Phosphorylation of Caldesmon

Vascular smooth muscle caldesmon is phosphorylated in resting muscle and the stoichiometry of phosphorylation increases in response to pharmacological stimulation (Adam et al., 1989). On the basis of these findings, phosphorylation mechanisms are proposed to modulate the physiological effects of caldesmon on either contractile or cytoskeletal function. There are 

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Apart from the phosphorylation theory, other regulatory mechanisms have also been suggested for smooth muscle contraction. A thin-filament protein that has been proposed as a regulatory component is caldesmon [102], Purified caldesmon is a potent inhibitor of actin-tropomyosin interaction with myosin. The mechanisms by which calcium removes this inhibition are controversial. Furthermore, phosphorylation of caldesmon by a caldesmon kinase in vitro has also been implicated in this... 

Phosphorylation of caldesmon in vitro by CaM-kinase II (or PKC) has been shown to interfere with caldesmon binding to F-actin, and results in a reversal of the caldesmon inhibitory effect on actin-activated myosin ATPase activity (Ngai and Walsh, 1987). This has led to speculation that these kinases may be in-... 

FIGURE 4 Schematic depicting the potential roles for MAPK in contractile versus proliferative (or cultured) smooth muscle. MAPK is activated in response to stimulation by growth factors, stretch, and pharmacological agents. This process can be inhibited by cAMP and cAMP-dependent protein kinase. Once activated, MAPK phos-phorylates a number of intracellular proteins (both cytoplasmic and nuclear) that result in an alteration of growth and proliferation in cultured cells. In contractile smooth muscle, MAPK phosphorylation of caldesmon may lead to alterations in muscle contractility or actin filament structure. 

A role for MAPK in the contractile responsiveness of smooth muscle may result from either of two possible mechanisms, both involving caldesmon phosphorylation. First, caldesmon phosphorylation by MAPK may lead directly to an alteration of actomyosin activity. Caldesmon may exert this effect alone, or in concert with other myofibrillar proteins such as cal-ponin. Second, phosphorylation of caldesmon may alter the dynamics of actin filament organization within the cell. Caldesmon phosphorylation may result in alterations of the cellular cytoskeleton that must occur during prolonged contractions. 

Yamashiro S, Matsumura F (1991) Mitosis-specific phosphorylation of caldesmon possible molecular mechanism of cell rounding during mitosis. Bioessays 13 563568 Zigmond SH (1996) Signal transduction and actin filament organization. Curr Opin Cell Biol 8 6673... 

Nixon GF, lizuka K, Haystead CM, Haystead TA, Somlyo AP, Somlyo AV (1995) Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca sensitivity in rabbit smooth muscle. J Physiol (Lond) 487 283-289 Noda S, Ito M, Watanabe S, Takahashi K, Maruyama K (1992) Conformational changes of actin induced by calponin. Biochem Biophys Res Commun 1992 May 29 185(l) 481-487... 

Adam LP, Haeberle JR, Hathaway DR (1989) Phosphorylation of caldesmon in arterial smooth muscle. J Biol Chem 264 7698-7703 Adam LP, Franklin MT, Raff GF, Hathaway DR (1995) Activation of mitogen-activated protein kinase in porcine carotid arteries. Circ Res 76 183-190 Alessi D, MacDougall KL, Sola MM, Ikebe M, Cohen P (1992) The control of protein phosphatase-1 by targeting subunits. Eur J Biochem 210 1023-1035 Amano M, Mukai H, Ono Y, Chihara K, Matsui T, Hamajima Y, Okawa K, Iwamatsu A, Kaibuchi K. (1996) Identification of a putative target forRho as the serine-threonine kinase protein kinase N. Science 271 648-650 Andrea JE, Walsh MP. (1997) Personal communication... 

In addition to the displacement of caldesmon, smooth muscle cell contraction requires kinase-induced phosphorylation of myosin. Smooth muscle has a unique type of myosin filament called p-light chains which are the target (substrate) for MLCK, but MLCK is only active when complexed with CaCM. Myosin light chain phosphatase reverses the PKA-mediated process and when cytosolic calcium ion concentration falls, CDM is released from CaCM and re-associates with the actin. The central role of calcium-calmodulin in smooth muscle contraction is shown in Figure 7.4. 

ATP -I- synapsin <1, 2> (<1> brain synapsin best substrate of chicken gizzard caldesmon kinase [1] <1> brain synapsin best substrate, phosphorylated at 950% the rate of caldesmon [5]) (Reversibility <1,2>... 

Fig. 4. The temporal sequence of events when a resting strip of tracheal smooth muscle is activated by carbacholamine addition at 10 min. There is a transient rise in [Ca2+]c (—) followed by a transient increase in the content (—) of phosphorylated myosin light chains (MLC-P) which lead in turn to the initiation of force development (—). Increased force is sustained even though the content of MLC-P declines. Preceding the sustained phase of force maintenance, there is an increase in the phosphorylation of desmin (D-P), synemin (S-P), caldesmon (CD-P) and a number of low molecular weight cytosolic proteins (X-P). These remain phosphorylated throughout the sustained phase of the response during which there is a sustained increase in Ca2+ cycling across the plasma membrane which regulates the activity of the membrane-associated protein kinase C.

In skeletal muscle, disinhibition of actin is necessary for contraction to occur, and control of contraction is said to be actin-linked. In smooth muscle, phosphorylation of myosin light chains (MLCs) is required for contraction. Several mechanisms alter MLC phosphorylation, and so in smooth muscle, control of contraction is primarily myosin-linked. Three control proteins have been identified in smooth muscle myosin light chain kinase (MLCK) caldesmon (CaD) and calponin (CaP). Figure 21-14... 

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. 

Caldesmon has also been shown to inhibit tension development in chemically permeabilized gizzard smooth muscle (Pfitzer etal., 1993). Furthermore, inhibition of caldesmon/F-actin interaction in permeabilized VSM resulted in contractile force generation independently of changes in [Ca +J, supporting the concept that caldesmon may function as a regulator in situ independently of LC20 phosphorylation (Katsuyama et al., 1992). Both caldesmon (Adam et al.,... 

These two phenotypes of smooth muscle, in addition to having marked differences in contractile activity, express different isoforms of several contractile proteins and certain soluble enzymes. In particular, proliferative smooth muscle contains at least three PDPKs p34 i 2 p42 , and p44 PK the contractile phenotype of smooth muscle, only p42 P and p44 mark have been identified. The precise function and a complete description of the substrates for MAPK in the contractile phenotype of smooth muscle are unknown however, one substrate that has been idenhfied is the actin and myosin binding protein, caldesmon. Because of the phosphorylation of cal-desmon, MAPK may be involved in either smooth muscle contractile regulation or the structural organization of actin filaments within smooth muscle cells. 

Katayama E, Scott-Woo G, Ikebe M (1995) Effect of caldesmon on the assembly of smooth muscle myosin. J Biol Chem 270 39193925 Kelley CA, Kawamoto S, Conti MA, Adelstein RS (1991) Phosphorylation of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells. J Cell Sci Suppl 14 4954... 

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