A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin.
ABSTRACT: Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.
Project description:Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation.
Project description:Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans. Although a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely unknown. Herein, we have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scp1, which most closely resembles transgelin and contains one calponin homology (CH) domain. We show that Scp1 is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains. Purified Scp1 binds directly to filamentous actin, cross-links actin filaments, and stabilizes filaments against disassembly. Sequences in Scp1 sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH domain. Overexpression of SCP1 suppresses sac6Delta defects, and deletion of SCP1 enhances sac6Delta defects. Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize the yeast actin cytoskeleton.
Project description:Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ~190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.
Project description:alpha-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of alpha-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous alpha-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained approximately 90% of their complement of alpha-calponin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca(2+)-induced contraction, with a half-maximal effect at approximately 3 microM alpha-calponin. Pretreatment of demembranated muscle strips with alpha-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca(2+)-independent, since exogenous alpha-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of alpha-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca(2+)-induced and Ca(2+)-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type alpha-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated alpha-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of alpha-calponin as a regulatory site of phosphorylation.
Project description:Calponin and caldesmon, constituents of smooth-muscle thin filaments, are considered to be potential modulators of smooth-muscle contraction. Both of them interact with actin and inhibit ATPase activity of smooth- and skeletal-muscle actomyosin. Here we show that calponin and caldesmon could bind simultaneously to F-actin when used in subsaturating amounts, whereas each one used in excess caused displacement of the other from the complex with F-actin. Calponin was more effective than caldesmon in this competition: when F-actin was saturated with calponin the binding of caldesmon was eliminated almost completely, whereas even at high molar excess of caldesmon one-third of calponin (relative to the saturation level) always remained bound to actin. The inhibitory effects of low concentrations of calponin and caldesmon on skeletal-muscle actomyosin ATPase were additive, whereas the maximum inhibition of the ATPase attained at high concentration of each of them was practically unaffected by the other one. These data suggest that calponin and caldesmon cannot operate on the same thin filaments. CA(2+)-calmodulin competed with actin for calponin binding, and at high molar excess dissociated the calponin-actin complex and reversed the calponin-induced inhibition of actomyosin ATPase activity.
Project description:Calponins form an evolutionary highly conserved family of actin filament-associated proteins expressed in both smooth muscle and non-muscle cells. Whereas calponin-1 and calponin-2 have already been studied to some extent, little is known about the role of calponin-3 under physiological conditions due to the lack of an appropriate animal model. Here, we have used an unbiased screen to identify novel proteins implicated in signal transduction downstream of the precursor B cell receptor (pre-BCR) in B cells. We find that calponin-3 is expressed throughout early B cell development, localizes to the plasma membrane and is phosphorylated in a Syk-dependent manner, suggesting a putative role in pre-BCR signaling. To investigate this in vivo, we generated a floxed calponin-3-GFP knock-in mouse model that enables tracking of cells expressing calponin-3 from its endogenous promoter and allows its tissue-specific deletion. Using the knock-in allele as a reporter, we show that calponin-3 expression is initiated in early B cells and increases with their maturation, peaking in the periphery. Surprisingly, conditional deletion of the Cnn3 revealed no gross defects in B cell development despite this regulated expression pattern and the in vitro evidence, raising the question whether other components may compensate for its loss in lymphocytes. Together, our work identifies calponin-3 as a putative novel mediator downstream of the pre-BCR. Beyond B cells, the mouse model we generated will help to increase our understanding of calponin-3 in muscle and non-muscle cells under physiological conditions.
Project description:Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131-228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution. Our data indicates that the free calponin peptide is largely unstructured in solution, although four short amino-acid stretches corresponding to residues 140-146, 159-165, 189-195, and 199-205 display the propensity to form ?-helices. The presence of four sequential transient helices probably provides the conformational malleability needed for the promiscuous nature of this region of calponin. We identified all amino acids involved in actin binding and demonstrated for the first time, to our knowledge, that the N-terminal flanking region of Lys(137)-Tyr(144) is an integral part of the actin-binding site. We have also delineated the second actin-binding site to amino acids Thr(180)-Asp(190). Ca(2+)-calmodulin binding extends beyond the previously identified minimal sequence of 153-163 and includes most amino acids within the stretch 143-165. In addition, we found that calmodulin induces chemical shift perturbations of amino acids 188-190 demonstrating for the first time, to our knowledge, an effect of Ca(2+)-calmodulin on this region. The spatial relationship of the actin and calmodulin contacts as well as the transient ?-helical structures within the regulatory region of calponin provides a structural framework for understanding the Ca(2+)-dependent regulation of the actin-calponin interaction by calmodulin.
Project description:The actin-binding protein calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood. To investigate this underlying physical mechanism, we studied an in vitro model system of cross-linked actin using bulk rheology. Networks with basic calponin exhibited a delayed onset of strain stiffening (10.0% without calponin, 14.9% with calponin) and were able to withstand a higher maximal strain before failing (35% without calponin, 56% with calponin). Using fluorescence microscopy to study the mechanics of single actin filaments, we found that calponin increased the flexibility of actin filaments, evident as a decrease in persistence length from 17.6 ?m without to 7.7 ?m with calponin. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, and suggest that calponin stabilization of actin networks can be explained purely by changes in single-filament mechanics. We propose a model in which calponin stabilizes actin networks against shear through a reduction of persistence length of individual filaments.
Project description:Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.
Project description:Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (?nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (??M) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4–6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1–3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4–6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.