Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

Structural and functional characterization of calponin fragments

Calponin from chicken gizzard consists of two principal components, possibly isoforms, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cleavage with 2-nitro-5-thiocyanobenzoic acid indicated that calponin contains 2 cysteine residues. Purified fragments of 30 and 21 kDa retained the following properties of the intact protein: actin-, tropomyosin- and calmodulin-binding, and ability to inhibit the actin-activated MgATPase activity of smooth muscle myosin. Both fragments, like intact calponin, were phosphorylated by protein kinase C which inhibited their binding to actin and relieved their inhibition of the ATPase. Tryptic digestion of calponin phosphorylated by protein kinase C generated 3 phosphopeptides with the following N-terminal sequences: FASQQGMTAYGTR, GASQQGMTVYGLP, and NHSGHVQ, each possessing a single phosphoserine.     

Regulation of the actin-activated ATPase of aorta smooth muscle myosin

Phosphorylation of the 20,000-Da light chains, LC20, of vertebrate smooth muscle myosins is thought to be the primary mechanism for regulating the actin-activated ATPase activities of these myosins and consequently smooth muscle contraction. While actin stimulates the MgATPase activities of phosphorylated smooth muscle myosins, it is generally believed that the MgATPase activities of the unphosphorylated myosins are not stimulated by actin. However, under conditions where both unphosphorylated (5% phosphorylated LC20) and phosphorylated calf aorta myosins are mostly filamentous, the maximum rate, Vmax, of the actin-activated ATPase of the unphosphorylated myosin is one-half that of the phosphorylated myosin. While LC20 phosphorylation causes only a modest increase in Vmax, in the presence of tropomyosin, this phosphorylation does cause up to a 10-fold decrease in Kapp, the actin concentration required to achieve 1/2 Vmax. In the presence of low concentrations of tropomyosin/actin, a linear relationship is obtained between the fraction of LC20 phosphorylated and stimulation of the actin-activated ATPase. The relatively high actin-activated ATPase activity of unphosphorylated aorta myosin suggests that other proteins may be involved in the regulation of smooth muscle contraction. In contrast to the results presented here for aorta myosin, it has been reported that actin does not activate the MgATPase activity of unphosphorylated gizzard myosin and that the actin-activated ATPase of gizzard myosin increases more slowly than LC20 phosphorylation.     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.     

Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon

Caldesmon, a major calmodulin- and actin-binding protein of smooth muscle (Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5652-5655), has been obtained in highly purified form from chicken gizzard by a modification of a previously published procedure (Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870) and was found to cause a significant inhibition of both superprecipitation and actin-activated myosin Mg2+-ATPase activity in a system reconstituted from the purified contractile and regulatory proteins without influencing the phosphorylation state of myosin. This inhibitory effect was seen both in the presence and absence of tropomyosin. A Ca2+-and calmodulin-dependent kinase which catalyzed phosphorylation of caldesmon was identified in chicken gizzard; this kinase is distinct from myosin light-chain kinase. Caldesmon prepared by calmodulin-Sepharose affinity chromatography was contaminated with caldesmon kinase activity and was unable to inhibit actomyosin ATPase activity or superprecipitation. Phosphatase activity capable of dephosphorylating caldesmon was also identified in smooth muscle. These results indicate that caldesmon can inhibit smooth muscle actomyosin ATPase activity in vitro, and this function may itself be subject to regulation by reversible phosphorylation of caldesmon.     

Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Regulation of embryonic smooth muscle myosin by protein kinase C

Phosphorylation of the 20-kDa light chain regulates adult smooth muscle myosin; phosphorylation by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase stimulates the actomyosin ATPase activity of adult smooth muscle myosin; the simultaneous phosphorylation of a separate site on the 20-kDa light chain by the Ca2+/phospholipid-dependent enzyme protein kinase C attenuates the myosin light chain kinase-induced increase in the actomyosin ATPase activity of adult myosin. Fetal smooth muscle myosin, purified from 12-day-old fertilized chicken eggs, is structurally different from adult smooth muscle myosin. Nevertheless, phosphorylation of a single site on the 20-kDa light chain of fetal myosin by myosin light chain kinase results in stimulation of the actomyosin ATPase activity of this myosin. Protein kinase C, in contrast, phosphorylates three sites on the fetal myosin 20-kDa light chain including a serine or threonine residue on the same peptide phosphorylated by myosin light chain kinase. Interestingly, phosphorylation by protein kinase C stimulates the actomyosin ATPase activity of fetal myosin. Moreover, unlike adult myosin, there is no attenuation of the actomyosin ATPase activity when fetal myosin is simultaneously phosphorylated by myosin light chain kinase and protein kinase C. These data demonstrate, for the first time, the in vitro activation of a smooth muscle myosin by another enzyme besides myosin light chain kinase and raise the possibility of alternate pathways for regulating smooth muscle myosin in vivo.     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.     

Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin

Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed.     

Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation

The inhibition of myosin phosphatase evokes smooth muscle contraction in the absence of Ca(2+), yet the underlying mechanisms are not understood. To this end, we have cloned smooth muscle zipper-interacting protein (ZIP) kinase cDNA. ZIP kinase is present in various smooth muscle tissues including arteries. Triton X-100 skinning did not diminish ZIP kinase content, suggesting that ZIP kinase associates with the filamentous component in smooth muscle. Smooth muscle ZIP kinase phosphorylated smooth muscle myosin as well as the isolated 20-kDa myosin light chain in a Ca(2+)/calmodulin-independent manner. ZIP kinase phosphorylated myosin light chain at both Ser(19) and Thr(18) residues with the same rate constant. The actin-activated ATPase activity of myosin increased significantly following ZIP kinase-induced phosphorylation. Introduction of ZIP kinase into Triton X-100-permeabilized rabbit mesenteric artery provoked a Ca(2+)-free contraction. A protein phosphatase inhibitor, microcystin LR, also induced contraction in the absence of Ca(2+), which was accompanied by an increase in both mono- and diphosphorylation of myosin light chain. The observed sensitivity of the microcystin-induced contraction to various protein kinase inhibitors was identical to the sensitivity of isolated ZIP kinase to these inhibitors. These results suggest that ZIP kinase is responsible for Ca(2+) independent myosin phosphorylation and contraction in smooth muscle.     

Phosphorylation of bovine platelet myosin by protein kinase C

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.     

Protein kinase C–catalyzed calponin phosphorylation in swine carotid arterial homogenate

Calponin, a thin filament–associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca²⁺/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca²⁺-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca²⁺- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol–dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca²⁺, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca²⁺/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of ³²P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation. J. Cell. Physiol. 176:545–552, 1998. © 1998 Wiley-Liss, Inc.     

Suppression of superprecipitation of contractile proteins from chicken gizzard muscle by preincubation in the presence of adenosine triphosphate without Ca2+

Superprecipitation of reconstituted actomyosin composed of smooth muscle myosin, skeletal muscle actin and smooth muscle native tropomyosin was studied. When the actomyosin solution was preincubated in the presence of ATP and the absence of Ca2+, or in the relaxed state, superprecipitation was markedly suppressed. The extent of suppression was correlated with the inhibition of the phosphorylation of the 20,000-dalton light chain of smooth muscle myosin. This is consistent with the theory that the interaction of smooth muscle actomyosin is regulated by the phosphorylation of myosin light chain through a system of myosin light chain kinase and phosphatase. However, further studies showed that the myosin light chain kinase and phosphatase system could not explain the present suppression of superprecipitation, even if a cyclic AMP-dependent protein kinase system was also involved. A new regulatory factor should be taken into account in the regulation of smooth muscle actomyosin interaction.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct association and translocation of PKC-alpha with calponin

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.