Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli

2010 ◽  
Vol 429 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Olga V. Shcherbakova ◽  
Daria V. Serebryanaya ◽  
Alexander B. Postnikov ◽  
Mechthild M. Schroeter ◽  
Stefan Zittrich ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Taenia Coli ◽  
Related Protein ◽  
Smooth Muscle Contractility ◽  
Regulatory Myosin Light Chain

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC.

Calcium-dependent mechanisms of regulation of smooth muscle contraction

1991 ◽  
Vol 69 (12) ◽  
pp. 771-800 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Smooth Muscle Contraction ◽  
Light Chains ◽  
Contractile State ◽  
Calmodulin Binding

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, α-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 Ca2+-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca2+-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 Ca2+-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 Ca2+- and phospholipid-dependent protein kinase (protein kinase C).Key words: smooth muscle, Ca2+, myosin phosphorylation, regulation of contraction.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

scholarly journals Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs

2002 ◽  
Vol 93 (4) ◽  
pp. 1296-1300 ◽  
Author(s):  
Debra J. Turner ◽  
Peter B. Noble ◽  
Matthew P. Lucas ◽  
Howard W. Mitchell
Keyword(s):  
Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Porcine Model ◽  
Smooth Muscle Contraction ◽  
Airway Wall ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Airway Narrowing

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0–20 cmH2O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls ( P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi ( P < 0.01) and smooth muscle strips ( P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.

Calmodulin-dependent myosin light chain kinases from cardiac and smooth muscle: a comparative study

1980 ◽  
Vol 58 (4) ◽  
pp. 299-308 ◽  
Author(s):  
Michael P. Walsh ◽  
Jean-Claude Cavadore ◽  
Bernard Vallet ◽  
Jacques G. Demaille
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Proteolytic Fragment ◽  
Cardiac Enzyme ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Various properties of cardiac and smooth muscle calmodulin-dependent myosin light chain kinases (MLCKs) have been compared. The enzymes exhibit the same isoelectric point (6.5) but differ markedly in molecular weight (Mr = 72 000 for both canine and bovine cardiac MLCK, and Mr = 130 000 for smooth muscle MLCK). Comparison of the tryptic peptide maps of bovine cardiac and turkey gizzard MLCKs indicates that the cardiac enzyme is a fragment of a protein homologous to the smooth muscle kinase. While the smooth muscle kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, the same is not true for either bovine or canine cardiac MLCK. Controlled tryptic hydrolysis of phosphorylated smooth muscle MLCK, followed by affinity chromatography on a column of calmodulin–Sepharose, enables separation of a phosphopeptide (Mr = 22 000) from a mixture of peptides of Mr = 50 000 and 24 000 which are bound to the column in the presence of Ca2+ and eluted with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The phosphorylation site, therefore, is distinct from the calmodulin-binding site. It appears that cardiac MLCK is proteolyzed during the isolation procedure. The purified cardiac enzyme represents a proteolytic fragment which retains Ca2+ and calmodulin dependence but only a fraction of the specific activity of the native enzyme, and has lost the site of phosphorylation by cAMP-dependent protein kinase. A protease is shown to exist in myocardium which is capable of digesting smooth muscle MLCK rapidly at low temperature, and which is resistant to classical antiproteases.

scholarly journals Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase

2002 ◽  
Vol 364 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Mitsuo MITA ◽  
Hayato YANAGIHARA ◽  
Shigeru HISHINUMA ◽  
Masaki SAITO ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
State Level ◽  
Smooth Muscle Contraction ◽  
Membrane Depolarization ◽  
Arterial Smooth Muscle ◽  
Tonic Component ◽  
Phasic Component ◽  
Steady Level

Depolarization of the sarcolemma of smooth muscle cells activates voltage-gated Ca2+ channels, influx of Ca2+ and activation of cross-bridge cycling by phosphorylation of myosin catalysed by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK). Agonist stimulation of smooth muscle contraction often involves other kinases in addition to MLCK. In the present study, we address the hypothesis that membrane depolarization-induced contraction of rat caudal arterial smooth muscle may involve activation of Rho-associated kinase (ROK). Addition of 60mM K+ to de-endothelialized muscle strips in the presence of prazosin and propranolol induced a contraction that peaked rapidly and then declined to a steady level of force corresponding to approx. 30% of the peak contraction. This contractile response was abolished by the Ca2+-channel blocker nicardipine or the removal of extracellular Ca2+. An MLCK inhibitor (ML-9) inhibited both the phasic and tonic components of K+-induced contraction. On the other hand, the ROK inhibitors Y-27632 and HA-1077 abolished the tonic component of K+-induced contraction, and slightly reduced the phasic component. Phosphorylation levels of the 20-kDa light chain of myosin increased rapidly in response to 60mM K+ and subsequently declined to a steady-state level significantly greater than the resting level. Y-27632 abolished the sustained and reduced the phasic elevation of the phosphorylation of the 20-kDa light chain of myosin, without affecting the K+-induced elevation of cytosolic free Ca2+ concentration. These results indicate that ROK activation plays an important role in the sustained phase of K+-induced contraction of rat caudal arterial smooth muscle, but has little involvement in the phasic component of K+-induced contraction. Furthermore, these results are consistent with inhibition of myosin light-chain phosphatase by ROK, which would account for the sustained elevation of myosin phosphorylation and tension in response to membrane depolarization.

Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle

1996 ◽  
Vol 271 (4) ◽  
pp. H1348-H1355 ◽  
Author(s):  
N. Jin ◽  
R. A. Siddiqui ◽  
D. English ◽  
R. A. Rhoades
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Tyrosine Kinases ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase Pathway

Two separate signal transduction pathways exist in vascular smooth muscle: one for cell growth, proliferation, and differentiation and the other for contraction. Although activation of protein tyrosine kinases is intimately involved in the signaling pathway that induces cell growth, proliferation, and differentiation, activation of myosin light chain kinase (MLCK) is an important step in the pathway leading to smooth muscle contraction. Indirect evidence suggests that “cross talk” exists between these two signaling pathways, but the common intermediates are not well defined. The purpose of this study was to determine whether a vasoconstrictor and a mitogen initiate crossover signaling between the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. Rat aorta and pulmonary arteries were isolated and stimulated with either fetal calf serum (FCS) or phenylephrine in the presence or absence of a tyrosine kinase inhibitor (genistein) or tyrosine phosphatase inhibitor [sodium o-vanadate (Na3 VO4)]. Isometric force was recorded as a function of time; myosin light chain phosphorylation, protein tyrosine phosphorylation, and mitogen-activated protein kinase (MAPK) mobility were determined by immunoblotting. The results demonstrate that FCS, which contains a variety of growth factors known to activate tyrosine kinases, induced myosin light chain phosphorylation and contraction in vascular smooth muscle. Phenylephrine, a vasoconstrictor known to activate MLCK, induced tyrosine phosphorylation of a 42-kDa protein identified as MAPK. Tyrosine phosphorylation of this protein was inhibited by genistein and enhanced by vanadate. Genistein significantly inhibited both serum- and phenylephrine-induced myosin light chain phosphorylation as well as the serum- and phenylephrine-induced force generation, whereas vanadate enhanced these responses. These data demonstrate interrelationship between activation of the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. These interactions may influence smooth muscle contraction and be important in the regulation of smooth muscle cell proliferation.

Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness

Science ◽  
1986 ◽  
Vol 232 (4746) ◽  
pp. 80-82 ◽  
Author(s):  
KE Kamm ◽  
JT Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Head ◽  
Isometric Force ◽  
Smooth Muscle Contraction ◽  
Myosin Phosphorylation ◽  
First Order ◽  
Cooperative Process ◽  
Pseudo First Order

Contraction and myosin light-chain phosphorylation were measured in electrically stimulated tracheal smooth muscle. Latencies for the onset of force, stiffness, and light-chain phosphorylation were 500 milliseconds. Myosin light chain was phosphorylated from 0.04 to 0.80 mole of phosphate per mole of light chain with a pseudo-first-order rate of 1.1 per second with no evidence of an ordered or negatively cooperative process. Following the period of latency, stiffness increased with phosphorylation and both increased more rapidly than isometric force. The linear relation between stiffness and phosphorylation during activation suggests independent attachment of each myosin head upon phosphorylation.

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