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Biomedical subjects

Masumi Eto

Publications and source records attributed to Masumi Eto.

At least 19 recordsLinked to original sources
PubMed2007

Assay for three-way interaction of protein phosphatase-1 (Glc7) with regulatory subunits plus phosphatase inhibitor-2.

A method is described using yeast conjugation to assay the interactions of a protein phosphatase-1 (PP1) inhibitor protein with holoenzymes formed in situ by expression of regulatory subunit fusion proteins that recruit endogenous Glc7, the yeast ortholog of PP1. Mutations in the canonical recognition motif VxF used to bind PP1 (Glc7) allow for analysis of direct from indirect (three-way) interactions.

Binding Sites↗
PubMed2006

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle.

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) 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 Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(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 Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-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 Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.

Adrenergic alpha-Agonists↗
PubMed2006

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells.

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+ -dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+ -channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+ -induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+ -influx or CPI-17-MLCP signaling is downregulated.

Angiotensin II↗
PubMed2005

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells.

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-delta, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Assembly of MYPT1 with protein phosphatase-1 in fibroblasts redirects localization and reorganizes the actin cytoskeleton.

Dephosphorylation of actin-binding proteins by a specialized form of protein Ser/Thr phosphatase type-1 (PP1) regulates smooth muscle contraction and morphology and motility of nonmuscle cells. This myosin and ezrin/radixin/moesin (ERM)-targeted phosphatase comprises the delta isoform PP1 catalytic subunit plus a primary regulatory subunit called myosin phosphatase targeting (MYPT1). We reconstructed myosin/ERM phosphatase in living rat embryo fibroblasts (REF52 cells) by transient expression of epitope-tagged MYPT1 (myc-MYPT1) plus HA-tagged PP1. Unexpectedly, wild-type myc-MYPT1 expressed alone accumulated predominantly in the nucleus, as visualized by immunofluorescent microscopy, whereas if coexpressed with HA-PP1, it was localized in the cytosol and deposited on cytoskeleton myofilaments. The F38A mutation of MYPT1 that eliminates PP1 binding gave nuclear localization of myc-MYPT1, even when coexpressed with HA-PP1. Thus, expression of both subunits was necessary to form myosin/ERM phosphatase in situ and mediate myofilament localization. The results indicate there is little endogenous PP1 available for interaction or interchange with ectopic regulatory subunits in living cells. We concluded that myosin binding by the C-terminal domain of MYPT1 is not sufficient to override nuclear import in fibroblasts, but the binding of PP1 to myc-MYPT1 neutralizes nuclear import. Full-length myc-MYPT1 plus HA-PP1 induced only subtle changes in organization of the actin cytoskeleton, however coexpression of myc-MYPT1(1-300) with HA-PP1 dispersed stress fibers without major alteration in morphology and myc-MYPT1(1-498) disrupted the cytoskeleton and produced radically extended cells that appeared like neurons. Based on these responses, we conclude that the MYPT1 C-terminus functions as an auto-inhibitory domain, and a central domain in MYPT1 can mediate extensive reorganization of the actin cytoskeleton.

Actins↗
PubMed2005

Phospho-pivot modeling predicts specific interactions of protein phosphatase-1 with a phospho-inhibitor protein CPI-17.

Phospho-amino acids in proteins are directly associated with phospho-receptor proteins, including protein phosphatases. Here we produced and tested a scheme for docking together interacting phospho-proteins whose monomeric 3D structures were known. The phosphate of calyculin A, an inhibitor for protein phosphatase-1 and 2A (PP1 and PP2A), or phospho-CPI-17, a PP1-specific inhibitor protein, was docked at the active site of PP1. First, a library of 186,624 virtual complexes was generated in silico, by pivoting the phospho-ligand at the phosphorus atom by step every 5 degrees on three rotational axes. These models were then graded for probability according to atomic proximity between two molecules. The predicted structure of PP1 x calyculin A complex fitted to the crystal structure with r.m.s.d. of 0.23 A, providing a validate test of the modeling method. Modeling of PP1 x phospho-CPI-17 complex yielded one converged structure. The segment of CPI-17 around phospho-Thr38 is predicted to fit in the active site of PP1. Positive charges at Arg33/36 of CPI-17 are in close proximity to Glu274 of PP1, where the sequence is unique among Ser/Thr phosphatases. Single mutations of these residues in PP1 reduced the affinity against phospho-CPI-17. Thus, the interface of the PP1 x CPI-17 complex predicted by the phospho-pivot modeling accounts for the specificity of CPI-17 against PP1.

Animals↗
PubMed2004

Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17.

Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2004

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits.

Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1.PP1C.P-CPI-17, leading to an increase in smooth muscle contraction.

Allosteric Regulation↗
PubMed2004

CPI-17-deficient smooth muscle of chicken.

Ca(2+) sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI-17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist-induced contractile Ca(2+) sensitization in mammalian artery. CPI-17 has not been detected in chicken artery, but is readily detectable in pigeon artery. To evaluate a role of CPI-17, we compared contractility of the arteries of 'CPI-17-deficient' chicken with those of CPI-17-rich rabbit and pigeon, and studied the effect of CPI-17-reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca(2+) sensitization are expressed in both chicken and rabbit arteries. Agonists, such as an alpha(1)-agonist and endothelin-1, produced significant contraction in arteries of all species under physiological Ca(2+)-containing conditions. Depletion of Ca(2+) abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI-17-rich tissues, chicken arteries exerted little Ca(2+) sensitization in response to alpha(1)-agonist or endothelin-1. GTPgammaS produced a slight Ca(2+) sensitizing effect in chicken artery, but this was significantly smaller compared with CPI-17-rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and alpha-toxin-permeabilized chicken artery in contrast to a large contraction in CPI-17-rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI-17 into beta-escin-permeabilized chicken artery restored PDBu-induced and enhanced GTPgammaS-induced Ca(2+) sensitization. Thus, CPI-17 is essential for G protein/PKC-mediated Ca(2+) sensitization in smooth muscle.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases.

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.

Animals↗
PubMed2003

Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue.

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.

Amino Acid Substitution↗
PubMed2003

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 Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr(695) of the MLCP regulatory subunit (MYPT1) and at Thr(38) of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr(38) 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 Thr(695) and CPI-17 Thr(38) was examined along with MYPT1 Thr(850), which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr(38) and MYPT1 Thr(850) were phosphorylated in response to agonists or GTPgammaS concurrently with contraction and myosin phosphorylation in alpha-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr(695) 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 Thr(850) and CPI-17 Thr(38), respectively, in intact VD while MYPT1 Thr(695) phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr(695) is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr(38) may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca(2+) sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.

Amides↗
PubMed2003

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 xenotransplants 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.

Amides↗
PubMed2002

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.

Animals↗
PubMed2002

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

Integrin-linked kinase (ILK) has been implicated in Ca(2+)- 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 17 kDa] 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 Thr(38) of CPI-17 and Thr(57) 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. Thr(38) of CPI-17 or Thr(57) of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr(38) or Thr(57) respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala, 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 Ca(2+) sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala treated with ILK in the presence of adenosine 5'-[gamma-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.

Animals↗