Casein kinase II phosphorylation of caldesmon downregulates myosin-caldesmon interactions

It is well-known that caldesmon (CaD) is a substrate for casein kinase II (CKII), and the phosphorylation of CaD by CKII regulates the interaction of CaD with myosin. However, the functionally relevant CKII phosphorylation site(s) on CaD and the precise role of CaD phosphorylation by CKII in mediating CaD's function have remained elusive. In this study, we demonstrate that Ser-26 is the major CKII phosphorylation site on CaD, while Ser-73 is of relatively minor importance. Moreover, the phosphorylation of Ser-26 and Ser-73 reduced CaD's ability to bind myosin by 45% and 27%, respectively, suggesting that the interaction of CaD with myosin is downregulated, at least in part, by the phosphorylation of these serine residues by CKII. Our results also demonstrate that there are at least four myosin-binding motifs within the amino-terminal region of CaD, located between residues 1-23, 34-43, 44-53, and 86-115, respectively. The myosin-binding motif between residues 44-53 contributes to strong myosin binding, while the three other myosin-binding motifs are responsible for weak myosin binding. The sequences between residues 24-33 and 54-85 on CaD are not required for the binding of CaD to myosin; thus, both Ser-26 and Ser-73 are located outside of the myosin-binding motifs. It is therefore likely that the downregulation of myosin-CaD interactions by CKII phosphorylation is due to phosphorylation-induced conformational changes in the adjacent myosin-binding motifs on CaD, rather than by the direct modification of these myosin-binding motifs by CKII.     

A novel regulatory protein that affects the functions of caldesmon and myosin light chain kinase.

A caldesmon (CaD)-binding protein of about 65 kDa (by SDS-PAGE) was purified from smooth muscle of chicken gizzard. The 65-kDa protein prevented the inhibitory effect of CaD on the ATP-dependent interaction between actin and myosin. Unlike the case with calmodulin (CaM), Ca2+ was not required for this effect. As reported in the preceding communication, myosin light chain kinase (MLCK), another well characterized protein that binds CaM, has CaD-like activity that modulates the interaction by binding to actin. The 65-kDa protein was also effective in relieving the modulation, while leaving unaffected the kinase activity that phosphorylates the light chain of smooth muscle myosin.     

Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration

Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.     

Macrophage caldesmon is an actin bundling protein.

A rapid purification procedure was developed for the isolation of caldesmon (CaD) from rabbit alveolar macrophage. The purified protein migrated with an apparent M(r) of 74,000 +/- 4000 on SDS-PAGE and cross-reacted with anti-gizzard CaD antibodies. A higher M(r) isoform was isolated from chicken gizzard. Their actin-binding parameters and effects on actomyosin-ATPase activity were investigated under identical experimental conditions. Electron microscope studies revealed that macrophage CaD was able to cross-link actin filaments into both networks and bundles. Compact F-actin bundles were predominantly or exclusively seen at cross-linker to actin molar ratios in the 1:20 to 1:10 range. Apparent K(a) at extrapolated saturation of the CaD-binding sites on F-actin was 1.2 x 10(6) M(-1) for macrophage CaD and 1.6 x 10(6) M(-1) for chicken gizzard CaD. CaD from either source was able to stimulate the actin-activated ATPase activity of macrophage myosin. Unexpectedly, chicken gizzard CaD also increased the ATPase activity of gizzard myosin. The degree of stimulation was approximately doubled in the presence of a large excess of Ca(2+)-calmodulin but was unaffected by the presence of macrophage tropomyosin. However, macrophage CaD did not behave as a Ca(2+)- and calmodulin-regulated actin-binding protein. These results, together with published data on other well-characterized actin bundling proteins, suggest that nonmuscle CaD could be essentially involved in the formation and organization of actin bundles at adhesion sites and cell surface projections. However, they afforded no evidence that the macrophage isoform might play a specific role in the Ca(2+)-dependent regulation of actin and myosin II interactions.     

The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.     

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

Background

Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.

Methods

To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (F_unb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin.

Results

F_unb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the F_unb (0.06 ± 0.01 pN). Inspection of the traces of the F_unb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.

Conclusions

CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.

General significance

This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.     

Phosphorylation of Caldesmon at Sites between Residues 627 and 642 Attenuates Inhibitory Activity and Contributes to a Reduction in Ca-Calmodulin Affinity

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca²⁺-calmodulin. We investigated the effects of phosphorylation by p²¹-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca²⁺-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca²⁺-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca²⁺-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca²⁺-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity.     

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

Summary The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.     

Tyrosine phosphorylation of caldesmon is required for binding to the Shc.Grb2 complex

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, transforming mutant of the epidermal growth factor receptor. This oncogene has been shown to be sarcomagenic in vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M. J., Lingle, W. L., Salisbury, J. L., and Maihle, N. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11351-11356) showed that expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protein. This phosphorylation is transformation-associated, and the phosphorylated form of CaD is associated with a signaling complex consisting of Shc, Grb2, and Sos in transformed fibroblasts. To identify the tyrosine phosphorylation site(s) in the CaD molecule and to further elucidate the functional role of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we have generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cells (an immortalized chicken embryo fibroblast cell line), and transient transfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major sites of tyrosine phosphorylation on CaD. Interestingly, Tyr-27 is located within the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies also show that the tyrosine phosphorylation of CaD enhances its binding to the Shc.Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-165 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc. Grb2 complex. Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc.Grb2 complex.     

Role of the basic C-terminal half of caldesmon in its regulation of F-actin: comparison between caldesmon and calponin

We previously reported that caldesmon (CaD), together with tropomyosin (TM), effectively protects actin filaments from gelsolin, an actin-severing protein. To elucidate the structure/function relationship of CaD, we dissected the functional domain of CaD required for the protection. The basic C-terminal half of rat nonmuscle CaD (D3) inhibits gelsolin activity to the same degree as intact CaD, although a smaller C-terminal region of D3 does not. This smaller C-terminal region contains the minimum regulatory domain responsible for the inhibition of actomyosin ATPase, and for the binding to actin, calmodulin and TM. These results suggest that the domain responsible for the inhibition of gelsolin activity lies outside the minimum regulatory domain, and that the positive charge possessed by the C-terminal half of CaD is important for its interaction with actin. Moreover, while the D3 fragment promotes the aggregation of F-actin into bundles as reported previously, this bundle formation is inhibited by the acidic N-terminal half of CaD, as well as by poly-l-glutamate. It seems likely that the acidic N-terminal half of CaD neutralizes the superfluous basic feature of the C-terminal half. A comparison between D3 and calponin, another actin-binding protein that is also basic and has similar actin-regulatory activities, is also discussed.     

Tropomyosin and caldesmon regulate cytokinesis speed and membrane stability during cell division

The contractile ring and the cell cortex generate force to divide the cell while maintaining symmetrical shape. This requires temporal and spatial regulation of the actin cytoskeleton at these areas. We force-expressed misregulated versions of actin-binding proteins, tropomyosin and caldesmon, into cells and analyzed their effects on cell division. Cells expressing proteins that increase actomyosin ATPase, such as human tropomyosin chimera (hTM5/3), significantly speed up division, whereas cells expressing proteins that inhibit actomyosin, such as caldesmon mutants defective in Ca(2+)/calmodulin binding (CaD39-AB) and in cdk1 phosphorylation sites (CaD39-6F), divide slowly. hTM5 and hTM5/3-expressing cells lift one daughter cell off the substrate and twist. Furthermore, CaD39-AB- and CaD39-6F-expressing cells are sensitive to hypotonic swelling and show severe blebbing during division, whereas hTM5/3-expressing cells are resistant to hypotonic swelling and produce membrane bulges. These results support a model where Ca(2+)/calmodulin and cdk1 dynamically control caldesmon inhibition of tropomyosin-activated actomyosin to regulate division speed and to suppress membrane blebs.     

Smooth muscle alpha-tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament

To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard alphaTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm). They were labeled with benzophenone maleimide (BPM) and UV-irradiated on thin filaments. One chain of BPM-56Tm and two chains of BPM-100Tm crosslinked to CaD. Only BPM-100Tm crosslinked to actin in the absence and presence of CaD and binding of low ratios of myosin subfragment 1 (S1) prevented the crosslinking. Tm-S1 crosslinks were produced when actin.Tm was saturated with S1. Thus, CaD on the actin.Tm filament is located <10 A away from Tm amino acids 56 and 100; in the closed state of the actin.Tm filament, Tm residue 100 is located close to the actin surface and is moved further away in the S1-induced open state; in the open state, S1 binds close to Tm.     

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.     

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.     

Caldesmon is a cytoskeletal target for PKC in endothelium

We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise.     

The major myosin-binding site of caldesmon resides near its N-terminal extreme

The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.     

C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.     

Overexpression of human fibroblast caldesmon fragment containing actin- , Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.