Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

Characterization of calcium-binding light chain as a Ca(2+)-receptive subunit of Physarum myosin.

Physarum myosin is uniquely under an inhibitory Ca(2+)-regulation in the ATP-dependent interaction with actin [Kohama (1990) Trends Pharmacol. Sci. 11, 433-435, for review]. Calcium-binding light chain (CaLc) has been suggested to be of primary importance to the control from its amino acid sequence [Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313]. To provide a biochemical basis for this suggestion, the Ca-binding capacity of CaLc and its Kd for Ca2+ were measured. The Ca-binding properties of CaLc allowed those of Physarum myosin to be explained in terms of CaLc. However, the mode of Ca(2+)-regulation by CaLc differs according to the enzyme upon which Ca-sensitivity is confered by CaLc, i.e., CaLc activated bovine phosphodiesterase activity and inhibited Physarum myosin ATPase activity, with the same Kd in microM levels. Thus, CaLc appears to work as a mere Ca-receptive subunit in Physarum myosin, with the secret of the inhibition lying in other subunits. CaLc was also shown to belong to a family of alkali light chains (AlLc) by allowing it to bind skeletal myosin as a substitute for its AlLc. Therefore, present study is the first biochemical indication that the AlLc family is involved in regulating the myosin function.     

Identification of an activator protein for myosin light chain kinase as the Ca2+-dependent modulator protein

Myosin light chain kinase which phosphorylates g2 light chain of skeletal muscle myosin requires an activator for the activity (Yazawa, M., and Yagi, K (1977) J. Biochem. (Tokyo) 82, 287-289). This activator has now been identified as the modulator protein known to be a Ca2+-dependent regulator for phosphodiesterase, adenylate cyclase, and ATPases. The identification is based on the quantitative cross-reactivity of muscle activator protein and brain modulator protein in activating myosin light chain kinase and brain phosphodiesterase and identical properties of both proteins in regard to sensitivities to Ca2+, UV absorption spectra, UV absorption difference spectra with or without Ca2+, and mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of modulator protein, the activity of myosin light chain kinase was reversibly controlled by the physiological concentration of Ca2+. We suggest that two Ca2+-receptive proteins, i.e. modulator protein and troponin-C, may play roles in the contraction-relaxation cycle of skeletal muscle.     

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.     

Ca2+ binding to myosin regulatory light chain affects the conformation of the N-terminus of essential light chain and its binding to actin

We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.     

Environmental pollutant Cd2+ biphasically and differentially regulates myosin light chain kinase and phospholipid/Ca2+-dependent protein kinase

Cd2+ was found to mimic effectively, potentiate and antagonize the stimulatory action of Ca2+ on myosin light chain kinase (MLCK) and phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK, or protein kinase C). PL-Ca-PK, however, was slightly less sensitive to Cd2+ regulation than was MLCK. Cd2+ also biphasically regulates (i.e., stimulation followed by inhibition) phosphorylation, in the homogenates of the rat caudal artery, of myosin light chain and other endogenous proteins catalyzed by MLCK and PL-Ca-PK. The activation by Cd2+ of MLCK was inhibited by anticalmodulins (e.g., R-24571), whereas the inhibition by a higher Cd2+ concentration of MLCK and PL-Ca-PK was reversed by thiol agents (e.g., cysteine). The present findings may provide one mechanism underlying the vascular toxicity of Cd2+, a major environmental pollutant.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.     

A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner

A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000- dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner

? Villin is one of the major actin filament bundling proteins in plants. The function of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this report, the biochemical activity and cellular function of AtVLN4 were examined. ? The biochemical property of AtVLN4 was characterized by co-sedimentation assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants. ? Recombinant AtVLN4 protein exhibited multiple activities on actin, including actin filament bundling, calcium (Ca(2+))-dependent filament severing and barbed end capping. Expression of AtVLN4 in tobacco pollen induced the formation of supernumerary actin cables and reduced pollen tube growth. Loss of function of AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles. ? Our results demonstrated that AtVLN4 is involved in root hair growth through regulating actin organization in a Ca(2+)-dependent manner.     

Requirement of phosphorylation of Physarum myosin heavy chain for thick filament formation, actin activation of Mg2+-ATPase activity, and Ca2+-inhibitory superprecipitation

In an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows: 1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-gamma-S. 2. In the reaction with ATP-gamma-S, only the myosin HC of myosin B was phosphorylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC. 3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested. 4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all. 5. In a gel filtration in 0.5 M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules. 6. In 40 mM KCl and 5-10 mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not. The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.     

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.     

Ca2+ activates actin-filament sliding on scallop myosin but inhibits that on Physarum myosin

The actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by microM levels of Ca2+, the mode of which is in contrast to the activating effect of Ca2+ on scallop myosin (Kohama, K. (1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.     

Thyroid hormones inhibit the Ca2+ calmodulin-induced activation of myosin light chain kinase

L-Thyroxine (T4) and L-triiodothyronine (T3) specifically, inhibited myosin light chain kinase (MLC-kinase) from various tissues whereas inhibitory effects of T4 and T3 on other protein kinases such as protein kinase C, cAMP-dependent protein kinase, casein kinase I, casein kinase II and calmodulin kinase II were much weaker. T4 was a more potent inhibitor of MLC-kinase than T3. Kinetic studies showed that T4 behaved as a competitive inhibitor of MLC-kinase toward calmodulin (CaM) and that Ki value was 2.5 microM. The activity of the catalytic fragment of MLC-kinase, which is active without CaM, was not inhibited by T4. 125I-T4 gel overlay revealed that CaM did not bind T4 but MLC-kinase had 125I-T4 binding activity. These observations suggest that T4 binds at or near CaM binding domain of MLC-kinase and inhibits CaM-induced activation of MLC-kinase.     

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner

The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa²⁺-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca²⁺ concentration([Ca²⁺]_i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca²⁺concentration. The elevation in[Ca²⁺]_iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa²⁺ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa²⁺-independent mechanism.

     

Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity

The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed.