Phosphorylation caldesmon

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. 

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>... 

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... 

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... 

Calponin is another polypeptide monomer (M.W. 32,000) that can inhibit actin-activated myosin ATPase activity. In contrast to CaD, CaP exerts its effect in the absence of tropomyosin and completely inhibits motility in a 2/3 ratio with actin. CaP inhibits myosin binding to actin, but does so by reducing the affinity of actin for myosin rather than competing for the same site. CaP can be phosphorylated by PKC and CaMKII, both of which reverse CaP s inhibitory activity. As with caldesmon, many questions remain. The ratio of CaP to actin actually observed in smooth muscle is in the range 1 10 to 1 16, far from the 2/3 ratio found to produce near-complete inhibition of motility. Therefore, the importance of CaP and its regulation by phosphorylation is still debatable. 

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. 

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-... 

To account for activation of arterial smooth muscle independently of LC20 phosphorylation, attention has been focused on the possible roles of the thin filament-associated regulatory proteins, caldesmon and calponin. Both proteins have been localized in the actomyosin domain of the smooth muscle cell and both have been shown to inhibit actin-activated myosin ATPase by interacting with F-actin, tropomyosin, and/or myosin (Clark et al., 1986 Takahashi et al.,... 

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. 

MBP occurs on specific threonine residues that are proline-directed that is, the phosphorylated amino acids are immediately followed by proline in the amino acid sequence of the protein. In studies of peptide substrates phosphorylated by MAPK it appears that the sequence Ser/Thr-Pro is a minimum consensus phosphorylation sequence for MAPK. However, the amino acids carboxyl- and amino-terminal to this sequence modify the ability of MAPK to covalently attach phosphate. In particular, the placement of proline at position -2 (relative to the phosphorylated amino acid) increases kinase activity. On the basis of these data, the optimal consensus phosphorylation sequence for MAPK is Pro-X-(Ser/Thr)-Pro (Clark-Lewis et al., 1991). Certain proteins, including caldesmon, are phosphorylated by MAPK on sites that do not match this optimal consensus sequence exactly (Adam and Hathaway, 1993). 

A2 or cPLA2), and proteins for which the function of phosphorylation is incompletely understood (caldesmon). In addition to cell growth and proliferation, the activation of MAPK is linked to physiological functions such as osmosensing in yeast, stretch sensing in cardiac tissue, and a potential role in smooth muscle contractile or cytoskeletal function. A complete description of the substrates phosphorylated by MAPK under physiological conditions in different cell types, and the responses controlled by those modifications, are areas of intense investigation. 

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... 

MAPK is more likely to be the physiologically relevant "caldesmon kinase" since there is no evidence to date for p34 i 2 or any PDPK other than MAPK, in arterial muscle (Adam and Hathaway, 1993 Adamef a/., 1995). With the use of a specific peptide substrate, the 42- and 44-kDa isoforms of MAPK (p42mapk and p44MAPK) were found to contain all the proline-directed protein kinase activity in contractile smooth muscle. However, it is possible that PDPKs other than MAPK exist in smooth muscle and that (1) the activity of these are not detected using this peptide substrate and (2) they phosphorylate caldesmon. 

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