Masumi Eto

Expression of CPI‐17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C‐induced contraction in rabbit smooth muscle

1 Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)‐induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI‐17, and the thin filament regulator h‐calponin. 2 Immunohistochemical and Western blot analyses indicated that CPI‐17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI‐17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI‐17 concentration of the phasic vas deferens. 3 In contrast to CPI‐17 expression patterns, phasic muscles contained more MLCP myosin‐targeting subunit than tonic tissues. Calponin expression was not statistically different. 4 Addition of phorbol ester to α‐toxin‐permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI‐17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several‐fold greater in femoral artery as compared to vas deferens. 5 We conclude that the expression ratio of CPI‐17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI‐17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI‐17 and high MLCP content. This implicates CPI‐17 inhibition of MLCP as an important component in modulating vascular muscle tone.

Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca2+ Sensitization in Rabbit Smooth Muscle

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr38 phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr695 and CPI‐17 Thr38 was examined along with MYPT1 Thr850, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI‐17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.

Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in Triton X-100-demembranated rabbit arterial smooth muscle

1 Triton X‐100‐demembranated smooth muscle loses Ca2+‐sensitizing responsiveness to protein kinase C (PKC) activators while intact and α‐toxin‐permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2 Western blot analyses showed that the content of the PKC α‐isoform (PKCα) was markedly reduced and that the smooth muscle‐specific protein phosphatase‐1 inhibitor protein CPI‐17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3 Unphosphorylated recombinant CPI‐17 alone induced a small but significant contraction at constant Ca2+. Isoform‐selective PKC inhibitors inhibited unphosphorylated but not pre‐thiophosphorylated CPI‐17‐induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI‐17. 4 Exogenously replenishing PKCα alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5 PKC in the presence of CPI‐17, but not the [T38A]‐CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI‐17 itself was phosphorylated. 6 In in vitro experiments, CPI‐17 was a much better substrate for PKCα than calponin, caldesmon, MLC and myosin. 7 Our results indicate that PKC requires CPI‐17 phosphorylation at Thr‐38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle.

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC20 phosphorylation and contraction.

Ca2+-Dependent Rapid Ca2+ Sensitization of Contraction in Arterial Smooth Muscle

Ca2+ ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca2+ with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca2+ transient, we compared Ca2+ signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during &agr;1 agonist–induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38±0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca2+ rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca2+ release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca2+-dependent protein kinase C. This was followed by a slow Ca2+-independent and Rho-kinase/protein kinase C–dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29±0.09 at rest to the peak of 0.68±0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca2+ sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca2+ rise induces a rapid inhibition of MLC phosphatase coincident with the Ca2+-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI‐17 and PHI‐1 by the MYPT1‐associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST‐rN‐ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI‐17 (but not PHI‐1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI‐17 were identified as Thr 38 and Ser 12 using Edman sequencing with 32P release and a point mutant of Thr 38.

Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors

The regulatory circuit controlling cellular protein phosphatase-1 (PP1), an abundant group of Ser/Thr phosphatases, involves phosphorylation of PP1-specific inhibitor proteins. Malfunctions of these inhibitor proteins have been linked to a variety of diseases, including cardiovascular disease and cancer. Upon phosphorylation at Thr38, the 17-kDa PP1 inhibitor protein, CPI-17, selectively inhibits a specific form of PP1, myosin light chain phosphatase, which transduces multiple kinase signals into the phosphorylation of myosin II and other proteins. Here, the mechanisms underlying PP1 inhibition and the kinase/PP1 cross-talk mediated by CPI-17 and its related proteins, PHI, KEPI, and GBPI, are discussed.

Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants

The purpose of this study was to determine the effects of inhibitors of Rho kinase (ROK) and matrix metalloproteinases (MMPs) on angiogenesis and tumor growth and to evaluate ROK activity in human prostate cancer PC3 cells and endothelial cells (HUVECs). Vacuolation by endothelial cells and lumen formation, the earliest detectable stages of angiogenesis, were inhibited by the ROK inhibitor Wf‐536. Combining Wf‐536 with the MMP inhibitor Marimastat greatly enhanced in vitro inhibition of endothelial vacuolation, lumen and cord formation, and VEGF‐ and HGF‐stimulated endothelial sprout formation from aorta. Inhibition of sprout formation by the two inhibitors was synergistic. Both agents inhibited migration of HUVECs. The regulatory subunit (MYPT1) of the myosin phosphatase was phosphorylated in PC3 cells and HUVECs, and phosphorylation of MYPT1 and the myosin regulatory light chain was reduced by Wf‐536, providing direct evidence of ROK activity. Early treatment of immuno‐incompetent mice bearing xenotrans‐plants of PC3 cells with a combination of Wf‐536 plus Marimastat with or without Paclitaxel, significantly inhibited tumor growth, prevented tumor growth escape after discontinuation of Paclitaxel, and increased survival.—Somlyo, A. V., Phelps, C., Dipierro, C., Eto, M., Read, P., Barrett, M., Gibson, J. J., Burnitz, M. C., Myers, C., Somlyo, A. P. Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants. FASEB J. 17, 223–234 (2003)

Cerebellar Long-Term Synaptic Depression Requires PKC-Mediated Activation of CPI-17, a Myosin/Moesin Phosphatase Inhibitor

Cerebellar LTD requires brief activation of PKC and is expressed as a functional downregulation of AMPA receptors. Modulation of vascular smooth-muscle contraction by G protein-coupled receptors (called Ca(2+) sensitization) also involves PKC phosphorylation and activation of a specific inhibitor of myosin/moesin phosphatase (MMP). This inhibitor, called CPI-17, is also expressed in brain. Here, we tested the hypothesis that LTD, like Ca(2+) sensitization, employs a PKC/CPI-17 cascade. Introduction of activated recombinant CPI-17 into cells produced a use-dependent attenuation of glutamate-evoked responses and occluded subsequent LTD. Moreover, the requirement for endogenous CPI-17 in LTD was demonstrated with neutralizing antibodies plus gene silencing by siRNA. These interventions had no effect on basal synaptic strength but blocked LTD induction. Thus, a biochemical circuit that involves PKC-mediated activation of CPI-17 modulates the distinct physiological processes of vascular contractility and cerebellar LTD.

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

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 (IC50 = 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 β-escin-permeabilized ileum SM at constant pCa 6.3 (EC50 = 2 μm), 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 Ca2+ sensitization of smooth muscle force.