Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

1990 ◽  
Vol 259 (4) ◽  
pp. C631-C639 ◽  
Author(s):  
H. A. Singer
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Activation Mechanism ◽  
Arterial Smooth Muscle ◽  
Maximal Force ◽  
Kinase C ◽  
Mlc Phosphorylation

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.

scholarly journals The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle

2000 ◽  
Vol 352 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Lynn P. WEBER ◽  
Minoru SETO ◽  
Yasuharu SASAKI ◽  
Karl SWÄRD ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphatase ◽  
Arterial Smooth Muscle ◽  
Kinase C ◽  
Detergent Treatment ◽  
Rat Tail

Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20kDa light chains of myosin (LC20). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC20 phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC20 phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC20 phosphorylation in intact RTA is mediated by MLCK; (ii) α1-adrenoceptor stimulation increases LC20 phosphorylation via MLCK and PKC; (iii) Ca2+-induced LC20 phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs α and β following detergent treatment; (iv) very little LC20 diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC20 phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension–LC20 phosphorylation relationship is markedly different between the two preparations such that the basal level of LC20 phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.

scholarly journals A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase.

1994 ◽  
Vol 104 (2) ◽  
pp. 265-286 ◽  
Author(s):  
M Masuo ◽  
S Reardon ◽  
M Ikebe ◽  
T Kitazawa
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Force Development ◽  
Kinase C ◽  
Sensitizing Effect ◽  
Mlc Phosphorylation ◽  
Gamma S

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.

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

1988 ◽  
Vol 255 (2) ◽  
pp. 423-429 ◽  
Author(s):  
P H Howe ◽  
A A Abdel-Latif
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscarinic Agonist ◽  
Sphincter Muscle ◽  
Kinase C ◽  
Mlc Phosphorylation ◽  
Rabbit Iris

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.

Inhibition of myosin light chain phosphatase during Ca(2+)-independent vasocontraction

1993 ◽  
Vol 265 (5) ◽  
pp. C1319-C1324 ◽  
Author(s):  
H. Itoh ◽  
A. Shimomura ◽  
S. Okubo ◽  
K. Ichikawa ◽  
M. Ito ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Water Soluble ◽  
Two Dimensional ◽  
Kinase C ◽  
Rat Thoracic Aorta ◽  
Phosphopeptide Mapping

Phorbol 12,13-dibutyrate (PDB) induced a sustained contraction of rat thoracic aorta strip in Ca(2+)-free buffer without significant change in intracellular free Ca2+ concentration. NKH477, a water-soluble forskolin derivative, markedly relaxed the PDB-induced contraction. The PDB-induced contraction was associated with the phosphorylation of 20-kDa myosin light chain (MLC). Two-dimensional phosphopeptide mapping of 20-kDa MLC revealed that approximately 90% of the phosphopeptides was derived from an MLC kinase-catalyzed reaction and approximately 10% was due to phosphorylation by protein kinase C. NKH477 inhibited the PDB-induced phosphorylation of 20-kDa MLC. MLC phosphatase activity of intact aorta strips was inhibited by the treatment with PDB, and the inhibition was recovered by the application of NKH477. These results suggest that the regulation of MLC phosphatase in vascular smooth muscle may play important roles in the PDB-induced contraction and the NKH477-induced relaxation in Ca(2+)-free buffer.

Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle

2001 ◽  
Vol 281 (2) ◽  
pp. G467-G478 ◽  
Author(s):  
U. D. Sohn ◽  
Weibiao Cao ◽  
Da-Chun Tang ◽  
J. T. Stull ◽  
J. R. Haeberle ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Circular Muscle ◽  
Kinase C ◽  
Pkc Inhibitors ◽  
Esophageal Sphincter ◽  
Mlc Phosphorylation

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

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