2012Co-Authors: Alexander S Khromov, Mykhaylo V Artamonov, Ko Momotani, Masumi Eto, John D Shannon, Li Jin, Avril V. SomlyoAbstract:Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain Phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain Phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.
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Reciprocal regulation controlling the expression of CPI-17, a specific inhibitor protein for the myosin light chain Phosphatase in vascular smooth muscle cells
American journal of physiology. Cell physiology, 2012Co-Authors: Jee In Kim, Mark W. Urban, Garbo D. Young, Masumi EtoAbstract:Cellular activity of the myosin light chain Phosphatase (MLCP) determines agonist-induced force development of smooth muscle (SM). CPI-17 is an endogenous inhibitor protein for MLCP, responsible for mediating G-protein signaling into SM contraction. Fluctuations in CPI-17 expression occur in response to pathological stresses, altering excitation-contraction coupling in SM. Here, we determined the signaling pathways regulating CPI-17 expression in rat aorta tissues and the cell culture using a pharmacological approach. CPI-17 transcription was suppressed in response to the proliferative stimulus with platelet-derived growth factor (PDGF) through the ERK1/2 pathway, whereas it was elevated in response to inflammatory, stress-induced and excitatory stimuli with tranforming growth factor-β, IL-1β, TNFα, sorbitol, and serotonin. CPI-17 transcription was repressed by inhibition of JNK, p38, PKC, and Rho-kinase (ROCK). The mouse and human CPI-17 gene promoters were governed by the proximal GC-boxes at the 5′-flanking region, where Sp1/Sp3 transcription factors bound. Sp1 binding to the region was more prominent in intact aorta tissues, compared with the SM cell culture, where the CPI-17 gene is repressed. The 173-bp proximal promoter activity was negatively and positively regulated through PDGF-induced ERK1/2 and sorbitol-induced p38/JNK pathways, respectively. By contrast, PKC and ROCK inhibitors failed to repress the 173-bp promoter activity, suggesting distal enhancer elements. CPI-17 transcription was insensitive to knockdown of myocardin/Kruppel-like factor 4 small interfering RNA or histone deacetylase inhibition. The reciprocal regulation of Sp1/Sp3-driven CPI-17 expression through multiple kinases may be responsible for the adaptation of MLCP signal and SM tone to environmental changes.
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Effects of a fluorescent Myosin light chain Phosphatase inhibitor on prostate cancer cells.
Frontiers in oncology, 2011Co-Authors: Scott Grindrod, Masumi Eto, Simeng Suy, Shannon Fallen, Jeffery Toretsky, Milton L. BrownAbstract:Myosin light chain Phosphatase is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of myosin light chain Phosphatase (MLCP) using structure based design in recombinant PP1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/32P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and independent (AI) prostate cancer cell lines were treated with the lead inhibitor resulting in decreased in growth rate, reduced DNA synthesis and G2/M cell cycle arrest. Moreover, AI cell lines showed an increased sensitivity to drug treatment having GI50 values four times lower than the AD prostate cancer line. This was reinforced by reduced BrdU DNA incorporation into AI cells compared to AD cells. Beta-actin disruption was also seen at much lower drug concentrations in AI cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently no clinical therapeutics targeting AI prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype.
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Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction
The Journal of biological chemistry, 2009Co-Authors: Alexander S Khromov, Andra S Stevenson, Avril V. Somlyo, Nandini Choudhury, Masumi EtoAbstract:The reversible regulation of myosin light chain Phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin Phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A Phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.
Frank V. Brozovich - One of the best experts on this subject based on the ideXlab platform.
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cGMP effects on vascular tone: modulating the activity of myosin light chain Phosphatase
BMC Pharmacology, 2009Co-Authors: Frank V. BrozovichAbstract:Background During flow-mediated vasodilatation, nitric oxide activates guanylate cyclase and the resultant increase cGMP leads to an activation of PKGI. PKGI activates a number of targets in the smooth muscle cell that result in smooth muscle relaxation, including MLC Phosphatase. MLC Phosphatase isolated from smooth muscle is a holoenzyme consisting of three subunits; a 20 kDa subunit, a 38 kDa catalytic subunit and a myosin targeting subunit (MYPT1). MYPT1 has two isoforms that differ by the presence of a an alternatively spliced 31 bp 3' exon; exon inclusion codes for a MYPT1 that lacks a COOH-terminus leucine zipper (LZ-), while exon exclusion shifts the reading frame and codes for a LZ+ MYPT1 isoform.
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PHI-1 interacts with the catalytic subunit of myosin light chain Phosphatase to produce a Ca2+ independent increase in MLC20 phosphorylation and force in avian smooth muscle
FEBS letters, 2006Co-Authors: Amr El-toukhy, Allison M. Given, Ozgur Ogut, Frank V. BrozovichAbstract:In avian smooth muscles, GTPγS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC20) phosphorylation at a constant [Ca2+]. P-PHI-1, but not PHI-1, increased MLC20 phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain Phosphatase by interacting with the active site of PP1c to produce a Ca2+ independent increase in MLC20 phosphorylation and force.
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Captopril prevents myosin light chain Phosphatase isoform switching to preserve normal cGMP-mediated vasodilatation.
Journal of molecular and cellular cardiology, 2006Co-Authors: Frank Chen, Albert Y. Rhee, Brian D. Hoit, Ozgur Ogut, Frank V. BrozovichAbstract:Abstract Congestive heart failure (CHF) is characterized by abnormal vasoconstriction and an impairment in nitric oxide (NO)-mediated vasodilatation. We have previously demonstrated that the decrease in sensitivity to NO lies at least partially at the level of the smooth muscle and is due to a reduction in the relative expression of the leucine zipper positive (LZ + ) isoform of the myosin targeting subunit (MYPT1) of myosin light chain Phosphatase. We hypothesized that since the attenuated vasodilatory response to NO in CHF has been shown to be secondary to an increased activity of the renin–angiotensin system, angiotensin converting enzyme (ACE) inhibition could affect MYPT1 isoform expression. To test this hypothesis, a rat myocardial infarction (MI) model of CHF was used; following left coronary artery ligation, rats were divided into control and captopril-treated groups. A third group of rats was given prazosin for 4 weeks. In the untreated control group, left ventricular function (LVF) was reduced at 2 weeks post-MI and remained at this level. Captopril treatment attenuated the fall in LVF. In the control aorta and iliac artery, the expression of the LZ + MYPT1 isoform fell 44–52% between 2 and 4 weeks post-MI, whereas in animals treated with captopril, MYPT1 isoform expression did not change. A decrease in the sensitivity to cGMP-mediated smooth muscle relaxation occurred coincident with the decrease in LZ + MYPT1 expression. The change in LZ + MYPT1 expression was not due to the decrease in afterload, as prazosin therapy produced an improvement in LVF but did not increase the relative expression of LZ + MYPT1 isoform. These data suggest that ACE inhibition, unique from pure afterload reduction, prevents MYPT1 isoform switching, which would preserve normal flow, or NO-mediated vasodilatation.
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Vascular Reactivity in Heart Failure. Role of Myosin Light Chain Phosphatase
Circulation research, 2004Co-Authors: Syed M. Karim, Albert Y. Rhee, Allison M. Given, Michael D. Faulx, Brian D. Hoit, Frank V. BrozovichAbstract:Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. During CHF, vascular tone is regulated by the interplay of neurohormonal mechanisms and endothelial-dependent factors and is characterized by both central and peripheral vasoconstriction as well as a resistance to nitric oxide (NO)–mediated vasodilatation. At the molecular level, vascular tone depends on the level of regulatory myosin light chain phosphorylation, which is determined by the relative activities of myosin light chain kinase and myosin light chain Phosphatase (MLCP). The MLCP is a trimeric enzyme with a catalytic, a 20-kDa and a myosin targeting (MYPT1) subunit. Alternative splicing of a 3′ exon produces leucine zipper positive and negative (LZ +/− ) MYPT1 isoforms. Expression of a LZ + MYPT1 has been suggested to be required for NO-mediated smooth muscle relaxation. Thus, we hypothesized that the resistance to NO-mediated vasodilatation in CHF could be attributable to a change in the relative expression of LZ +/− MYPT1 isoforms. To test this hypothesis, left coronary artery ligation was used to induce CHF in rats, and both the dose response relationship of relaxation to 8-Br-cGMP in skinned smooth muscle and the relative expression of LZ +/− MYPT1 isoforms were determined. In control animals, the expression of the LZ + MYPT1 isoform predominated in both the aorta and iliac artery. In CHF rats, LVEF was reduced to 30±5% and there was a significant decrease in both the sensitivity to 8-Br-cGMP and expression of the LZ + MYPT1 isoform. These results indicate that CHF is associated with a decrease in the relative expression of the LZ + MYPT1 isoform and the sensitivity to 8-Br-cGMP–mediated smooth muscle relaxation. The data suggest that the resistance to NO-mediated relaxation observed during CHF lies at least in part at the level of the smooth muscle and is a consequence of the decrease in the expression of the LZ + MYPT1 isoform.
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unzipping the role of myosin light chain Phosphatase in smooth muscle cell relaxation
Journal of Biological Chemistry, 2004Co-Authors: Qi Quan Huang, Steven A Fisher, Frank V. BrozovichAbstract:Abstract Recently, it has been hypothesized that myosin light chain (MLC) Phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC Phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3′-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC Phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p < 0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC Phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.
Justin A. Macdonald - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Smooth Muscle Myosin Light Chain Phosphatase by Multisite Phosphorylation of the Myosin Targeting Subunit, MYPT1.
Cardiovascular & hematological disorders drug targets, 2018Co-Authors: Justin A. Macdonald, Michael P. WalshAbstract:BACKGROUND Smooth muscle contraction is triggered primarily by activation of Ca2+/calmodulin-dependent myosin light chain kinase leading to phosphorylation of the regulatory light chains of myosin II. Numerous contractile stimuli also induce inhibition of myosin light chain Phosphatase thereby prolonging the contractile response. The Phosphatase is a trimeric enzyme containing a catalytic subunit, a regulatory, myosin-binding subunit (MYPT1) and a third subunit of uncertain function. MYPT1 is phosphorylated at multiple sites by several kinases, which regulate Phosphatase activity, protein-protein interactions and subcellular localization. The best-characterized phosphorylation events involve phosphorylation by Rho-associated coiled-coil kinase (ROCK) at T697 and T855, which inhibits Phosphatase activity, and phosphorylation by cAMP- or cGMPdependent protein kinases (PKA and PKG, respectively) at S696, T697, S854 and T855, which has no effect on Phosphatase activity. Furthermore, phosphorylation by ROCK at T697 and T855 prevents phosphorylation by PKA or PKG at the neighboring serine residues. Some 30 phosphorylation sites have been identified in MYPT1 with many more suggested by large-scale phosphoproteomic studies. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation of MYPT1 functions in part because of their involvement in pathological processes. For example, dysfunctional MYPT1 phosphorylation has been implicated in the pathogenesis of several vascular disorders, including type 2 diabetes. CONCLUSION Much effort is now being devoted to the development of novel therapeutics targeting MYPT1 and specific kinases involved in the phosphorylation of MYPT1.
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Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain Phosphatase by Phos-tag SDS-PAGE
The FASEB Journal, 2016Co-Authors: Michael P. Walsh, Justin A. Macdonald, Cindy SutherlandAbstract:Phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain Phosphatase plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylatio...
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Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain Phosphatase by Phos-tag SDS-PAGE
American journal of physiology. Cell physiology, 2016Co-Authors: Cindy Sutherland, Justin A. Macdonald, Michael P. WalshAbstract:Phosphorylation of the myosin-targeting subunit 1 of myosin light chain Phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant Phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant Phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain Phosphatase activity and smooth muscle contraction.
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ERK and p38MAPK pathways regulate myosin light chain Phosphatase and contribute to Ca2+ sensitization of intestinal smooth muscle contraction
Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society, 2014Co-Authors: Eikichi Ihara, Mona Chappellaz, Justin A. MacdonaldAbstract:Background Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK) and p38MAPK, are known regulators of smooth muscle contractility. The contraction of smooth muscle is mainly regulated by the phosphorylation of regulatory light chains of myosin II (LC20), which is driven by the balance between myosin light chain kinase (MLCK) and myosin light chain Phosphatase (MLCP). We hypothesized that one possible mechanism for MAPK-dependent modulation of intestinal smooth muscle contractility is via the regulation of MLCP activity. Methods Contractile responses to carbachol (CCh) and effects of MAPK inhibitors on CCh-induced contractions were assessed with isolated rat ileal longitudinal smooth muscle strips. Biochemical assessments of MLCP activity and myosin phosphatse targeting subunit (MYPT1) and CPI-17 phosphorylations were completed. Key Results Treatment of ileal smooth muscle with PD98059 (10 μM; MEK inhibitor) or SB203580 (10 μM; p38MAPK inhibitor) significantly inhibited CCh-induced contractile force. Decreased MLCP activity was observed during sustained contractions induced by CCh; the MLCP activity was recovered by treatment with PD98059 and SB203580. However, MYPT1 (Thr697 and Thr855) and CPI-17 (Thr38) phosphorylations were not affected. Application of ML-7 (MLCK inhibitor) during CCh-induced sustained contraction elicited an MLCP-dependent relaxation, the rate of which was accelerated by application of PD98059 and SB203580 with proportional changes in LC20 phosphorylation levels but not MYPT1 phosphorylation (Thr697 or Thr855). Conclusions & Inferences ERK and p38MAPK contribute to CCh-induced sustained contraction in a LC20 phosphorylation dependent manner. Moreover, both kinases inhibit MLCP activity possibly by a novel mechanism.
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the myosin Phosphatase targeting protein mypt family a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein Phosphatase type 1δ
Archives of Biochemistry and Biophysics, 2011Co-Authors: Michael E Grassie, Michael P. Walsh, Lori D. Moffat, Justin A. MacdonaldAbstract:Abstract The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine Phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of Phosphatase activity and Ca2+ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain Phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain Phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain Phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.
