G protein-mediated Ca-sensitization of CPI-17 phosphorylation in arterial smooth muscle

CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain phosphatase in smooth muscle and plays an essential role in agonist-induced contraction. To elucidate the in situ mechanism for G protein-mediated Ca²⁺-sensitization of CPI-17 phosphorylation, α-toxin-permeabilized arterial smooth muscle strips were used to monitor both force development and CPI-17 phosphorylation in response to GTPγS with varying Ca²⁺ concentrations. CPI-17 phosphorylation increased at unphysiologically high Ca²⁺ levels of pCa ? 6. GTPγS markedly enhanced the Ca²⁺ sensitivity of CPI-17 steady-state phosphorylation but had no enhancing effect under Ca²⁺-free conditions, while the potent PKC activator PDBu increased CPI-17 phosphorylation regardless of Ca²⁺ concentration. CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A, a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with time even under Ca²⁺-free conditions. Furthermore, as Ca²⁺ concentration increased, so did CPI-17 phosphorylation rate. GTPγS markedly enhanced the rate of phosphorylation of CPI-17 at a given Ca²⁺. In the absence of calyculin A, either steady-state phosphorylation of CPI-17 under Ca²⁺-free conditions in the presence of GTPγS or at pCa 6.7 in the absence of GTPγS was negligible, suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion, cooperative increases in Ca²⁺ and G protein activation are required for a significant activation of total kinases that phosphorylate CPI-17, which together overcome CPI-17 phosphatase activity and effectively increase the Ca²⁺ sensitivity of CPI-17 phosphorylation and smooth muscle contraction.     

Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase

Phosphorylation of CPI-17 by Rho-associated kinase (Rho-kinase) and its effect on myosin phosphatase (MP) activity were investigated. CPI-17 was phosphorylated by Rho-kinase to 0.92 mol of P/mol of CPI-17 in vitro. The inhibitory phosphorylation site was Thr(38) (as reported previously) and was identified using a point mutant of CPI-17 and a phosphorylation state-specific antibody. Phosphorylation by Rho-kinase dramatically increased the inhibitory effect of CPI-17 on MP activity. Thus, CPI-17 as a substrate of Rho-kinase could be involved in the Ca(2+) sensitization of smooth muscle contraction as a downstream effector of Rho-kinase.     

Association of CPI-17 with protein kinase C and casein kinase I

The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.     

A functional interaction between CPI-17 and RACK1 proteins in bronchial smooth muscle cells

CPI-17 is a phosphorylation-dependent inhibitor of smooth muscle myosin light chain. Using yeast two-hybrid system, we have identified the receptor for activated C kinase 1 (RACK1) as a novel interaction partner of CPI-17. The direct interaction and co-localization of CPI-17 with RACK1 were confirmed by immunoprecipitation and confocal microscopy analysis, respectively. An in vitro assay system using recombinant/purified proteins revealed that the PKC-mediated phosphorylation of CPI-17 was augmented in the presence of RACK1. These results suggest that RACK1 may play a role in PKC/CPI-17 signaling pathway.     

蛋白激酶C对大鼠支气管平滑肌KV通道的影响

用全细胞膜片钳、Western印迹法和逆转录—PCR技术,观察蛋白激酶C(protein kinase C,PKC)对大鼠支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)电压依赖性延迟整流钾通道(Kv)活性及其亚型Kvl．5表达的影响。结果为：(1)PKC激活剂豆蔻酰佛波醇乙酯(phorbol 12-myristate 13-acetate,PMA)显著抑制急性分离大鼠BSMCs的Kv通道电流,该效应被PKC阻断剂Ro31—8220显著抑制;(2)PMA显著抑制体外培养大鼠BSMCs的Kvl．5 mRNA和蛋白质的表达,该效应被Ro31—8220显著抑制。上述观察结果提示,PKC活化可抑制大鼠BSMCs的Kv通道电流活性,下调Kvl．5亚型的表达水平。     

Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C

Smooth muscle caldesmon was phosphorylated by protein kinase C up to 1.90 mol P/mol caldesmon. Phosphorylated caldesmon was completely digested by trypsin and the produced phosphopeptides were purified by C-8 and C-18 reverse phase chromatography. Four phosphopeptides were determined and two phosphoserines were identified. Both were localized in the C-terminal domain at serine-587 and serine-726. By following the time course of phosphorylation, serine-587 was found to be the preferred site. Effects of the phosphorylation of caldesmon by protein C on the inhibition of acto-H-meromyosin ATPase activity was also examined. While unphosphorylated caldesmon inhibited the ATPase activity by 60%, phosphorylated caldesmon hardly inhibited the ATPase activity. Therefore, it was concluded that the phosphorylation at serine-726 and serine-587 reverses the inhibitory activity of caldesmon.     

Prolactin stimulation of protein kinase C activity in rat aortic smooth muscle

Prolactin (PRL) activated protein kinase C (PKC) in a dose dependent manner in rat aortic smooth muscle. Aortic strips incubated with sub-nanomolar concentrations of ovine PRL for 25 min. at 37 degrees C showed a significant stimulation of PKC activity in both cytosolic and particulate fractions. This activation could be blocked using either anti-PRL antibodies or 1-(5- isoquinolinesulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The results further support the role of PKC in the signal transduction pathway for PRL action and suggest that this activation may be involved in vascular smooth muscle function.     

Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation

Ca²⁺ sensitivity of smooth muscle (SM) contraction is determined by CPI-17, an inhibitor protein for myosin light chain phosphatase (MLCP). CPI-17 is highly expressed in mature SM cells, but the expression level varies under pathological conditions. Here, we determined the expression of CPI-17 in embryonic SM tissues and arterial neointimal lesions using immunohistochemistry. As seen in adult animals, the predominant expression of CPI-17 was detected at SM tissues on mouse embryonic sections, whereas MLCP was ubiquitously expressed. Compared with SM α-actin, CPI-17 expression doubled in arterial SM from embryonic day E10 to E14. Like SM α-actin and other SM marker proteins, CPI-17 was expressed in embryonic heart, and the expression was down-regulated at E17. In adult rat, CPI-17 expression level was reduced to 30% in the neointima of injured rat aorta, compared with the SM layers, whereas the expression of MLCP was unchanged in both regions. Unlike other SM proteins, CPI-17 was detected at non-SM organs in the mouse embryo, such as embryonic neurons and epithelium. Thus, CPI-17 expression is reversibly controlled in response to the phenotype transition of SM cells that restricts the signal to differentiated SM cells and particular cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution of active protein kinase C in smooth muscle.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.     

Modulation of smooth muscle calponin by protein kinase C and calmodulin

When smooth muscle calponin was incubated with protein kinase C, 1 mole of phosphate was incorporated per mole of calponin. The apparent Km value for calponin of the protein kinase was about 0.4 microM. The phosphorylation of calponin by protein kinase C was inhibited markedly by calmodulin in a calcium-dependent manner. Kinetic analysis of calmodulin-induced inhibition of calponin phosphorylation by protein kinase C revealed that calmodulin inhibited the phosphorylation in a noncompetitive fashion with calponin and the determined Ki value was 0.4 microM. These results suggest that interaction of calmodulin with calponin may play a regulatory role in the phosphorylation by protein kinase C and smooth muscle contraction.     

Intestinal inflammation downregulates smooth muscle CPI-17 through induction of TNF-alpha and causes motility disorders

Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1beta decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders by using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by proinflammatory cytokines. After the induction of acute ileitis with 2,4,6,-trinitrobenzensulfonic acid, CPI-17 expression declined in a time-dependent manner. This decrease in CPI-17 expression was parallel with the reduction of cholinergic agonist-induced contraction of smooth muscle strips and sensitivity of permeabilized smooth muscle fibers to Ca(2+). Among the various proinflammatory cytokines tested, TNF-alpha and IL-1beta were observed to directly inhibit CPI-17 expression and contraction in cultured rat intestinal tissue. Moreover, both TNF-alpha and IL-1beta inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1alpha/beta double-knockout mice. However, IL-1beta treatment failed to inhibit CPI-17 expression and contraction in TNF-alpha knockout mice. In beta-escin-permeabilized ileal tissues, pretreatment with anti-phosphorylated CPI-17 antibody inhibited the carbachol-induced Ca(2+) sensitization in the presence of GTP. These findings suggest that CPI-17 was downregulated during intestinal inflammation and that TNF-alpha plays a central role in this process. Downregulation of CPI-17 may play a role in motility impairments in inflammation.     

Purification and characterization of protein kinase C from rabbit iris smooth muscle. Myosin light-chain phosphorylation in vitro and in intact muscle.

Protein kinase C of rabbit iris smooth muscle was purified by the sequential use of three chromatographic steps, i.e. anion-exchange (DEAE-cellulose), gel filtration (Sephadex G-150) and substrate affinity (protamine-agarose), and its properties were investigated by using as substrate myosin light-chain protein (MLC) isolated from the same tissue. The enzyme appeared as a single band on SDS/polyacrylamide-gel electrophoresis, with a molecular mass of approx. 80 kDa. Histone H-1 and iris muscle MLC, but not rabbit skeletal-muscle MLC, were effective substrates for the enzyme, with apparent Km values of 3.0 and 16.6 microM respectively. The enzyme, with MLC as substrate, had the following characteristics. (a) Its activity was dependent on Ca2+ and phosphatidylserine (PS). In the presence of Ca2+ and PS, diolein and phorbol dibutyrate (PDBu) increased its activity by 61 and 65% respectively. Half-maximal activation of the enzyme (Ka) occurred at 10 microM free Ca2+, and in the presence of diolein and PDBu the apparent Ka for Ca2+ was decreased to 3 microM and 2 microM respectively. (b) Studies on the relative potency of various cofactors in activating the enzyme revealed that PS, phorbol myristate acetate and 1-stearoyl-2-arachidonylglycerol were the most potent of the phospholipids, phorbol esters and diacylglycerols respectively. (c) H-7, a protein kinase C inhibitor, inhibited MLC phosphorylation in a dose-dependent manner, with 50% inhibition at 10 microM. (d) Addition of carbamoylcholine (for 1 min) or PDBu (for 25 min) to iris sphincter muscle prelabelled with [32P]Pi specifically increased MLC phosphorylation, and only the stimulatory effect of the muscarinic agonist was blocked by atropine. The data provide additional support for a role for protein kinase C in the contractile response of the iris smooth muscle.     

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle

The contribution of protein kinase C to the contraction by oxytocin of rat uterine longitudinal smooth muscle in Ca(2+)-free solution was investigated. Immunological analysis revealed that type II (beta) and III (alpha) protein kinase C subspecies were present in rat uterine smooth muscle. The pretreatment of a diacylglycerol kinase inhibitor R59022 to accumulate diacylglycerol potentiated the Ca(2+)-independent contraction. The contractile activity was diminished with the depletion of protein kinase C, when the contraction was evoked repeatedly by oxytocin during the prolonged exposure to a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggested the involvement of protein kinase C in oxytocin-induced contraction in Ca(2+)-free solution.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Contraction-associated translocation of protein kinase C in rat skeletal muscle

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism.     

CPI-17-mediated contraction of vascular smooth muscle is essential for the development of hypertension in obese mice

Several factors have been implicated in obesity-related hypertension, but the genesis of the hypertension is largely unknown. In this study, we found a significantly upregulated expression of CPI-17(C-kinasepotentiated protein phosphatase 1 inhibitor of 17 kDa) and protein kinase C(PKC) isoforms in the vascular smooth muscles of high-fat diet(HFD)-fed obese mice. The obese wild-type mice showed a significant elevation of blood pressure and enhanced calcium-sensitized contraction of vascular smooth muscles. However, the obese CPI-17-deficient mice showed a normotensive blood pressure, and the calcium-sensitized contraction was consistently reduced. In addition, the mutant muscle displayed an abolished responsive force to a PKC activator and a 30%-50% reduction in both the initial peak force and sustained force in response to various G protein-coupled receptor(GPCR) agonists. Our observations showed that CPI-17-mediated calcium sensitization is mediated through a GPCR/PKC/CPI-17/MLCP/RLC signaling pathway. We therefore propose that the upregulation of CPI-17-mediated calcium-sensitized vasocontraction by obesity contributes to the development of obesity-related hypertension.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.