Smooth Muscle Caldesmon Research Articles

Over‐expression of smooth muscle caldesmon in mouse fibroblasts

Caldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have investigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK-) cells were transfected stably with plasmids carrying the TK+ gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+ gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immunofluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress-fibers and in membrane ruffles. The immunofluorescence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control.

Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin.

We have demonstrated that caldesmon does not alter the affinity of weak binding actomyosin complexes when it inhibits actin-tropomyosin activation at physiological ratios (1 per 14 actins), and we proposed that it acts upon the strong binding complexes in the same way that troponin-tropomyosin does. We therefore compared the effect of caldesmon, caldesmon fragments, and troponin upon the interaction of the strongly bound complexes S-1.ADP, S-1.adenylyl imidodiphosphate (AMP.PNP), and N-ethylmaleimide-treated myosin subfragment-1 (NEM-S-1) with actin-tropomyosin. In 0.17 M ionic strength buffer [14C]iodoacetamide-labeled S1.ADP bound to actin-smooth muscle tropomyosin with no evidence of cooperativity; Kd = 0.8 +/- 0.3 microM (n = 5). Inhibitory concentrations of sheep aorta caldesmon or rabbit skeletal muscle troponin made the binding highly cooperative. At low levels of saturation the apparent Kd was 10-40 microM with 10 microM caldesmon and 8-20 microM with 6 microM troponin; at > 50% saturation the binding was indistinguishable from actin-tropomyosin alone. A similar result was obtained for the binding of [14C]iodoacetamide-labeled S-1.AMP.PNP to actin-smooth muscle tropomyosin at 0.03 M ionic strength (Kd = 0.47 +/- 0.05 microM). Binding was slightly cooperative and became highly cooperative in the presence of inhibitory concentrations of troponin, caldesmon, and the human caldesmon fragments H7 (amino acids 622-767) and H9 (amino acids 726-793). We conclude that caldesmon and troponin both act as allosteric effectors of the "on"/"off" equilibrium of actin-tropomyosin. 0.1 NEM-S-1/actin potentiated actin-smooth muscle tropomyosin activation of myosin MgATPase 7-fold at 0.03 M ionic strength. Caldesmon inhibited the ATPase in the presence and absence of 0.5 microM NEM-S-1. NEM-S-1 reactivated actin-tropomyosin, which had been inhibited by troponin, caldesmon, H7, or H9. This is compatible with opposing effects of NEM-S-1 and caldesmon or troponin upon the actin-tropomyosin on/off equilibrium.

Calmodulin-sensitive interaction of human nebulin fragments with actin and myosin.

Nebulin is a giant protein ruler of the actin filament of skeletal muscle. This modular protein primarily consists of a repeating sequence (module) of 35 residues and a superrepeat of seven modules. These modules are thought to be actin binding domains along the length of nebulin. Cloned human nebulin fragments of 7-8 modules bind with high affinity to calmodulin, actin, myosin, and myosin head. The stoichiometry of high-affinity binding is approximately one actin monomer bound per nebulin fragment and one myosin bound per nebulin fragment, as determined by cosedimentation binding assays. These observations raise the intriguing possibility that nebulin might have regulatory functions on actomyosin interactions. Nebulin fragments from the N-terminal half situated in the actomyosin overlap region of the sarcomere inhibit actomyosin ATPase activity and the sliding velocity of actin over myosin in motility assays, while a nebulin fragment near the C-terminus, which is localized to the Z-line, does not prevent actin sliding. Calmodulin reverses the inhibition of ATPase and accelerates actin sliding in calcium-dependent manner. Calmodulin with calcium greatly reduces the binding of nebulin fragments to both actin and myosin. The data suggest that the nebulin can interact with both actin and myosin in a calcium/calmodulin-dependent manner and might have regulatory functions in skeletal muscle contraction in a fashion analogous to caldesmon in smooth muscle.

Myosin I from mammalian smooth muscle is regulated by caldesmon-calmodulin.

A single-headed monomeric myosin (myosin I) was isolated from pig urinary bladder smooth muscles and purified to homogeneity. Myosin I from smooth muscle is composed of a 110-kDa heavy chain and three 17-kDa light chains. The heavy chain from smooth muscle myosin I does not cross-react with the antibody against conventional myosin (myosin II) from smooth muscle, but it does show antigenic similarity to adrenal medulla myosin I heavy chain. The light chain from smooth muscle myosin I is similar to calmodulin in molecular weight, amino acid composition, and migration on SDS-polyacrylamide gel electrophoresis in the presence of Ca2+. The high salt ATPase activity of myosin I in the presence of CaCl2 is higher than that in K(+)-EDTA. Smooth muscle actin causes a 5-10-fold activation of the Mg-ATPase activity of myosin I. In the presence of Ca2+, exogenous calmodulin enhances the actin-activated ATPase activity of myosin I, and the increased activity is associated with the binding of exogenous calmodulin to myosin I heavy chain. A maximum of 4 mol of light chains/mol of myosin I heavy chain is observed in the presence of exogenous calmodulin. Caldesmon, a calmodulin/actin-binding protein, inhibits the actin-activated ATPase activity of myosin I. This inhibition is reversed by exogenous calmodulin in the presence of Ca2+. The actin activation of myosin I ATPase exhibits around 50% Ca2+ sensitivity in the presence of exogenous calmodulin. When caldesmon is bound to actin, Ca2+ sensitivity is increased to 80% in the presence of calmodulin. Therefore, smooth muscle caldesmon, which is thought to play a role in the regulation of actin activation of myosin II, also regulates the actin activation of myosin I ATPase in smooth muscle.

Localization of phospholipid-binding sites of caldesmon

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C-terminal part of caldesmon (so-called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606–756), H7 (566–710) and H2 (626–710) form tight complexes with phosphatidylserine, whereas fragments H8 (658–737), H9 (669–737) and fragment H4 (566–624) interact with phospholipids less effectively. It is concluded that the phospholipid-binding site is located in the sequence 626–710 of caldesmon. This sequence contains calmodulin-binding sites and serine residues phosphorylated by protein kinase C and pro-directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon-phospholipid interaction described earlier.

Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.

Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon.

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.

Effect of 67 kDa calcimedin on caldesmon functioning

Interaction of smooth muscle caldesmon with calmodulin, troponin C, S-100 protein and 67 kDa calcimedin was analyzed. Native gel electrophoresis and crosslinking revealed the complex formation between caldesmon and three EF-hand Ca-binding proteins, whereas calcimedin did not interact with caldesmon. In the presence of Ca 2+, calcimedin binds to actin-tropomyosin without affecting the interaction of caldesmon with this complex. Although calcimedin reversed the inhibitory action of caldesmon on the actomyosin ATPase activity at a lower concentration than three other Ca-binding proteins, this effect only slightly depends on Ca 2+ and was observed at the concentration of calcimedin comparable to that of actin. It is concluded that calcimedin itself cannot be responsible for Ca-dependent regulation of caldesmon functioning, but actin bundling induced by calcimedin (or by other actin binding proteins) decreases the inhibitory action of caldesmon on the actomyosin ATPase activity.

MAP kinases from bovine brain: purification and characterization.

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.

Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments

Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development.

Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments

It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.

Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase.

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.

Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon

We expressed the following fragments of chicken gizzard caldesmon in the pMW 172/BL21 (DE3) system at 0.4-2.2 mg of pure protein/liter of culture: full-length smooth muscle caldesmon (CDh) (amino acids 1-756), nonmuscle caldesmon (CDl), amino acids 1-128 (N128), 1-578 (N578), 230-419, 606-756 (606C), and 658-756 (658C). CDh bound tropomyosin with a Kd of 1.5 microM; N578, 230-419, and 606C bound with affinities at least 2-5 fold lower; N128 bound weakly; and 658C did not bind. Only N128 and N578 bound to smooth muscle myosin, both about 10-fold weaker than CDh and CDl. Only 606C and 658C bound to actin-tropomyosin with affinities CDh = 606C > 658C. The binding to actin-tropomyosin was biphasic, whereas the binding to actin was monophasic, corresponding to the weak binding component found in the presence of tropomyosin. Calmodulin bound only to the C-terminal fragments with the same affinity as CDh. CDh, 606C, and 658C inhibited actin-tropomyosin-activated myosin ATPase, with maximal inhibition correlated with 1 caldesmon bound/14 actins, and inhibition was reversed by Ca(2+)-calmodulin. Thus, the actomyosin ATPase regulatory function, calmodulin binding, and most actin binding is located within the C-terminal 99 amino acids, whereas tropomyosin binding is distributed throughout the rest of the molecule.

Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin

Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin.

Caldesmon

Recent research has led to an understanding of the in vitro properties of caldesmon, including the regulation of actomyosin ATPase activity, cross-linking between actin and myosin, enhancement of microfilament stability and stimulation of polymerization of actin. While it remains to be established whether caldesmon functions similarly in vivo, recent studies have suggested that smooth muscle caldesmon regulates the inhibition of vascular smooth muscle tone, and that non-muscle caldesmon plays roles in the regulation of cell motility and cytoskeletal organization in three biological activities: granule movement, hormone secretion and reorganization of microfilaments during mitosis.

Synthesis and Expression of Smooth Muscle Phenotype Markers in Primary Culture of Rabbit Aortic Smooth Muscle Cells: Influence of Seeding Density and Media and Relation to Cell Contractility

Rabbit aortic smooth muscle cells (SMC) were seeded at moderate or high densities and grown either in the presence of serum or in the serum-substitution formula Monomed. Expression and synthesis of marker proteins caldesmon, calponin, smooth muscle myosin, and vinculin were monitored during SMC cultivation. Contractility was tested by the ability of cultured SMC to deform silicone membranes following ionomycin treatment. The results show that cells of moderate density grown in Monomed, as opposed to those grown in 5% serum, have the smooth muscle isoform of caldesmon 1.6-fold higher, calponin 1.4-fold and smooth muscle myosin 1.4-fold higher on Day 14 of cultivation. Synthesis of these proteins corresponded to their expression in SMC. The metavinculin:vinculin ratio slightly decreased over the first days with a following reestablishment on Day 8. Contraction was observed until Day 13, compared with Day 7 for cells grown in the presence of serum. High seeding density also prevented a decrease in the expression of smooth muscle markers with the exception of smooth muscle caldesmon whose content in the high density SMC culture was not significantly different from that in the moderate density culture. The period of contractility of SMC in the high density culture was also similar to that in the moderate density culture in the presence of serum. We conclude that cultivation of primary SMC in Monomed allows the maintenance of cells in the contractile phenotype more effectively than high initial seeding density.

Calcium-dependent regulation of caldesmon by an 11-kDa smooth muscle calcium-binding protein, caltropin.

Caldesmon from chicken gizzard muscle has been examined for its ability to interact with caltropin using affinity chromatography and the fluorescent probe acrylodan. The action of caltropin on the inhibitory effect of caldesmon on actomyosin ATPase was also studied. Like calmodulin, caltropin could release the inhibitory effect of caldesmon in the presence of Ca2+. Complete reversal was obtained when 1 mol of caltropin was added per mol of caldesmon. When caldesmon was applied to caltropin-Sepharose in the presence of Ca2+, most of the caldesmon was bound to the column and could be eluted with EGTA, indicating that there is a direct interaction between caldesmon and caltropin. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 504 nm. Addition of caltropin in the presence of Ca2+ resulted in a nearly 50% increase in fluorescence intensity, and this was accompanied by a blue shift in the emission maximum (i.e., lambda em,max 492 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of caltropin with labeled caldesmon indicated a strong affinity for this protein (Kd was in the order of 8 x 10(-8)-2 x 10(-7) M). However, when caltropin was added to labeled caldesmon in the presence of EGTA, there was no indication of any interaction. Caltropin was at least as potent as calmodulin, if not better, in reversing the inhibitory effect of caldesmon in the presence of calcium, making it a potential Ca2+ factor in regulating caldesmon in smooth muscle.

Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.

The effects of smooth muscle caldesmon on actin filament motility

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.

Phosphorylation of vascular smooth muscle caldesmon by endogenous kinase

Caldesmon was phosphorylated up to 1.2 molP i/mol using a partially purified endogenous kinase fraction. The phosphorylation site was within the C-terminal 99 amino acids. We were also able to phosphorylate caldesmon incorporated into native and synthetic smooth muscle thin filaments. Phosphorylation did not alter caldesmon binding to actin or inhibition or actomyosin ATPase. It also did not change Ca 2+ sensitivity in native thin filaments. Phosphorylated caldesmon bound to myosin less than unphosphorylated caldesmon, especially when the myosin was also not phosphorylated. This work did not support the hypothesis that caldesmon function is modulated by phosphorylation.