This minireview will cover current concepts on the identity and mechanistic function of smooth muscle actin binding proteins that may regulate actin-myosin interactions. The potential roles of tropomyosin, caldesmon, calponin, and SM22 will be discussed. The review, for purposes of brevity, will be nonexhaustive but will give an overview of available information on the in vitro biochemistry and potential in vivo function of these proteins. Preterm labor is discussed as a possible example of where thin filament regulation may be relevant. Considerable controversy surrounds the putative physiological significance of these proteins, and emphasis will be placed on the need for more experimental work to determine the degree to which tissue- and species-specific effects have clouded the interpretation of functional data.

Smooth muscle contractile activity is a major regulator of function of the vascular system, respiratory system, gastrointestinal system and the genitourinary systems. Malfunction of contractility in these systems leads to a host of clinical disorders, and yet, we still have major gaps in our understanding of the molecular mechanisms by which contractility of the differentiated smooth muscle cell is regulated. This review will summarize recent advances in the molecular understanding of the regulation of smooth muscle myosin activity via phosphorylation/dephosphorylation of myosin, the regulation of the accessibility of actin to myosin via the actin-binding proteins calponin and caldesmon, and the remodelling of the actin cytoskeleton. Understanding of the molecular ‘players’ should identify target molecules that could point the way to novel drug discovery programs for the treatment of smooth muscle disorders such as cardiovascular disease, asthma, functional bowel disease and pre-term labour.

Abstract-Subcellular targeting of kinases controls their activation and access to substrates. Although Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the ␥ isoform of CaMKII that contains a potential targeting sequence in its association domain . Antisense knockdown of CaMKII␥ G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKII␥ G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKII␥ G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKII␥ G-2 activation, and before CaMKII␥ G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKII␥ G-2 inhibited targeting. Furthermore, prevention of CaMKII␥ G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization. Key Words: CaMKII Ⅲ smooth muscle Ⅲ contractility Ⅲ targeting Ⅲ extracellular signal-regulated kinase C a 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase that is expressed as 4 different isoforms (␣, ␤, ␥, and ␦). Each member of this kinase family possesses 3 major domains ( Figure 1A): an N-terminal catalytic/regulatory domain containing a Ser/Thr kinase region and overlapping autoinhibitory and Ca 2ϩ /calmodulin binding regions, a central-linker domain containing variable regions, and a C-terminal association domain that promotes oligomerization. 1 CaMKII is not active until Ca 2ϩ /calmodulin binds to its regulatory domain. This binding allows CaMKII to undergo autophosphorylation at Thr287 (numbering according to the ␥ isoform), which, in turn, increases its affinity for Ca 2ϩ /calmodulin and allows kinase activity even in the absence of Ca 2ϩ /calmodulin. The ␣ and ␤ isoforms of CaMKII are predominantly expressed in neural tissue, 2 where they have been demonstrated to be involved in synaptic plasticity, memory, and learning. 3,4 In contrast, differentiated smooth muscle cells (dSMCs) express mainly the ␥ isoform of CaMKII, 5 which has been linked to contractile activity by the use of antisense and phar...

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase C⑀ (PKC⑀) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKC⑀ and PKC␣ with possible involvement of C1B. The C2 region of PKC⑀ binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKC␤II, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKC␣, -⑀, and -␤II severalfold compared with control conditions. Similarly, calponin was found to increase the amount of 32 P-labeled phosphate incorporated into PKC from [␥-32 P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

The present study was undertaken to determine whether calponin (CaP) participates in the regulation of vascular smooth muscle contraction and, if so, to investigate the mechanism. By PCR homology cloning, the cDNA sequence of ferret basic (h1) CaP was determined and phosphorothioate antisense and random oligonucleotides were synthesized and introduced into strips of ferret aorta by a chemical loading procedure. Treatment of ferret aorta with CaP antisense oligonucleotides resulted in a decrease in protein levels of CaP to 54 % of that in random sequence‐loaded muscles, but no change in the protein levels of caldesmon (CaD), actin, desmin or extracellular regulated protein kinase (ERK). Contraction in response to phenylephrine or a phorbol ester was significantly decreased in antisense‐treated muscles compared to random sequence‐loaded controls. Neither basal intrinsic tone nor the contraction in response to 51 mm KCl was significantly affected by antisense treatment. During phenylephrine contractions, phospho‐ERK levels increased, as did myosin light chain (LC20) phosphorylation. Phenylephrine‐induced ERK phosphorylation and CaD phosphorylation at an ERK site were significantly decreased by CaP antisense. Increases in myosin light chain phosphorylation were unaffected. The data indicate that CaP plays a significant role in the regulation of contraction and suggest that in a tonically active smooth muscle CaP may function as a signalling protein to facilitate ERK‐dependent signalling, but not as a direct regulator of actomyosin interactions at the myofilament level.