scholarly journals Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase

2002 ◽  
Vol 367 (2) ◽  
pp. 517-524 ◽  
Author(s):  
Jing Ti DENG ◽  
Cindy SUTHERLAND ◽  
David L. BRAUTIGAN ◽  
Masumi ETO ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Kinase Assay ◽  
Myosin Light Chain Phosphatase ◽  
Phosphatase Inhibitors ◽  
Phosphatase Inhibitor ◽  
Integrin Linked Kinase ◽  
Phosphopeptide Mapping

Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.

scholarly journals Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE

2016 ◽  
Vol 310 (8) ◽  
pp. C681-C691 ◽  
Author(s):  
Cindy Sutherland ◽  
Justin A. MacDonald ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Coiled Coil ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Tissue Samples ◽  
Phosphatase Inhibitor ◽  
Sds Page

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 Thr697 and Thr855, PKA phosphorylates these two sites and the neighboring Ser696 and Ser854, and prior phosphorylation at Thr697 and Thr855 by ROCK precludes phosphorylation at Ser696 and Ser854 by PKA. Furthermore, phosphorylation at Thr697 and Thr855 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.

scholarly journals The latch-bridge hypothesis of smooth muscle contraction

2005 ◽  
Vol 83 (10) ◽  
pp. 857-864 ◽  
Author(s):  
Richard A Murphy ◽  
Christopher M Rembold
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Striated Muscle ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Shortening Velocity ◽  
General Applicability ◽  
Cross Bridge ◽  
Mechanical Transduction

In contrast to striated muscle, both normalized force and shortening velocities are regulated functions of cross-bridge phosphorylation in smooth muscle. Physiologically this is manifested as relatively fast rates of contraction associated with transiently high levels of cross-bridge phosphorylation. In sustained contractions, Ca2+, cross-bridge phosphorylation, and ATP consumption rates fall, a phenomenon termed "latch". This review focuses on the Hai and Murphy (1988a) model that predicted the highly non-linear dependence of force on phosphorylation and a directly proportional dependence of shortening velocity on phosphorylation. This model hypothesized that (i) cross-bridge phosphorylation was obligatory for cross-bridge attachment, but also that (ii) dephosphorylation of an attached cross-bridge reduced its detachment rate. The resulting variety of cross-bridge cycles as predicted by the model could explain the observed dependencies of force and velocity on cross-bridge phosphorylation. New evidence supports modifications for more general applicability. First, myosin light chain phosphatase activity is regulated. Activation of myosin phosphatase is best demonstrated with inhibitory regulatory mechanisms acting via nitric oxide. The second modification of the model incorporates cooperativity in cross-bridge attachment to predict improved data on the dependence of force on phosphorylation. The molecular basis for cooperativity is unknown, but may involve thin filament proteins absent in striated muscle.Key words: chemo-mechanical transduction, activation-contraction coupling, cross-bridge, myosin light chain kinase, myosin light chain phosphatase, phosphorylation, cooperativity.

scholarly journals Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red

2000 ◽  
Vol 349 (3) ◽  
pp. 797-804 ◽  
Author(s):  
Aki YAMADA ◽  
Osamu SATO ◽  
Minoru WATANABE ◽  
Michael P. WALSH ◽  
Yasuo OGAWA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Direct Interaction ◽  
Smooth Muscle Contraction ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
Myosin Light Chain Phosphatase ◽  
Concentration Dependent Manner

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca2+ release channels, but has additional effects such as the induction of Ca2+ sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca2+] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC50 23µM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca2+]. Ca2+-independent contraction was induced by RuR (EC50 843µM) and by microcystin (EC50 59nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300µM RuR enhanced the contraction induced by 30nM microcystin but markedly decreased that induced by 1µM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca2+ sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.

scholarly journals Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle

2005 ◽  
Vol 392 (3) ◽  
pp. 641-648 ◽  
Author(s):  
David P. Wilson ◽  
Cindy Sutherland ◽  
Meredith A. Borman ◽  
Jing Ti Deng ◽  
Justin A. MacDonald ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Cultured Cells ◽  
Kinase Inhibitor ◽  
Smooth Muscle Contraction ◽  
Integrin Linked Kinase

Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63±0.05 μM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 μM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.

scholarly journals Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle

2003 ◽  
Vol 369 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Naohisa NIIRO ◽  
Yasuhiko KOGA ◽  
Mitsuo IKEBE
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Kinase C ◽  
Agonist Stimulation ◽  
Myosin Binding ◽  
Mlc Phosphorylation

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both α-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

scholarly journals Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium.

1992 ◽  
Vol 267 (30) ◽  
pp. 21492-21498
Author(s):  
M.C. Gong ◽  
A Fuglsang ◽  
D Alessi ◽  
S Kobayashi ◽  
P Cohen ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphatase

Modulation of uterine resistance artery lumen diameter by calcium and G protein activation during pregnancy

1994 ◽  
Vol 267 (3) ◽  
pp. H952-H961 ◽  
Author(s):  
G. D'Angelo ◽  
G. Osol
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
G Proteins ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Lumen Diameter ◽  
Maximal Response ◽  
Adrenergic Stimulation ◽  
Myosin Light Chain Phosphatase ◽  
Arterial Lumen

The purpose of this study was to determine whether the increased sensitivity of uterine resistance arteries from late pregnant (LP) rats to alpha-adrenergic stimulation is due to an alteration in the fundamental relationship between cytosolic calcium (Ca2+) and arterial lumen diameter. Uterine arcuate arteries were permeabilized with Staphylococcus aureus alpha-toxin under optimal conditions and constricted to varying degrees with discrete Ca2+ concentrations at a distending pressure of 50 mmHg. Arterial segments from nonpregnant (NP) and LP rats exhibited similar Ca2+/lumen diameter characteristics. Ca2+ (0.1 microM) produced appreciable constriction, and lumen diameter decreased steeply between 0.175 and 0.25 microM Ca2+; maximal responses were attained with 0.5 microM Ca2+. Activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-triphosphate (GTP; 1-100 microM), as reportedly occurs during alpha-adrenergic stimulation, potentiated the Ca(2+)-induced constriction by 121 and 79% in arteries from LP and NP rats, respectively. No significant differences between the two animal groups were noted. Guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S; 0.1-10 microM), a nonhydrolyzable analogue of GTP, effected a larger potentiating effect over that maximal response caused by GTP in arteries from NP rats. Ca(2+)- and Ca2+/GTP-induced constrictions were more potently reversed by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S)., a competitive inhibitor of GTP, in arteries from NP rats. These data suggest that pregnancy-induced increases in sensitivity to alpha-adrenergic stimulation may be related to altered G protein cycling rates, such that G proteins in smooth muscle cells in arcuate arteries from NP rats are more susceptible to deactivation. Alternatively, consistent with the model of G protein-mediated inhibition of myosin light chain phosphatase, myosin light chain phosphatase activity may be enhanced in uterine vascular smooth muscle from NP rats relative to that from LP rats.

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