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.

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 Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697

2005 ◽  
Vol 389 (3) ◽  
pp. 763-774 ◽  
Author(s):  
David P. Wilson ◽  
Marija Susnjar ◽  
Enikő Kiss ◽  
Cindy Sutherland ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Thromboxane A2 ◽  
Myosin Light Chain Phosphatase ◽  
Kinase C

The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in α-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.

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 ◽  
Author(s):  
Lynn P. WEBER ◽  
Minoru SETO ◽  
Yasuharu SASAKI ◽  
Karl SWÄRD ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Arterial Smooth Muscle ◽  
Myosin Phosphorylation ◽  
Force Development ◽  
Kinase C ◽  
Rat Tail

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.

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