Myosin activation

In this chapter we will discuss the various forms of myosin and the roles they play in living systems. We will compare and contrast the function and regulation of myosin activity in different cellular environments. Finally, we will examine the clinical aspects of myosin strucmre and function. 

Tobacman, L. S., Nihli, M., Butters, C., Heller, M., Hatch, V., Craig, R., Lehman, W., and Homsher, E. (2002). The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity. /. Biol. Chem. 277, 27636-27642. 

Figure 6.10. Calcium-dependent signalling by adrenergic receptors. a p-Adrenergic receptors activate adenylate cyclase. cAMP activates protein kinase A (PKA). In heart muscle, PKA phospho-rylates several Ca transporters and charmels, so that the amount of Ca available for contraction is increased. PL Phospholam-ban SERCA SR/ER Ca transporter, b In smooth muscle, myosin activation in works by way of phosphorylation, which is performed by myosin light chain kinase (MLCK). Inactivation is accomplished by myosin light chain phosphatase (MLCP). c aj-Adrenergic receptors stimulate phospholipase C, which releases inositoltriphosphate (IP3). IP3 binds to a cognate ligand-gated Ca chaimel in the ER and releases Ca, which with calmodulin activates MLCK.

Phosphorylation by PKA decreases the activity of MLCK, considered to be intimately involved in ASM tension development (Gerthoffer, 1991). Phosphorylation of MLCK, at certain sites on this enzyme, decreases its affinity for the Ca /CaM complex. Ultimately this means that irrespective of the concentration of the ICzM complex, there will be fewer activated MLCK molecules, reduced myosin activation, and thus less contractile force. 

Other signaling pathways activate Rho kinase, which can stimulate myosin activity in two ways. First, Rho kinase can phosphorylate myosin LC phosphatase (see Figure 19-25b), thereby Inhibiting Its activity. With the phosphatase inactivated, the level of myosin LC phosphorylation and thus myosin activity Increase. In addition, Rho kinase direcdy activates myosin by phosphorylating the regulatory light chain. Note that Ca plays no role in the regulation of myosin activity by Rho kinase. 

Benian GM, Kiff JE, Neckelmann N, Moerman DG, Waterson RH (1989) Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegatis. Nature 342 45-50... 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

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. 

The molecular events of contraction are powered by the ATPase activity of myosin. Much of our present understanding of this reaction and its dependence on actin can be traced to several key discoveries by Albert Szent-Gyorgyi at the University of Szeged in Hungary in the early 1940s. Szent-Gyorgyi showed that solution viscosity is dramatically increased when solutions of myosin and actin are mixed. Increased viscosity is a manifestation of the formation of an actomyosin complex. 

Szent-Gyorgyi further showed that the viscosity of an actomyosin solution was lowered by the addition of ATP, indicating that ATP decreases myosin s affinity for actin. Kinetic studies demonstrated that myosin ATPase activity was increased substantially by actin. (For this reason, Szent-Gyorgyi gave the name actin to the thin filament protein.) The ATPase turnover number of pure myosin is 0.05/sec. In the presence of actin, however, the turnover number increases to about 10/sec, a number more like that of intact muscle fibers. 

However, release of ADP and P from myosin is much slower. Actin activates myosin ATPase activity by stimulating the release of P and then ADP. Product release is followed by the binding of a new ATP to the actomyosin complex, which causes actomyosin to dissociate into free actin and myosin. The cycle of ATP hydrolysis then repeats, as shown in Figure 17.23a. The crucial point of this model is that ATP hydrolysis and the association and dissociation of actin and myosin are coupled. It is this coupling that enables ATP hydrolysis to power muscle contraction. 

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

The ETa receptor activates G proteins of the Gq/n and G12/i3 family. The ETB receptor stimulates G proteins of the G and Gq/11 family. In endothelial cells, activation of the ETB receptor stimulates the release of NO and prostacyclin (PGI2) via pertussis toxin-sensitive G proteins. In smooth muscle cells, the activation of ETA receptors leads to an increase of intracellular calcium via pertussis toxin-insensitive G proteins of the Gq/11 family and to an activation of Rho proteins most likely via G proteins of the Gi2/i3 family. Increase of intracellular calcium results in a calmodulin-dependent activation of the myosin light chain kinase (MLCK, Fig. 2). MLCK phosphorylates the 20 kDa myosin light chain (MLC-20), which then stimulates actin-myosin interaction of vascular smooth muscle cells resulting in vasoconstriction. Since activated Rho... 

This kinase specifically phosphorylates the regulatory light chain of myosin after activation by calcium-calmodulin. Several isozymes of approximately 135 kDa exist. 

A subfamily of Rho proteins, the Rnd family of small GTPases, are always GTP-bound and seem to be regulated by expression and localization rather than by nucleotide exchange and hydrolysis. Many Rho GTPase effectors have been identified, including protein and lipid kinases, phospholipase D and numerous adaptor proteins. One of the best characterized effector of RhoA is Rho kinase, which phosphorylates and inactivates myosin phosphatase thereby RhoA causes activation of actomyosin complexes. Rho proteins are preferred targets of bacterial protein toxins ( bacterial toxins). 

Calcium-dependent regulation involves the calcium-calmodulin complex that activates smooth muscle MLCK, a monomer of approximately 135 kDa. Dephosphorylation is initiated by MLCP. MLCP is a complex of three proteins a 110-130 kDa myosin phosphatase targeting and regulatory subunit (MYPT1), a 37 kDa catalytic subunit (PP-1C) and a 20 kDa subunit of unknown function. In most cases, calcium-independent regulation of smooth muscle tone is achieved by inhibition of MLCP activity at constant calcium level inducing an increase in phospho-rMLC and contraction (Fig. 1). 

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