Regulation of vascular smooth muscle tone by caldesmon.

Caldesmon is an actin-binding protein present in smooth muscle cells that also inhibits actin-activated myosin ATPase activity. To assess the possible role of caldesmon in the regulation of smooth contraction, we investigated the effects of synthetic peptides on force directly recorded from single hyperpermeable smooth muscle cells of ferret aorta and portal vein. GS17C, a peptide that contains the residues from Gly651 to Ser667 of the caldesmon sequence plus an added cysteine at the C terminus, binds calmodulin in a Ca(2+)-dependent manner and also binds to F-actin but does not inhibit actomyosin ATPase activity (Zhan, Q., Wong, S.S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814). In cells in which Ca2+ was clamped at pCa 7.0, GS17C induced a dose-dependent contraction (EC50 = 0.92 microM) in aorta cells, whereas it evoked little or no contraction in portal vein cells. The GS17C-induced contraction in aorta cells was inhibited at higher Ca2+ concentrations (above pCa 6.6) and by pretreatment with calmodulin. Another peptide, C16AA, which contains the residues from Ala594 to Ala609 and does not bind actin or calmodulin, did not induce contraction. Our results strongly suggest that GS17C induces contraction by the displacement of the inhibitory region of endogenous caldesmon and, furthermore, that caldesmon present in these smooth muscle cells regulates contraction by providing a basal resting inhibition of vascular tone.     

Phosphorylation of caldesmon by cdc2 kinase

A recent report that mitosis-specific phosphorylation causes the nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678) suggests that this process may contribute to the major structural reorganization of the eukaryotic cell at mitosis. In this study we have demonstrated that smooth muscle caldesmon is phosphorylated in vitro by cdc2 kinase from mitotic phase HeLa cells to 1.2 mol of phosphate/mol of caldesmon. Tryptic maps showed three major phosphorylated spots and approximately equal amounts of phosphorylated Ser and Thr were identified. F-actin or calmodulin in the presence of Ca2+ blocks the phosphorylation of caldesmon. Phosphorylation of caldesmon greatly reduced its binding to F-actin. The phosphorylation sites were located in a 10,000-Da CnBr fragment at the COOH-terminal end of the caldesmon molecule known to house the binding sites for actin and calmodulin (Bartegi A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238). Our finding supports the model that phosphorylation of caldesmon by cdc2 kinase at mitosis may contribute to the disassembly of the microfilament bundles during prophase.     

Localization of the calmodulin- and the actin-binding sites of caldesmon

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Th?gersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.     

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.     

Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region

A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.     

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.     

Dissociation of the effect of caldesmon on the ATPase activity and on the binding of smooth heavy meromyosin to actin by partial digestion of caldesmon

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.     

Interaction between chicken gizzard caldesmon and tropomyosin

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.     

Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin

We have previously shown that inhibition of the ATPase activity of skeletal muscle myosin subfragment 1 (S1) by caldesmon is correlated with the inhibition of S1 binding in the presence of ATP or pyrophosphate (Chalovich, J., Cornelius, P., and Benson, C. (1987) J. Biol Chem. 262, 5711-5716). In contrast, Lash et al. (Lash, J., Sellers, J., and Hathaway, D. (1986) J. Biol. Chem. 261, 16155-16160) have shown that the inhibition of ATPase activity of smooth muscle heavy meromyosin (HMM) by caldesmon is correlated with an increase in the binding of HMM to actin in the presence of ATP. We now show, in agreement, that caldesmon does increase the binding of smooth muscle HMM to actin-tropomyosin while decreasing the ATPase activity. The effect of caldesmon on the binding of smooth HMM is reversed by Ca2+-calmodulin. Caldesmon strengthens the binding of smooth S1.ATP and skeletal HMM.ATP to actin-tropomyosin but to a lesser extent than smooth HMM.ATP. Furthermore, this increase in binding of smooth S1.ATP and skeletal HMM.ATP does not parallel the inhibition of ATPase activity. In contrast, in the absence of ATP, all smooth and skeletal myosin subfragments compete with caldesmon for binding to actin. Thus, the effect that caldesmon has on the binding of myosin subfragments to actin-tropomyosin depends on the source of myosin, the type of subfragment, and the nucleotide present. The inhibition of actin-activated ATP hydrolysis by caldesmon, however, is not greatly different for different smooth and skeletal myosin subfragments. Evidence is presented that caldesmon inhibits actin-activated ATP hydrolysis by attenuating the productive interaction between myosin and actin that normally accelerates ATP hydrolysis. The increased binding seen by some myosin subfragments, in the presence of ATP, may be due to binding of these subfragments to a nonproductive site on actin-caldesmon. The subfragments which show an increase in binding in the presence of ATP and caldesmon appear to bind directly to caldesmon as demonstrated by affinity chromatography.     

Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon

Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high Mr TMs) with higher Mr values and greater affinity for actin than the other (low Mr TMs); 2) presaturation of F-actin with high Mr TMs, but not with low Mr TMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high Mr TMs but also makes low Mr TMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high Mr TMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low Mr TMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high Mr TMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high Mr TMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.     

Caldesmon has two calmodulin-binding domains

Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. C. & Bryan, J., J. Biol. Chem. 261, 16155-16160, 1987; Yazawa, M., Yagi, K. & Sobue, K., J. Biochem. 102, 1065-1073, 1987). In the case of CNBr cleavage a 37 kDa calmodulin-binding fragment (CB40) was obtained. Both CT40 and CB40 contain a reactive thiol group, but these thiols are apparently in different environments as judged by the responses of attached fluorescent labels to calmodulin-binding. A comparison of the N-terminal sequences of CB40 and CT40 with the complete sequence of caldesmon shows that the two calmodulin-binding fragments in fact originate from different parts of the parent molecule. Thus there exist two calmodulin-binding sites in caldesmon, one in the N-terminal half and the other in the C-terminal half of the molecule. This is consistent with the recent finding that up to two calmodulin molecules can be crosslinked to each caldesmon molecule (Wang, C.-L.A., Biochem. Biophys. Res. Commun., 156, 1033-1038, 1988).     

Turkey gizzard caldesmon: molecular weight determination and calmodulin binding studies

Sedimentation equilibrium and sedimentation velocity measurements demonstrate that turkey gizzard caldesmon is an elongated molecule of molecular mass 75 +/- 2 kDa. The frictional ratio (2.14) is consistent with a prolate ellipsoid of axial ratio 24, corresponding to an apparent length and width of 516 and 21.5 A, respectively. As was previously determined for chicken gizzard caldesmon [Graceffa, P., Wang, C.-L.A., & Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202], this molecular weight is appreciably smaller than the value (approximately 135,000) estimated from the results of NaDodSO4 gel electrophoresis experiments. However, a significant difference between the true molecular weights of turkey and chicken gizzard caldesmons--75,000 versus 93,000--also points to probable molecular weight variations within the subclass. Binding measurements, based on perturbation of the intrinsic tryptophan fluorescence of caldesmon in the presence of calmodulin, show that the interaction between the two proteins is strongly ionic strength and temperature dependent. Dissociation constants of 0.075 and 0.38 microM were determined in solutions containing 0.1 and 0.2 M KCl, respectively, at 24.3 degrees C. Fluorescence emission spectra and fluorescence anisotropy excitation spectra indicate that the tryptophanyl residues of caldesmon are located in solvent-accessible regions of the molecule, where they exhibit a high degree of mobility even when calmodulin is bound.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells

alpha-Spectrin, myosin light chain kinase (MLCK), and caldesmon have been detected in the nuclei of rat liver cells by 125I-calmodulin overlay, immunoblotting, and immunocytochemical methods. alpha-Spectrin is localized in the nuclear matrix, nuclear envelope, and nuclear pores. It has also been detected inside the nuclei in the form of small aggregates. MLCK is present in the nuclear matrix, envelope, nucleoli, and in a nuclease extract (S1 subfraction) but not in the nuclear pores. Caldesmon shows a diffuse distribution pattern inside the nuclei but it is not present in the nucleoli. Since all these proteins are components of the actin-myosin motility systems the presence of actin in the different nuclear subfractions has also been investigated: actin is present in the nuclear matrix, nuclear envelope, nucleoli, and nuclear pores. Proliferative activation of rat liver cells in vivo by partial hepatectomy induces the increase of alpha-spectrin, MLCK, and actin in different nuclear subfractions. This, together with the increase of nuclear calmodulin at the same time after hepatectomy (Pujol, M. J., Soriano, M., Aligúe, R., Carafoli, E., and Bachs, O. (1989) J. Biol. Chem. 264, 18863-18865), indicates that nuclear calmodulin could activate a nuclear contractile system during proliferative activation. A 62-kDa protein (p62) which binds to calmodulin columns and shows immunological similarities to caldesmon is specifically located in the region surrounding the nuclear envelope and is associated with the heterochromatin.     

Cloning and expression of a smooth muscle caldesmon

Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data.     

Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.     

The N-terminal region of twitchin binds thick and thin contractile filaments: redundant mechanisms of catch force maintenance

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.     

Characterization of mitotically phosphorylated caldesmon.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.     

Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].