Myosin phosphorylation

Pfitzer G (2001) Invited review regulation of myosin phosphorylation in smooth muscle. J Appl Physiol... 

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. 

Hellstrand That is what I am getting at. There are a lot of phase shifts in this system. One observation we have made is that under hypoxia we see a decrease in amplitude but an increase in frequency of the waves. We are trying to model a case where this would account for reduction of force simply on the basis of non-linearity of the [Ca2+] versus myosin phosphorylation versus force reactions. It seems intuitively that this could explain why there can be a reduction in force although there is no reduction in the overall level of global Ca2+. Is amplitude modulation something that people have seen ... 

Paul I was thinking that when there is a high level of myosin phosphorylation, with maximum activation and then turning it off, that there would be a lot of... 

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]... 

Danid JL, Molish IR, Rigmaiden M, Stewart G. Evidence for a role of myosin phosphorylation in file initiation of the platelet shape Aange response. J Biol Chem 1984 259 9826-31... 

Daniel JL, MtXISH IR, RiGMAIDEN M, Steward G. Evidence fiar a role of myosin phosphorylation in the initiation of the platelet sh e change response. JBiol Chan 259 9826-9831,1984. 

Daniel, J. L, Holmsea H. and Adelsteia R. S. Thrombin-stimulated myosin phosphorylation in intact... 

Dillon, P.F., Aksoy, M.O., Driska, S.P. and Murphy, RA. (1981). Myosin phosphorylation and the cross-bridge cycle in smooth muscle. Science (Wash). DC211, 495-497. 

Stull JT, Bowman BF, Gallagher PJ, et al. Myosin phosphorylation in smooth and skeletal muscles regulation and function. Prog Clin Biol Res 1990 327 107-126. 

An alternative mechanism by which cAMP may act to inhibit platelet function was proposed by Hathaway et al. (81). In this model (Figure 12), it is suggested that an increase in cAMP results in inhibition of myosin phosphorylation and consequent inhibition of platelet contractile activity (since unphos-phorylated myosin cannot interact with actin). Thus, it was proposed that cAMP causes the activation of a protein kinase which in turn phosphorylates myosin kinase. In the phosphory-lated form, myosin kinase is less capable of binding calmodulin and therefore is not as effective in phosphorylating myosin. 

The greatest challenge ahead, because it is the most difficult experimentally, is to determine the physiological role of caldesmon. In vitro experiments show how caldesmon might regulate smooth muscle contractility in concert with myosin phosphorylation, but they can never demonstrate that it does. For this we need new tools that can manipulate caldesmon within the intact cell. It is to be hoped that modern antisense RNA and transgenic techniques could provide the answer. 

FIG U R E 6 Inhibition of the actin-activated myosin MgATPase by CP. Actomyosin ATPase rates in the presence ( ) and the absence ( ) of Ca +-myosin phosphorylation levels were quantified in the presence (O) and the absence ( ) of Ca +. Actin concentration was 6 pM. Reproduced with permission from Winder and Walsh (1990c), Fig. 1, p. 10150, Copyright 1990, The American Society for Biochemistry and Molecular Biology, Inc. 

The identification and development of specific inhibitors of MLCK could provide valuable experimental tools for exploring physiological functions of MLCK and myosin phosphorylation in smooth muscle and nonmuscle cells. Although inhibitors that act by binding calmodulin have been used experimentally, their usefulness is limited because they inhibit other calmodulin-dependent enzymes in tissues and cells (Asano and Stull, 1985 Nakanishi et al., 1992). Therefore, attempts have been made to develop novel reagents that inhibit MLCK activity directly. ML-9 [l-(5-chloronaphthalenesulfonyl)-lH-hexahydro-l, 4-diazepine] inhibits purified smooth muscle MLCK competitively with respect to ATP (Saitoh et al., 1987). Predictably, it inhibits RLC phosphorylation in ac-tomyosin, skinned fibers, and smooth muscle strips (Ishikawa et al., 1988). However, ML-9 also inhibits other protein kinases, so its specificity under different experimental conditions needs to be established. 

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. 

Endothelial-derived relaxing factor (EDRF) (e.g., nitric oxide) and the atrial natriuretic factor(s) increase [cGMP] in smooth muscle. They relax arterial smooth muscle by three mechanisms decreasing [Ca +Jj, decreasing the [Ca2+]i sensitivity of phosphorylation, and uncoupling force from myosin phosphorylation (the latter two are pharmacomechanical mechanisms ... 

Modulation of myosin phosphorylation independent of changes in [Ca2+]j is another form of pharmacomechanical coupling. 

The [Ca +]i sensitivity of phosphorylation was determined from the relation between aequorin estimated [Ca +]j and myosin phosphorylation levels (Rembold, 1990, 1992). PDBu, phorbol dibutyrate FSK, forskolin [K+], depolarization. 

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... 

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