Phosphorylation of the 20,000-Da myosin light chain isoforms of arterial smooth muscle by myosin light chain kinase and protein kinase C

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.4.c631 ◽

1990 ◽

Vol 259 (4) ◽

pp. C631-C639 ◽

Cited By ~ 47

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.

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Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)39445-0 ◽

1985 ◽

Vol 260 (15) ◽

pp. 8978-8983 ◽

Cited By ~ 20

Author(s):

M Nishikawa ◽

S Shirakawa ◽

R S Adelstein

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Comparative Study ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Smooth Muscle Myosin ◽

Kinase C ◽

Muscle Myosin

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Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42382-9 ◽

1994 ◽

Vol 269 (1) ◽

pp. 536-541 ◽

Cited By ~ 3

Author(s):

O.H. Choi ◽

R.S. Adelstein ◽

M.A. Beaven

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Light Chains ◽

Myosin Light Chains ◽

Kinase C

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The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible

Blood ◽

10.1182/blood.v83.12.3562.3562 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3562-3573 ◽

Cited By ~ 56

Author(s):

AD Michelson ◽

SE Benoit ◽

MH Kroll ◽

JM Li ◽

MJ Rohrer ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Surface Expression ◽

Platelet Surface ◽

Kinase C ◽

Von Willebrand ◽

Willebrand Factor

Abstract Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

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