Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.     

An efficient exchange method for alkali light chain in myosin subfragment

The alkali light chain, A2, in subfragment-1 (S-1) was exchanged with A1 added externally in NH4 + -NH3 buffer (pH 9.9). The exchange yield was higher than 80% using only 2-fold molar excess of A1 over S-1 containing A2. The ATPase activities of the exchanged S-1 (A1) were the same as those of untreated S-1 (A1). The method was also applicable to exchanging the alkali light chains in myosin.     

Selective binding of L-thyroxine by myosin light chain kinase

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.     

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.     

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.     

Differential binding of rabbit fast muscle myosin light chain isoenzymes to regulated actin

The direct binding of S1(A1) and S1(A2) to regulated actin has been investigated by centrifugation. Binding was measured in the presence of either Mg·AdoPP[NH]P or Mg·ADP at 24°C at various ionic strengths. At low ionic strength, in either the presence or absence of Ca²⁺, the binding of S1(A1) to regulated actin was always stronger than for S1(A2). As the ionic strength was increased the differential binding between S1(A1) and S1(A2) was still maintained in the presence of Ca²⁺ but not in its absence. These data are discussed in terms of a modifying role for the N-terminal region of the A1 light chain in regulation of the contractile process.     

Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber

The Ca²⁺ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca²⁺-activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca²⁺ (10^-5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca²⁺ insensitive tension. In contrast, pretreatment with low Ca²⁺ (10^-8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca²⁺-sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca²⁺-activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP-dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca²⁺-activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.     

MSAP is a novel MIR-interacting protein that enhances neurite outgrowth and increases myosin regulatory light chain

Dynamic interactions between the actin cytoskeleton and specific proteins are crucial for changes in cell shape and motility. Here we describe a novel protein MSAP (MIR-interacting saposin-like protein) that is a positive regulator of neurite outgrowth. MSAP is expressed in different tissues, including brain, and has an apparent molecular weight of 21 kDa. MSAP interacts with the ezrin-radixin-moesin (ERM)-like myosin regulatory light chain-interacting protein (MIR), and the two proteins are co-localized in cell lines and in primary neurons. Overexpression of MSAP enhances neurite out-growth in neuroblastoma and PC12 cells, whereas down-regulation of MSAP using RNA silencing led to inhibition of neurite formation. The stimulation of neurite outgrowth by MSAP was abrogated by the overexpression of MIR, which induced a decrease in the levels of myosin regulatory light chain (MRLC). This reduction in MRLC by MIR was inhibited by blocking the activity of proteasome and by overexpression of MSAP, suggesting an effect on protein stability. Evidence was obtained that MIR decreases MRLC by inducing its ubiquitination. The results show that the levels of MRLC are controlled by MIR via ubiquitination and that the effect of MIR on MRLC is counteracted in the presence of MSAP. MSAP can stabilize MRLC and thus bring about an increase in neurite outgrowth.     

Preparation of the alkali and P light chains of chicken gizzard myosin. Amino acid sequence of the alkali light chain

1. A simple method is described for the purification of the alkali and P light chains from chicken gizzard myosin. 2. The sequence of the alkali light chain has been unequivocally determined, except for the N-terminal dipeptide, by using the tryptic and CNBr peptides. 3. No evidence was obtained for any specific high-affinity Ca2+-binding sites on the alkali light chain. 4. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50120 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1983) 209, 5.     

Isolation and sequencing of porcine fast skeletal muscle alkali myosin light chain 3 cDNA

Abstract

A cDNA coding for alkali myosin light chain 3 (MLC3_F) was isolated from a porcine skeletal muscle library. This clone has an insert of 859 bp encompassing the complete CDS (coding sequence) plus the 5’ and 3’ untranslated regions. Computer analysis showed that porcine MLC3_F cDNA is highly homologous to the corresponding cDNAs of human, rabbit and rat. Moreover, Northern analysis showed the presence of two bands that represent the mature mRNAs of the MLCl_F and MLC3_F isoforms according to data observed in other species.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.