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PubMed1992

Phosphorylation by casein kinase II alters the biological activity of calmodulin.

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1992

Chronic low frequency stimulation reduces myosin phosphorylation in rabbit fast twitch muscle.

The effect of 1-12 days of electrical stimulation (10 Hz) on the ability to phosphorylate the P-light chain of myosin was studied in rabbit tibialis anterior muscle. Myosin phosphorylation was induced by exposure of the stimulated muscle and that of the contralateral leg to a single conditioning stimulus train (5 Hz) for 25 s via the motor nerve. Isometric tension was measured as were the myosin light chain composition and the activities of the enzymes responsible for phosphorylation and dephosphorylation. A computer simulation of the potential effect of a stimulation-induced disruption of Ca2+ metabolism on phosphorylation was also performed. Chronic stimulation for as little as 1 day eliminated light chain phosphorylation and reduced the myosin light chain kinase activity by approximately 36%. Conversely, phosphatase activity and light chain composition were unaffected. The model demonstrated that a slight depression in the magnitude of the Ca2+ transient could potentially attenuate phosphorylation. The data suggest that phosphorylation of myosin is extremely sensitive to prolonged muscle activity. Furthermore, it appears more likely that this sensitivity is related to regulation of intracellular free Ca2+ than to the other elements of the calmodulin-dependent system for myosin phosphorylation examined.

Animals↗

PubMed1992

Modulation of neutrophil superoxide generation by inhibitors of protein kinase C, calmodulin, diacylglycerol and myosin light chain kinases, and peptidyl prolyl cis-trans isomerase.

To assess the role of protein kinase C (PKC) in the respiratory burst of adherent human polymorphonuclear leukocytes (PMNL), reduction of ferricytochrome C by cells triggered with a phorbol ester (PMA), ionophore A23187, serum-treated zymosan (STZ) or three lipid derivatives, 3-decanoyl-sn-glycerol (G-3-OCOC9), (R,R)-1,4-diethyl-2-O-decyl-L-tartrate (Tt-2-OC10) and 3-decyloxy-5-hydroxymethylphenol (DHP) was examined in a microtiter plate procedure in the presence of inhibitors of PKC and, for comparison, inhibitors of calmodulin, diacylglycerol and myosin light chain kinases and the peptidyl-prolyl cis-trans isomerase activity of fujiphilin. 1) Of the protein kinase inhibitors examined, Ro 31-7549 and staurosporine reduced responses to all stimuli except possibly STZ; in contrast, K252a and the myosin light chain kinase inhibitors ML-7 and ML-9 blocked responses to A23187 and STZ better than those triggered by PMA. H-7 reduced responses to A23187, DHP and G-3-OCOC9, and calphostin, palmitoyl carnitine, sphingosine and the multifunctional drugs TMB-8 and W-7 reduced A23187; they also, when examined, reduced decane derivative-induced O2- production more effectively than PMA- and STZ-triggered responses. Polymyxin B, 4 alpha-PMA and retinal displayed no inhibitory capacity. 2) Of the selective calmodulin antagonists, CGS 9343B, Ro 22-4839 and calmidazolium did not inhibit the oxidative response irrespective of the stimulus used, whereas metofenazate reduced those evoked by A23187, DHP, G-3-OCOC9 and STZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗

PubMed1992

Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer.

We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and the in vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (alpha-myosin heavy chain) and low (beta-myosin heavy chain) enzymatic activity. Enzymatic activity of alpha- and beta-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the alpha-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the beta-myosin heavy chain isoenzyme. Summer animals expressed predominantly the beta-myosin heavy chain (79% of total myosin) while during hibernation the alpha-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22 degrees C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. The in vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.

Animals↗

PubMed1992

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.

Animals↗

PubMed1992

Cytokinesis.

The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.

Actomyosin↗

PubMed1992

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.

Animals↗

PubMed1992

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.

Amino Acid Sequence↗

PubMed1992

Relaxant effect of phalloidin on Triton-skinned microvascular and other smooth muscle preparations.

Guinea pig mesenteric microarteries (diameter 60-100 microns), the main branch of the mesenteric artery and taenia coli were skinned with 1% Triton X-100 for 4 h at 4 degrees C. Microarteries, mounted for circumferential force measurement, developed maximal active force in response to elevation of the free Ca2+ (pCa = 4.52, in EGTA buffer) in the presence of ATP (7.5 mM) and calmodulin (0.1-0.3 microM). In these preparations, addition of phalloidin (1-100 microM) slowly (greater than 1 h) relaxed submaximal contractions (pCa greater than 4.52) in a dose-dependent manner. Relaxation was irreversible as, after phalloidin wash-out, subsequent active force to pCa = 4.52 was also reduced. By contrast, phalloidin preincubation and wash-out under relaxed conditions (pCa greater than 8) only reduced subsequent force to pCa = 4.52 on prolonged stimulation. The extent of phalloidin-induced relaxation was not dependent on free Ca2+ between pCa 6.40 and 4.52. Phalloidin-induced relaxation did not occur during rigor contractions (i.e. absence of ATP and Ca2+). These mechanical effects of phalloidin were accompanied by a decreased leak of actin out of the skinned preparations and by the prevention of guanidine extraction of actin from these preparations. Phalloidin did not inhibit the myosin light chain kinase or phosphatase activity isolated from these preparations. In addition, the relaxant effects were also noted in taenia coli and the main branch of the mesenteric artery but not in skinned porcine ventricular heart muscle. These experiments suggest the possible participation of actin filament dynamics on the maintenance of active force in Triton-skinned smooth muscle.

Animals↗

PubMed1992

Inhibition of protein kinase C by annexin V.

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.

Annexin A5↗

PubMed1992

Myosin light chain kinase is expressed in neurons and glia: immunoblotting and immunocytochemical studies.

The contractile protein myosin is thought to subserve motility-related functions in a wide range of eukaryotic non-muscle cells including both neurons and glia. To determine if the Ca2+/calmodulin-dependent enzyme, myosin light chain kinase (MLCK) is involved in the regulation of neural myosin we investigated the presence and localization of MLCK in a variety of neural tissues by immunoblotting and immunocytochemistry. A specific immunoreactive protein (M(r) = 146,000) was detected in blotted homogenates from many regions of rat brain and from primary cultures of either astrocytes or cerebellar granule cells grown in the absence of other cell types. At the light microscopic level, MLCK-immunoreactivity was evident in many regions of rat brain, as well as in the cultured astrocytes and cerebellar granule cells. MLCK-immunoreactivity was observed to be largely cytosolic in astrocytes but with a proportion associated with the cytoskeleton. In the cerebellar granule cells immunoreactivity was present in neuronal processes as well as somata. The detection of MLCK in neural cells suggests that MLCK-catalyzed myosin phosphorylation may couple changes in intracellular calcium concentrations to motility-related functions of neurons and glia.

Animals↗

PubMed2026

Phytolacca acinosa Roxb. induces intestinal toxicity through the histamine-MLCK-tight junction axis: Integrated evidence from proteomics, metabolomics, intestinal organoids and epithelial barrier validation.

Phytolacca acinosa Roxb. (PR) is a saponin-rich medicinal plant associated with gastrointestinal toxicity, but the mechanisms underlying PR-induced intestinal barrier injury remain unclear. In this study, raw PR extract was analytically characterized by UPLC-ZenoTOF-MS/MS, confirming triterpenoid saponins as the predominant constituents. C57BL/6 J mice were orally exposed to characterized PR extract (1.20 or 12.0 g/kg for 5 h), and Caco-2 cells and mouse intestinal organoids were used to assess epithelial toxicity and barrier disruption. Histopathology, ELISA, FITC-dextran permeability assays, immunofluorescence, CCK-8, LDH release, western blotting, DIA-based proteomics and untargeted metabolomics were integrated to define toxicological mechanisms. PR induced dose-dependent intestinal inflammation and barrier dysfunction, with the ileum as the most sensitive target. PR increased serum DAO and D-lactate and intestinal TNF-α and IL-1β, disrupted organoid morphology, enhanced epithelial permeability, and reduced ZO-1 expression. Proteomics revealed changes in inflammatory, lipid-metabolic, cytoskeletal and tight-junction pathways, including upregulation of MLCK3 and phospholipase-related proteins and downregulation of ZO-1 and ZO-2. Metabolomics identified histidine metabolism disturbance and histamine accumulation. Integrated multi-omics and pharmacological validation indicated that histamine activated the PLC/IP₃/Ca²⁺/CaM/MLCK cascade, promoting MLC phosphorylation, tight-junction disassembly and epithelial leakiness. MLCK inhibition partially restored ZO-1/ZO-2 expression and attenuated PR-induced epithelial injury. These findings identify the histamine-MLCK-tight junction axis as a key mechanism of PR-induced intestinal toxicity and support hazard identification of saponin-rich PR exposure.

Animals↗

PubMed1992

Astrocyte process growth induction by actin breakdown.

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.

Actin Cytoskeleton↗

PubMed1992

GTP gamma S-dependent regulation of smooth muscle contractile elements.

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increases the sensitivity of the contractile response to activation by Ca2+ in permeabilized tracheal smooth muscle. Increased tension was associated with a proportional increase in myosin light chain phosphorylation. The site of phosphorylation was determined to be serine-19, which corresponds to the site rapidly phosphorylated by myosin light chain kinase. GTP gamma S did not affect the contraction induced by the protein phosphatase inhibitor okadaic acid but did enhance contraction produced by Ca(2+)-independent myosin light chain kinase. In tracheal homogenates Ca(2+)-dependent myosin light chain kinase activity was not affected by GTP gamma S; however, dephosphorylation of 32P-labeled heavy meromyosin by phosphatase was inhibited. Thus GTP gamma S may increase the Ca2+ sensitivity of contractile elements in tracheal smooth muscle by inhibition of protein phosphatase activity toward myosin light chain.

Animals↗

PubMed1992

An axial gradient of transgene methylation in murine skeletal muscle: genomic imprint of rostrocaudal position.

We previously used mice bearing a myosin light chain-chloramphenicol acetyltransferase (MLC1-CAT) transgene to show that adult muscle cells bear a heritable, cell autonomous memory of their rostrocaudal position. CAT mRNA and protein are expressed in a > 100-fold rostrocaudal gradient in skeletal muscles of developing and adult MLC1-CAT mice (Donoghue, M. J., Merlie, J. P., Rosenthal, N. and Sanes, J. R. (1991). Proc. Natl. Acad. Sci. USA 88, 5847-5851; Donoghue, M. J., Alvarez, J. D., Merlie, J. P. and Sanes, J. R. (1991). J. Cell Biol. 115, 423-434). Moreover, both in primary cultures and in myogenic cell lines prepared from individual muscles of these mice, CAT levels reflect the body position from which the myoblasts were derived (Donoghue, M.J., Morris-Valero, R., Johnson, Y.R., Merlie, J.P. and Sanes, J. R. (1992). Cell 69, 67-77). Here, we show that the methylation state of the MLC1-CAT transgene in skeletal muscles is also graded along the rostrocaudal axis: methylation levels decrease and expression levels increase in the order, jaw-->neck-->chest and forelimb-->hindlimb. Methylation levels are also approx. 10-fold higher in rostrally derived than in caudally derived myogenic cell lines, which express low and high levels of CAT, respectively. Within each cell line, undifferentiated cells (myoblasts), which do not express the transgene, and differentiated cells (myotubes), which do, are indistinguishable in methylation state. Thus, differentiation-related changes in transgene expression do not affect position-related levels of transgene methylation. On the other hand, treatment of rostrally derived lines with the demethylating agent, 5-azacytidine, decreases methylation and increases expression of the transgene. Thus, perturbation of methylation affects expression. Taken together, these results suggest that methylation provides a genomic imprint of rostrocaudal body position that may serve as a component of the positional memory that mammalian cells retain into adulthood.

Animals↗

PubMed1992

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

Arachidonic acid (AA) increased, at constant Ca2+, the levels of force and 20-kDa myosin light chain (MLC20) phosphorylation in permeabilized smooth muscle, and slowed relaxation and MLC20 dephosphorylation. The Ca(2+)-sensitizing effect of AA was not inhibited by inhibitors of AA metabolism (indomethacin, nordihydroguaiaretic acid, or propyl gallate), of protein kinase C (pseudopeptide) or by guanosine-5'-O-(beta-thiodiphosphate) and was abolished by oxidation of AA in air. A non-metabolizable AA analog, 5,8,11,14-eicosatetraynoic acid) also had Ca(2+)-sensitizing effects. Extensive treatment with saponin abolished the Ca(2+)-sensitizing effects of phorbol 12,13-dibutyrate and guanosine-5'-O-(gamma-thiotriphosphate), but not that of AA. A purified, oligomeric MLC20 phosphatase isolated from gizzard smooth muscle was dissociated into subunits by AA, and its activity was inhibited toward heavy meromyosin but not phosphorylase. We conclude that AA may act as a messenger-promoting protein phosphorylation through direct inhibition of the form of protein phosphatase(s) that dephosphorylate MLC20 in vivo.

Animals↗

PubMed1992

Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle.

Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.

Animals↗

PubMed1992

Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function.

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.

Actins↗