Contraction myosin phosphorylation

Obara, K. de Lanerolle, P. (1989). Isoproterenol attentuates myosin phosphorylation and contraction of tracheal muscle. J. Appl. Physiol. 66,2017-2022. 

If MLCK activates contraction by increasing myosin phosphorylation, then an increase in the activity of myosin light chain phosphatase, MLCP, by decreasing the fraction of myosin which is phosphorylated, should lead to relaxation from the active (contractile) state. Cyclic adenosine monophosphate (AMP) is a strong inhibitor of smooth muscle contraction and it has been suggested that activation of MLCP could result from its phosphorylation via cAMP activated protein kinase (see Figure 5). 

One should note overall, that while some of these suggested mechanisms may in the future prove to have a role in the control of smooth muscle contraction, in chemically skinned preparations maximum force development follows activation by the MLCK active subunit in extremely low Ca " ion concentrations. The conclusion can hardly be avoided that phosphorylation alone is sufficient for activation, and if another mechanism is involved, it is not necessary for the initial genesis of force. If such mechanisms are operative, then they might be expected to run in parallel or consequent to myosin phosphorylation. A possible example of this category of effect is that a GTP-dependent process (G-protein) shifts the force vs. Ca ion concentration relationship to lower Ca ion concentrations. This kind of mechanism calls attention to the divergence of signals along the intracellular control pathways. 

S ATP + myosin hght chain <1-12> (<1, 2, 8> event in initiation of smooth-muscle contraction [5] <8> involved in regulation of actin-myosin contractile activity in adrenal medulla [7] <8> obhgatory step in development of active tension in smooth muscle [13] <5> involved in myosin phosphorylation and enzyme secretion [16] <2, 3, 5, 6, 8, 10, 11> involved in muscle contractility and motility of non-muscle cells [33] <2> inhibition of actin-myosin ineraction [36,37]) (Reversibihty 1-12 [1-33]) [1-33]... 

Ca2+ regulation of smooth muscle contraction. Myosin light-chain kinase (MLCK) phosphorylates myosin and causes contraction, in response to calcium, as shown in this signaling pathway. 

It has been established for several years that the major mechanism for regulation of contraction in smooth muscle is myosin phosphorylation (Hart-shorne, 1987). Phosphorylation of the two 20,000-dalton light chains of myosin (LC20) activates the actin-dependent ATPase activity of myosin and this initiates the contractile response. Dephosphorylated myosin is associated with relaxed muscle. In this scheme there are two key enzymes the myosin light chain kinase (MLCK) and the myosin light chain phosphatase (MLCP). Obviously a balance of these two activities determines the level of myosin phosphorylation. 

B. Activation of Contraction by Phosphorylation of Myosin Light Chain... 

In canine tracheal muscles contracted with 1 jjlM methacholine, at 37°C, and relaxed with 0.4 jjlM atro-pin or 40 pM forskolin, myosin phosphorylation and force decayed simultaneously (de Lanerolle, 1988). In rabbit tracheal smooth muscle stimulated with carbachol, at 37°C, LC20 dephosphorylation occurred at about the same rate as the decline in stress... 

Since the determination of LC20 phosphate content varies among laboratories by two- to threefold, one cannot establish a stoichiometry of the phosphorylation in either the initial or steady state of contraction. Measurements in this laboratory with arterial and uterine muscles, which produced maximal stress and phosphorylation, suggest that both myosin heads are phosphorylated in the initial and one head in the sustained phase of contraction. The other extreme, contraction without phosphorylation, does not occur in our experience. 

The sufficiency of myosin phosphorylation for contraction in smooth muscle is indisputably demonstrated in experiments where the addition of pro-teolyzed CaM-independent MLCK to permeabilized fibers brings about contraction at very low [Ca +] (Walsh et al, 1982). Furthermore, injection of CaM-independent MLCK into single smooth muscle cells results in contraction under conditions where [Ca +]j remains at resting values (Itoh et al, 1989). These results tend to rule out a model in which thin filaments are fully inhibited in resting muscle, and require Ca2+-dependent disinhibition to support contraction. However, these experiments do not rule out the possibility that thin filament-binding proteins modulate the contractile response. Alterations in the RLC phosphorylation-force relation in intact muscle indicate that collateral regulation can occur. Moreover, experiments with permeabilized fibers demonstrate that thin filament-binding proteins can inhibit force independent of RLC phosphorylation (Pfitzer et al,... 

Asano M, Stull JT (1985) Effects of calmodulin antagonists on smooth muscle contraction and myosin phosphorylation. In Hidaka H, Hartshome DJ (eds) Calmodulin antagonists and cellular physiology. Orlando pp225-260... 

Gerthoffer WT, Murphy RA (1983) Ca , myosin phosphorylation, and relaxation of arterial smooth muscle. Am J Physiol 245 C271-C277 Gerthoffer WT, Pohl J (1994) Caldesmon and calponin phosphorylation in regulation of smooth muscle contraction. Can J Physiol Pharmacol 72 1410-1414 Gerthoffer WT, Trevethick MA, Murphy RA (1984) Myosin phosphorylation and cyclic adenosine 3, 5 -monophosphate in relaxation of arterial smooth muscle by vasodilators. Circ Res 54 83-89... 

Hori M, Sato K, Sakata K, Ozaki H, Takano-Ohmuro H, Tsuchiya T, Sugi H, Kato I, Karaki H (1992) Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle. J Pharmacol Exp Ther 261 506-512... 

Jiang MJ, Morgan KG (1989) Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. Pfliigers Arch 413 637-643... 

Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K (1995) Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375 500-503 Kamm KE, Stull JT (1985) The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Ann Rev Pharmacol Toxicol 25 593-620 Kamm KE, Stull JT (1986) Activation of smooth muscle contraction relation between myosin phosphorylation and stifftiess. Science 232 80-82 Kanamori M, Naka M, Asano M, Hidaka H (1981) Effects of N-(6-aminohexyl)-5-chloro-l-naphtalene ulfonamide and other calmodulin antagonists (calmodulin interacting scents) on calcium-induced contraction of rabbit aortic strips. J Pharmacol Exp Ther 217 494-499... 

Murphy RA (1982) Myosin phosphorylation and crossbridge regulation in arterial smooth muscle. State-of-the-art review. Hypertension 4 3-7 Murphy RA (1989) Contraction in smooth muscle cells. Annu Rev Physiol 51 275-283 Murphy RA (1994) What is special about smooth muscle The significance of covalent crossbridge regulation. FASEB J 8 311-318... 

Tanaka T, Ohta H, Kanda K, Tanaka T, Hidaka H, Sobue K (1990) Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction betvreen actin and myosin. Eur J Biochem 188 495-500 Tanner JA, Haeberle JR, Meiss RA (1988) Regulation of glycerinated smooth muscle contraction and relaxation by myosin phosphorylation. Am J Physiol 255 C34-C42 Tansey MG, Hori M, Karaki K, Kamm KE, Stull JT (1990) Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle. FEBS Lett 270 219-221... 

Pozzan T, Rizzuto R, Volpe P, Meldolesi J (1994) Molecular and cellular physiology of intracellular calcium stores. Physiol Rev 74 595-636 Raeymakers L, Wuytack F (1996) Calcium pumps. In Barany M (ed) Biochemistry of smooth muscle contraction. Academic Press, San Diego, pp 241-253 Rembold CM (1990) Modulation of the [Ca " ] sensitivity of myosin phosphorylation in intact swine arterial smooth muscle. J Physiol 429 77-94 Rembold CM, Weaver BA (1990) [Ca ], not diacylglycerol, is the primary regulator of sustained swine arterial smooth muscle contraction. Hypertension 15 692-698 Shimada T, Somlyo AP (1992) Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle. J Gen Physiol 100 27-44... 

Goeckeler ZM, Wysolmerski RB Myosin light chain kinase-regulated endothelial cell contraction The relationship between isometric tension, actin polymerization, and myosin phosphorylation. J Cell Biol 1995 130 613-627. 

In the presence of calcium, the primary contractile protein, myosin, is phosphorylated by the myosin light-chain kinase initiating the subsequent actin-activation of the myosin adenosine triphosphate activity and resulting in muscle contraction. Removal of calcium inactivates the kinase and allows the myosin light chain to dephosphorylate myosin which results in muscle relaxation. Therefore the general biochemical mechanism for the muscle contractile process is dependent on the avaUabUity of a sufficient intraceUular calcium concentration. 

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