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Molecular chaperones are commonly identified by their ability to suppress heat-induced protein aggregation. The muscle-specific molecular chaperone UNC-45B is known to be involved in myosin folding and is trafficked to the sarcomeres A-band during thermal stress. Here, we identify temperature-dependent structural changes in the UCS chaperone domain of UNC-45B that occur within a physiologically relevant heat-shock range. We show that distinct changes to the armadillo repeat protein topology result in exposure of hydrophobic patches, and increased flexibility of the molecule. These rearrangements suggest the existence of a novel thermosensor within the chaperone domain of UNC-45B. We propose that these changes may function to suppress aggregation under stress by allowing binding to a wide variety of aggregation prone loops on its client., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC-45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Unconventional myosin VIIA (Myo7a) has been known to associate with hereditary deafness. Here we present a novel function of Myo7a by identifying that Myo7a directly interacts with integrin β5 subunit and regulates cell adhesion and motility in an integrin-dependent manner. We found that Myo7a bound to the cytoplasmic tail of integrin β5. Further, we pinpointed an integrin-binding domain at F3 of the first FERM domain and F1 of the second FERM domain. Functionally, Myo7a-induced cell adhesion and migration were mediated by integrin αvβ5. These findings indicated that Myo7a interacts with integrin β5 and selectively promotes integrin αvβ5-mediated cell migration., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cell-generated traction forces induce integrin activation, leading to focal adhesion growth and cell spreading. It remains unknown, however, whether integrin activation feeds back to impact the generation of cytoskeletal tension. Here, we used elastomeric micropost arrays to measure cellular traction forces in wildtype and integrin-null cells. We report that activation of β1 but not β3 integrin, by either increasing density of immobilized fibronectin or treating with manganese, elicited fibroblast spreading and cytoskeletal tension. Furthermore, this force generation required Rho kinase and myosin activity. These findings suggest that integrin activation and cell traction forces comprise a bi-directional signaling unit of cell adhesion., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Rho-family small GTPases regulate epithelial cell sheet migration by organizing actin and myosin during wound healing. Here, we report that Pak3, but not Pak1, is a downstream target protein for Rac1 in wound closure of the Drosophila larval epidermis. Pak3-deficient larvae failed to close a wound hole and this defect was not rescued by Pak1 expression, indicating differential functions of the two proteins. Pak3 localized to the wound margin, which selectively required Rac1. Pak3-deficient larvae showed severe defects in actin-myosin organization at the wound margin and in submarginal cells, which was reminiscent of the phenotypes of Rac1-deficient larvae. These results suggest that Pak3 specifically mediates Rac1 signaling in organizing actin and myosin during Drosophila epidermal wound healing., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cell movement requires forces generated by non-muscle myosin II (NM II) for coordinated protrusion and retraction. The Cdc42/Rac effector MRCK regulates a specific actomyosin network in the lamella essential for cell protrusion and migration. Together with the Rho effector ROK required for cell rear retraction, they cooperatively regulate cell motility and tumour cell invasion. Despite the increasing importance of ROK inhibitors for both experimental and clinical purposes, there is a lack of specific inhibitors for other related kinases such as MRCK. Here, we report the identification of chelerythrine chloride as a specific MRCK inhibitor. Its ability to block cellular activity of MRCK resulted in the specific loss of NM II-associated MLC phosphorylation in the lamella, and the consequential suppression of cell migration., (Copyright © 2011. Published by Elsevier B.V.)

Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B(+/-) strain (8.73+/-1.10 Hz vs 16.56+/-1.26 Hz, P<0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B(+/-)) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V(max) during force maintenance., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex., (2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin-1 at epithelial adherens junctions. Nectin-1 serves as a mediator of cell-to-cell spread for Herpes simplex virus 1 (HSV-1). We analyzed the role of AF6 protein in the viral spread and nectin-1 clustering at cell-cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV-1 spreading, however, the clustering of nectin-1 at cell-cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV-1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E-cadherin-dependent cell adhesion.

During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.

In a gradient of chemoattractant, Dictyostelium cells are orientated with their front directed toward the source and their tail pointing into the opposite direction. The front region is specified by the polymerization of actin and the tail by the recruitment of filamentous myosin-II. We have dissected these front and tail responses by exposing cells to an upshift of cyclic AMP. A sharp rise and fall of polymerized actin within 10s is accompanied by the recruitment of proteins involved in turning actin polymerization on or off. The cortical accumulation of myosin-II starts when the front response has declined, supporting the concept of divergent signal transmission and adaptation pathways.

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.

The formation of transition state mimics of phosphoryl transfer reactions with the metal oxoanion vanadate is a powerful technique in macromolecular crystallography. The tendency of vanadate to form pentacovalent complexes exhibiting trigonal bipyramidal geometry makes this compound a close approximation of the transition state for such reactions. In many cases, vanadate complexes provide the most accurate visualization of the transition state that can be reasonably achieved. A survey of the Protein Data Bank reveals that a relatively small number of structures (39, representing 23 unique proteins) include vanadate, yet these structures represent four of the six E.C. categories of enzymes, and were obtained in crystals with pH values ranging from 5.0 to 7.8. Vanadate has additional advantages over other compounds such as aluminum fluoride, beryllium fluoride and nitrate used for visualization of transition state mimics in that vanadate readily forms covalent bonds with a variety of ligands and has produced a wider variety of transition state mimics. Given the hundreds of crystal structures that have been solved for phosphoryl transfer enzymes, it is surprising that vanadate has not been used more frequently for visualization of transition state analogs. We propose that an opportunity exists for vanadate to become a more commonly utilized component of the macromolecular crystallographer's toolbox.

Carp and scallop myosin and their subfragments (S-1 and rod) were reacted with glucose to investigate the effect of non-enzymatic glycosylation on the functionality of myosin. The filament-forming ability of the myosin rod diminished with the progress of non-enzymatic glycosylation and myosin became soluble in 0.1 M NaCl. The inhibition of the self-assembly of myosin molecules occurred chemically as a result of the increase in negative charge repulsion among myosin molecules and, further, physically as a result of the introduction of the glycosyl units into the surface of the rod region.

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.

The LIM only protein Lmo2 plays an important role in hematopoiesis and leukemogenesis. Lmo2 acts as a bridging molecule between components of hematopoietic gene regulatory protein complexes. We used the yeast two-hybrid system to identify novel Lmo2 interacting proteins and found that the AF6 protein binds to Lmo2. AF6 is a recurrent fusion partner of MLL, the human homolog of Drosophila trithorax chromatin remodeling protein that is involved in childhood leukemia and mixed lineage leukemia. Our data support the notion that recurrent fusion partners of chimeric MLL proteins recruit hematopoietic gene regulatory complexes.

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.

The study addressed the functional link between remodelling of the actomyosin cytoskeleton in pancreatic beta-cells and the regulation of insulin secretion. Confocal microscopy revealed that myosin heavy chain (MHC) IIA co-localized very well with filamentous (F)-actin in RINm5F cells but MHCIIB did not. Subcellular localization of MHCIIB was not altered by stimulation with 30 mM KCl (which evokes Ca(2+)-dependent insulin secretion). In contrast MHCIIA redistributed in a manner similar to F-actin, especially towards the apical surface, but also away from peripheral regions towards cell contact points on the basal surface. Finally, Ca(2+)-dependent insulin secretion was inhibited by stabilization of actin filaments with jasplakinolide. The results support a role for the MHCIIA/actin cytoskeleton in regulating insulin secretion.

Myosin Is are associated with specific membranes, however, the mechanism for regulating their intracellular localization is unclear. As a first step towards understanding this mechanism, membrane rebinding assays using Dictyostelium myoB were performed. Crude, cytosolic myoB bound to intact, but not to NaOH-treated plasma membranes. In contrast, partially purified myoB binds to both intact and NaOH-treated plasma membranes. Chemical cross-linking of cytosolic myoB yielded several products, whereas none were found with the partially purified myoB. These results suggest a model where proteins regulating the specific binding of myoB to the plasma membrane may exist both in the cytosol and on the plasma membrane.

Recent structural evidence indicates that the light chain domain of the myosin head (LCD) bends on the motor domain (MD) to move actin. Structural models usually assume that the actin-MD interface remains static and the possibility that part of the myosin working stroke might be produced by rotation about the acto-myosin interface has been neglected. We have used an optical trap to measure the movement produced by proteolytically shortened single rabbit skeletal muscle myosin heads (S-1(A1) and S-1(A2)). The working stroke produced by these shortened heads was more than that which the MD-LCD bend mechanism predicts from the full-length (papain) S-1's working stroke obtained under similar conditions. This result indicates that part of the working stroke may be caused by motor action at the actin-MD interface.

The dynamic assembly/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization. Phosphorylation of three threonines, 1823, 1833 and 2029, in the tail of Dictyostelium discoideum myosin II has been implicated in control of myosin filament assembly. By systematically replacing the three threonines to aspartates, mimicking a phosphorylated residue, we found that position 1823 is the most critical one for the regulation of myosin filament formation and in vivo function. Surprisingly, a single charge change is able to perturb filament formation and in vivo function of myosin II.

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with NG-nitro-L-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.

Experiments were performed on glycerol-extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin-labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.Vi-->AM.ADP in the ATP hydrolysis cycle. It was found that the essential -SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long-lived free radicals located partly on SH-1 sulphydryls.

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.

Ras interacts with a number of effector molecules to achieve its prolific signalling. Based on iterative sequence profile and motif searches of databases a novel family of Ras-binding domains was recently identified (Ponting and Benjamin, Trends Biochem. Sci. 21: 422-425, 1996). Among them the rat unconventional myosin and Rho-GTPase-activating protein myr 5 was predicted to contain a Ras-binding domain at its N-terminus. Here we report that direct binding experiments between the proposed Ras-binding domain of myr 5 and Ras failed to demonstrate any interaction. Molecular modelling suggests that this domain in myr 5 adopts a similar folding topology as the Ras-binding domain of Raf kinase. However, unlike the Ras-binding domain of Raf kinase, the myr 5 domain lacks the positive surface charges necessary for binding the negatively charged Ras contact site. This result exemplifies the functional diversity of similar structures and suggests that the identified Ras-binding motif does not reliably predict Ras-binding domains.

Myosin accelerates plasminogen activation by tissue-type plasminogen activator (tPA), and is degraded extensively by plasmin. Myosin binds both tPA and plasminogen, and enhances activation of des1-77-plasminogen by tPA but not by urokinase-type plasminogen activator (uPA). Myosin decreases K(M) and increases k(cat) for des1-77-plasminogen activation by tPA, to yield catalytic efficiencies in excess of 8000 M-1 s-1. The effect of myosin is attributed to its C-terminal portion, the myosin rod. With a K(M) of 3 microM, myosin is a high-affinity substrate for plasmin. The findings indicate that myosin is a cofactor for plasminogen activation and a substrate for plasmin.

Titin ( also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show -- using in vitro motility and binding assays -- that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin-thin filament interaction occurs in a calcium-dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.

The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.

The functional significance of myosin light chains in vertebrate striated muscle is an issue of interest any myosin species selectivity modified by papain or trypsin in their LC1 and LC2 light chains are potentially useful for further investigation. We therefore determined the cleavage sites resulting in the (T)-LC1', (P)-LC1' and (T)-LC2'species. Sequence analysis of (T)-LC1' indicated that the cleavage point in LC1 is at Lys7. Under appropriate conditions papain rapidly cleaves a short N-terminal segment from myosin light chain 1 and produces a new isozyme specifically modified in its essential light chain 1. The cleavage occurred at either Ala11, Ala12, or Ala13, the Ala11 cleavage being the most frequent. Trypsin was used to produce a myosin species with a regulatory light chain 2 specifically truncated of a short N-terminal segment. The cleavage was specific at Arg8 with no indication of other significant cleavage sites in this LC2. The effects of trypsin and papain on myosin light chains are different, indicating different proteolytic specificities. None of these modifications, including (CT)-LC2" cleavage at Phe19, changed the K(+)-EDTA- and Ca(2+)-ATPase activities of monomeric myosin significantly, indicating that LC1 and LC2 N-terminal have little or no direct influence on the active site. An electric birefringence study also showed that these modified species retained their average shape and flexibility. These observations are essential in showing that the role of light chain extremities is expressed only in the presence of a minimum of structural organization (filament or acto-myosin complex).

In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca(2+)-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC20). GTP gamma S does not potentiate thiophosphorylation of MLC20, but does inhibit its dephosphorylation. Pretreatment with C. botulinum exotoxin C3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S. These results indicate that rho is involved in the GTP gamma S-induced enhancement of Ca(2+)-dependent MLC20 phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC20.

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)-actin complexed with BeFx. The cleavage had little effect on actin-actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F-actin was reduced by about 10 degrees C by this cleavage. The in vitro motility and Vmax, but not Km, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F-actin. The binding of tropomyosin to actin was unchanged by this modification.

Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.

The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of Pi formation, due to AM.ADP.Pi or Pi, as shown in the scheme: AM+ATP<-->A.M.ATP<-->AM.ADP.Pi<-->AM.ADP+Pi<-->AM+ADP. Therefore, in the steady state, either AM.ADP.Pi or AM.ADP or both predominate. To determine which, we studied the reaction using a Pi binding protein (from E. coli) labeled with a fluorophore such that it is specific and sensitive to free Pi [Brune, M. et al. (1994) Biochemistry 33, 8262-8271]. We show that the Pi bursts with myofibrillar ATPases (calcium-activated or not, or crosslinked) are due entirely to protein bound Pi. Thus, with myofibrillar ATPases the AM.ADP.Pi state predominates.

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect cells using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln945-Asp991 are critical for the formation of the two-headed structure. The actin activated ATPase activity of myosin mutants having a two-headed structure was activated by phosphorylation while that of myosin mutants that failed to form the two-headed structure was completely independent of phosphorylation. These results suggest that the two-headed structure is critical for the phosphorylation-dependent regulation.

Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies. Alternatively, the interaction between the two heads of the myosin molecule may influence the inhibition of LC20 exchange. These results suggest that LC20 interacts with both LC17 and the heavy chain, and the interaction between LC20 and LC17 is involved in the activation of actin-activated ATPase activity of smooth muscle myosin.

Twenty-five years after its proposal, the swinging theory of muscular contraction, in which the majority of scientists in the field have blindly believed, has not yet been verified. Rapidly growing experimental evidence indicates that the myosin heads do not swing. It is time to look for an alternative mechanism. Data is presented indicating that water is liberated during tension development and the extent to which it is released appears to affect the degree of tension. Since water can move (because of acquired extra energy, involvement in hydration forces etc.), it might cause protein movement.

14-3-3 proteins appear to play a critical role in Ca(2+)-stimulated secretion in permeabilized chromaffin cells. 14-3-3 proteins have been reported to be both stimulators and inhibitors of protein kinase C (PKC). We have found that 14-3-3 proteins, isolated on the basis of their ability to enhance secretory activity, stimulated histone phosphorylation by PKC, but they had no effect on myosin light chain phosphorylation by PKC. 14-3-3 proteins were also found to inhibit the rate of [32P]histone dephosphorylation but not the rate of [32P]myosin light chain dephosphorylation. Cross-linking experiments and affinity chromatography demonstrated that 14-3-3 proteins bind to histones. These results suggest that at least some of the reported effects of 14-3-3 proteins on PKC activity may result from 14-3-3 proteins binding to histone.

Purified chicken gizzard myosin light chain kinase (MLCK) analyzed by anion-exchange high-performance liquid chromatography (HPLC) can be consistently resolved into three well separated peaks, termed alpha, beta, gamma. These peaks are shown to correspond to differently charged forms of MLCK with the charge difference between alpha and beta twice as large as between beta and gamma. The isoelectric point and elution position of the peaks as well as their amplitudes are modified by phosphorylation or by autophosphorylation of MLCK suggesting that the observed charge differences are related to their different phosphate content. The three forms appear to have similar apparent affinity for both the substrates, ATP and the isolated regulatory light chain, but their specific activities are different.

The majority of protozoan myosins I possess tail domains composed of three distinct and conserved regions of sequence, referred to as tail homology regions 1, 2 and 3 (TH.1, TH.2 and TH.3). While the N-terminal approximately half of the tail (corresponding to TH.1) has been implicated in membrane binding, all or some portion of the C-terminal approximately half of the tail (corresponding to TH.2 plus TH.3) has been implicated in binding to F-actin in a nucleotide-insensitive fashion. Here we show, using fusion proteins containing portions of the Dictyostelium myosin IC (myoC) tail domain and F-actin sedimentation assays, that the ability of the myoC tail to bind to actin resides entirely within the glycine- and proline-rich TH.2 domain. The src-like TH.3 domain does not bind to actin, nor does it augment the binding properties of the TH.2 domain. In addition to defining more precisely the location of the actin binding site in the tail domain of a protozoan myosin I, these results have implications for the function of the src-like TH.3 domain in myosins I and other proteins.

A 3.5 kb cDNA clone was isolated from bovine adrenal gland cDNA library. The clone contained a full-length 3.1 kb open reading frame, encoding a novel myosin I. The deduced amino acid sequence was highly homologous to other known myosin Is in the N-terminal 2 kb region which corresponds to the myosin head domain, while no strong homology was detected in the tail region. The head-tail junction contained the Ca(2+)-independent calmodulin binding consensus sequence, suggesting that the novel myosin I binds calmodulin. This was confirmed by calmodulin overlay which showed the binding of 125I-calmodulin to the recombinant myosin I expressed in E. coli. Northern blots with probes from head and tail regions of this myosin I revealed that this novel myosin I is widely distributed among various tissues.

Calponin is a thin filament-associated protein in smooth muscle that has been shown to bind actin, tropomyosin and calmodulin, and has been implicated to play a role in regulation of smooth muscle contractility. Using a centrifugation assay we found that calponin interacts with unphosphorylated filamentous smooth muscle myosin. We found that this calponin-myosin interaction is reversed by Ca(2+)-CaM, and depends on ionic strength. At 50 mM NaCl the binding constant and the stoichiometry of this interaction were estimated to be 2 x 10(6) M-1, and 1.2-2.4 calponin per myosin, respectively. We suggest that the calponin-myosin interaction could be involved in regulation of smooth muscle contractility by anchoring myosin to actin.

Phosphorylation of myosin light chain kinase by a Ca(2+)-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTP gamma S and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca(2+)-dependent phosphorylation of these two contractile proteins in smooth muscle.

Two beta-actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine-374 replaced with serine (C374S), as well as the wild-type beta-actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro 'motility assay'. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild-type actins.

Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for periods of 55-61 days by means of an implantable neuromuscular stimulator. Gel electrophoresis clearly demonstrated the presence in stimulated muscles of slow myosin light and heavy chains, although fast isoforms were still present in all cases. Thus, contrary to previous reports, induction of slow myosin isoforms does occur in this, as in other, mammalian species. The time course of the response to stimulation appears to be more extended than that seen in the rabbit.

The addition of 15 mM caffeine to saponin-skinned human muscle fibers from M. vastus lateralis caused in the presence of 2 mM ATP an approx. 2-fold stimulation of respiration with glutamate+malate. This effect can be abolished by either the addition of the Ca2+ chelator EGTA, the inhibitor of Ca2+ transport Ruthenium red and the inhibitor of the myosin ATPase vanadate. The caffeine concentration dependency of respiration of fibers coincided with the caffeine-caused stimulation of myosin ATPase activity. The activation of oxidative phosphorylation in saponin-skinned human muscle fibers by caffeine can be explained by a stimulation of myosin ATPase caused by Ca2+ release from sarcoplasmic reticulum.

The finding that V1 cardiac myosin is expressed in masticatory skeletal muscles of the rabbit provided a unique opportunity for comparing the hormonal regulation of V1 in skeletal and cardiac muscles. Thyroid hormones had no significant effect on the postnatal expression of V1 in masticatory muscles, but increased this expression in cardiac ventricles. In contrast, androgenic hormones reduced V1 expression in masticatory muscles, but did not affect it significantly in cardiac ventricles. Modulation of V1 gene transcription in striated muscle is thus shown here to depend both on the target muscle and on the hormone.

Antibodies, obtained from myasthenia gravis patients, with reactivity for an immunodominant region of the nicotinic acetylcholine receptor were found to also react with muscle myosin. Since amino acid sequence analyses have previously suggested possible serological relationships between AChR and a head region sequence of myosin heavy chain, cross-reactive antibodies were examined for their ability to interfere with ATPase activities associated with this region of myosin. Results indicated that AChR-specific antibodies purified from MG patient serum by binding to and elution from antigen columns were found to inhibit Ca(2+)-dependent, myosin-associated ATPase activity; interestingly, this inhibition appeared to be relatively selective in that neither K+(EDTA)-dependent nor Mg(2+)-dependent ATPase activities were sensitive to antibody-mediated interference.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.