Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide is one of a new class of activators for Ca2+-activated, phospholipid-dependent protein kinase

N-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) activated Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). SC-9 acted as a substitute for phosphatidylserine, which is one of the endogenous factors in activating protein kinase C. SC-9 was also effective in regulating the physiological functions at the whole-cell level. For example, SC-9 stimulated hexose transport activity in mouse fibroblasts, a protein kinase C-regulated cellular function. Thus, SC-9 may be useful to study the molecular basis of the regulation of protein kinase C activity, and the biological significance of this enzyme.     

The Ca2+ -activated protease (calpain) modulates Ca2+/calmodulin dependent activity of smooth muscle myosin light chain kinase

The Ca2+ -activated neutral protease can proteolyze both Ca2+ -dependent cyclic nucleotide phosphodiesterase and smooth muscle myosin light chain kinase. Ca2+ -dependent cyclic nucleotide phosphodiesterase from rat brain was converted to the Ca2+ -independent active form by Ca2+ -activated protease. The proteolytic effects on myosin light chain kinase of Ca2+-activated protease differed in the presence and absence of the Ca2+-calmodulin (CaM) complex. In the presence of bound CaM, myosin light chain kinase (130k dalton) was degradated to a major fragment of 62 kDa, which had Ca2+/CaM-dependent enzyme and CaM-binding activity. When digestion occurred in the absence of bound CaM, myosin light chain kinase cleaved to a fragment of 60 kDa. This peptide had no enzymatic activity in the presence or absence of the Ca2+-CaM complex. Available evidence suggests that the Ca2+-activated proteases may recognize the conformational change of smooth muscle myosin light chain kinase induced by Ca2+-CaM complex.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.     

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.     

Substrate specificity of rat brain calcium-activated and phospholipid-dependent protein kinase

A synthetic peptide ArgThrProProProSerGly with sequence similar to the threonine sites of phosphorylation in both myelin basic protein and simian virus 40 T antigen could be phosphorylated in vitro by a purified rat brain Ca2+-activated and phospholipid-dependent protein kinase, protein kinase C. The apparent Km and Vm values of this heptapeptide for the enzyme were determined to be 240 microM and 60 nmol/min/mg, respectively. Up to 0.8 mol 32P could be incorporated into the peptide, mainly at the threonine residue. Substitution of the L-threonine residue in the heptapeptide by its D-enantiomer abolished the phosphorylatability of the peptide by protein kinase C. However, this (D)Thr-containing peptide could act as a competitive inhibitor for the kinase with an apparent Ki value of approximately 320 microM. These findings suggest that a triprolyl sequence may act as a recognition site for protein kinase C.     

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Phosphorylation of p36 in vitro with pp60src. Regulation by Ca2+ and phospholipid

P36 is a major substrate of the tyrosine protein kinases. P36 isolated from bovine intestine was used in phosphorylation reactions with pp60src. Phosphorylation was stimulated 3-5-fold by Ca2+, however the Km was the same (2.5 microM) at high or low Ca2+. Although the level of free Ca2+ needed for this enhanced phosphorylation was 10(-4)-10(-3) M, phosphatidylserine shifted the Ca2+ sensitivity to the 10(-6)-10(-5) M range. Independent evidence suggested that p36 interacts directly with liposomes containing phosphatidylserine. This raises the possibility that p36, like c-kinase, is a Ca2+-activated, phospholipid-dependent protein.     

Conformational studies of myosin phosphorylated by protein kinase C

Smooth muscle myosin from chicken gizzard is phosphorylated by Ca2+-activated phospholipid-dependent protein kinase, protein kinase C, as well as by Ca2+/calmodulin-dependent kinase, myosin light chain kinase (Endo, T., Naka, M., and Hidaka, H. (1982) Biochem. Biophys. Res. Commun. 105, 942-948). We have now demonstrated the effect of phosphorylation by protein kinase C on the smooth muscle myosin molecule. In glycerol/urea polyacrylamide gel electrophoresis the 20,000-dalton light chain phosphorylated by protein kinase C co-migrated with that phosphorylated by myosin light chain kinase. Moreover, the light chain phosphorylated by both kinases migrated more rapidly than did the light chain phosphorylated by either myosin light chain kinase or protein kinase C alone. Myosin phosphorylated by protein kinase C formed a bent 10 S monomer while that phosphorylated by myosin light chain kinase was an unfolded and extended 6 S monomer in the presence of 0.2 M KCl. In addition, myosin phosphorylated by kinases had a sedimentation velocity of 7.3 S, thereby suggesting that the myosin was partially unfolded. The unfolded myosin was visualized electron microscopically. The fraction in the looped form was higher when for myosin phosphorylated by both kinases higher than for that phosphorylated by light chain kinase alone. Therefore, phosphorylation by protein kinase C does not lead to the change in myosin conformation seen with myosin light chain kinase.     

Dual calcium-dependent protein phosphorylation systems in pancreas and their differential regulation by polymyxin B1

Both phospholipid/calcium (PL/Ca2+) activated and calmodulin/Ca2+ (CaM/Ca2+)activated protein kinase systems were found in rat pancreatic extracts treated with Sephadex G-25. At least four substrate proteins for PL/Ca2+-activated kinase and one for a CaM/Ca2+-activated kinase were noted. Polymyxin B, an amphipathic antibiotic, was over 100-fold more potent as an inhibitor of PL/Ca2+-dependent protein phosphorylation than of the CaM/Ca2+-dependent system (Ki = app. 7 microM v. 950 microM). Fluphenazine inhibited both PL/Ca2+- and CaM/Ca2+-dependent protein kinases with equal potency, as did dibucaine. Inhibition by polymyxin B of PL/Ca2+-dependent phosphorylation could be overcome by increased amounts of phosphatidylserine. Low concentrations (10(-5)M) of polymyxin B completely inhibited carbachol-stimulated amylase release from intact pancreatic acini. These results indicate that polymyxin B may be useful in delineating the relative roles of PL/Ca2+-dependent and CaM/Ca2+-dependent protein phosphorylation in biological systems and suggest a potential role for the PL/Ca2+-activated kinase in regulation of pancreatic exocrine function.     

Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction.

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).     

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.     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Inhibition of phospholipid/Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine

CP-46,665-1, an antineoplastic lipoidal amine, was found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), with an IC50 (concentration causing a 50% inhibition) of 10 microM. Its inhibition of the enzyme was reversed by phosphatidylserine, but not by Ca2+. The agent also inhibited the enzyme activity which was further augmented by 12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein or diolein. Phosphorylation of endogenous proteins from HL-60 cells by the enzyme, with or without being further augmented by TPA, was inhibited by CP-46,665-1 as well as by alkyllysophospholipid (an antineoplastic agent). CP-46,665-1, while without effect on cyclic AMP-dependent protein kinase, also inhibited myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase). The present findings suggest that inhibition of the Ca2+-effector enzymes may be related in part to the antimetastatic activity of the lipoidal amine.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Purified rabbit brain protein kinase C relaxes skinned vascular smooth muscle and phosphorylates myosin light chain

To clarify the role of protein kinase C in the mechanical response, the effects of exogenous protein kinase C and its cofactors were investigated on skinned smooth muscle preparations of the rabbit mesenteric artery. Addition of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine (PS) caused slow inactivation of a maximal Ca2+ contraction of the muscle fiber and correspondingly increased protein kinase C phosphorylation of myosin light chain. Neither protein kinase C nor enzyme cofactors (PS and TPA) produced relaxation of this tissue and all three components caused significant relaxation. Furthermore, when the muscle fiber was activated by Ca2+-insensitive fragment of MLC-kinase, addition of protein kinase C with PS and TPA decreased the tension and increased protein kinase C phosphorylation of myosin light chain. This evidence suggests that protein kinase C phosphorylation of myosin light chain may play an inhibitory role in the contraction of vascular smooth muscle.