The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

The Dictyostelium essential light chain is required for myosin function.

A Dictyostelium mutant (7-11) that expresses less than 0.5% of wild-type levels of the myosin essential light chain (EMLC) has been created by overexpression of antisense RNA. Cells from 7-11 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (RMLC). Myosin isolated from 7-11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7-11 myosin displays calcium-activated ATPase activity with a Vmax about 15%-25% of that of wild type, and a Km for ATP of 27 +/- 5 microM versus 83 +/- 30 microM for wild type. At actin concentrations as high as 17 microM, 7-11 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7-11 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7-11 cells take several hours longer than wild-type cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the EMLC are phenotypically similar to MHC null mutants, the EMLC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity.     

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.     

Physico-chemical studies on the light chains of myosin. 3. Evidence for a regulatory role of a rabbit myosin light chain

Treatment of myosin with DTNB causes a decrease in the calcium sensitivity of actomyosin, concurrently with the release of the DTNB light chains. The removal of the calcium-binding DTNB light chains is accompanied by a loss of the calcium binding capacity of myosin. A regulatory role is ascribed to these light chains.     

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head–head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head–head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837–854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.     

An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical--chemical and immunological characterization of the light chains.

The light chains of chicken breast muscle myosin (alkali 1 and 2, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 1.c.) have been isolated in pure form and characterized with respect to amino acid composition, uv and circular dichroism (CD) spectral properties, and molecular weight. Antibodies specific for each of the light chains have been used to demonstrate the similarity of alkali 1 and 2 (mol wt 21,000 and 16,000, respectively), and the distinctness of these from DTNB 1.c. (mol wt of 18,000). The DTNB 1.c. isolated by a variety of methods were all immunologically identical. Significant cross-reactivity was observed between corresponding rabbit and chicken light chains, confirming other indications of homology between these proteins in the two species. The immunological difference between alkali 1 and 2 was largely accounted for by an N-terminal peptide, rich in proline, alanine, and lysine, which is unique to alkali 1. The presence of antibodies to this peptide in anti-alkali 1 serum suggests an immunological approach to the question of how alkali 1.c. are distributed in myosin.     

Abnormal cardiac structure and function in mice expressing nonphosphorylatable cardiac regulatory myosin light chain 2.

A role for myosin phosphorylation in modulating normal cardiac function has long been suspected, and we hypothesized that changing the phosphorylation status of a cardiac myosin light chain might alter cardiac function in the whole animal. To test this directly, transgenic mice were created in which three potentially phosphorylatable serines in the ventricular isoform of the regulatory myosin light chain were mutated to alanines. Lines were obtained in which replacement of the endogenous species in the ventricle with the nonphosphorylatable, transgenically encoded protein was essentially complete. The mice show a spectrum of cardiovascular changes. As previously observed in skeletal muscle, Ca(2+) sensitivity of force development was dependent upon the phosphorylation status of the regulatory light chain. Structural abnormalities were detected by both gross histology and transmission electron microscopic analyses. Mature animals showed both atrial hypertrophy and dilatation. Echocardiographic analysis revealed that as a result of chamber enlargement, severe tricuspid valve insufficiency resulted in a detectable regurgitation jet. We conclude that regulated phosphorylation of the regulatory myosin light chains appears to play an important role in maintaining normal cardiac function over the lifetime of the animal.     

Examining the in vivo role of the amino terminus of the essential myosin light chain.

The functional significance of the actin binding region at the amino terminus of the cardiac essential myosin light chain (ELC) remains obscure. Previous experiments carried out in vitro indicated that modulation of residues 5-14 could induce an inotropic effect, increasing maximal ATPase activity at submaximal Ca(2+) concentrations (Rarick, H. M., Opgenorth, T. J., von Geldern, T. W., Wu-Wong, J. R., and Solaro, R. J. (1996) J. Biol. Chem. 271, 27039-27043). Using transgenesis, we effected a cardiac-specific replacement of ELC with a protein containing a 10-amino acid deletion at positions 5-14. Both the ventricular (ELC1vDelta5-14) and atrial (ELC1aDelta5-14) isoforms lacking this peptide were stably incorporated into the sarcomere at high efficiencies. Surprisingly when the kinetics of skinned fibers isolated from the ELC1vDelta5-14 or ELC1aDelta5-14 mice were examined, no alterations in either unloaded shortening or maximum shortening velocities were apparent. Myofibrillar Mg(2+)-ATPase activity was also unchanged in these preparations. No significant changes in the fiber kinetics in the cognate compartments were observed when either deletion-containing protein replaced endogenous ELC1v or ELC1a. The data indicate that the previously postulated importance of this region in mediating critical protein interactions between the cardiac ELCs and the carboxyl-terminal residues of actin in vivo should be reassessed.     

Fine tuning the myosin motor: the role of the essential light chain in striated muscle myosin

It has long been known that the essential light chain isoform of striated muscle affects the function of the myosin motor. There are two isoforms: A1-type and A2-type that differ by the presence of an extra 40 amino acids at the N-terminus of A1-type light chains. Evidence has accumulated from a variety of experimental techniques that this extension of A1-type light chains makes a direct contact with actin, increasing the overall affinity between myosin and actin and that this interaction is responsible for the modulation of myosin motor function. Some recent work, however, has provided some contradictory data. Experiments using more physiologically relevant forms of myosin have suggested that the effect of the N-terminal region of A1-type light chains may, in some circumstances, be to weaken, rather than strengthen the actin-myosin interaction. Work with transgenic mice in which this region was mutated showed no measurable phenotypic effects on either muscle or whole organism function questioning the in vivo significance of the light chain-actin interaction. It is also possible that the essential light chain has other functions in the cell. There is evidence that the protein may interact with IQGAP, a regulator of the actin cytoskeleton. The consequences of this interaction are unknown. This review aims to summarise the biochemical data on striated muscle myosin essential light chain isoform function and to reconcile it with these recent discoveries.     

Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase.

Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP.     

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.     

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.     

Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.     

Functions of the Caenorhabditis elegans regulatory myosin light chain genes mlc-1 and mlc-2.

Caenorhabditis elegans contains two muscle regulatory myosin light chain genes, mlc-1 and mlc-2. To determine their in vivo roles, we identified deletions that eliminate each gene individually and both genes in combination. Functions of mlc-1 are redundant to those of mlc-2 in both body-wall and pharyngeal muscle. mlc-1(0) mutants are wild type, but mlc-1(0) mlc-2(0) double mutants arrest as incompletely elongated L1 larvae, having both pharyngeal and body-wall muscle defects. Transgenic copies of either mlc-1(+) or mlc-2(+) rescue all defects of mlc-1(0) mlc-2(0) double mutants. mlc-2 is redundant to mlc-1 in body-wall muscle, but mlc-2 performs a nearly essential role in the pharynx. Approximately 90% of mlc-2(0) hermaphrodites arrest as L1 larvae due to pharyngeal muscle defects. Lethality of mlc-2(0) mutants is sex specific, with mlc-2(0) males being essentially wild type. Four observations suggest that hermaphrodite-specific lethality of mlc-2(0) mutants results from insufficient expression of the X-linked mlc-1(+) gene in the pharynx. First, mlc-1(0) mlc-2(0) double mutants are fully penetrant L1 lethals in both hermaphrodites and males. Second, in situ localization of mlc mRNAs demonstrates that both mlc-1 and mlc-2 are expressed in the pharynx. Third, transgenic copies of either mlc-1(+) or mlc-2(+) rescue the pharyngeal defects of mlc-1(0) mlc-2(0) hermaphrodites. Fourth, a mutation of the dosage compensation gene sdc-3 suppresses hermaphrodite-specific lethality of mlc-2(0) mutants.     

Ca2+ regulation not associated with phosphorylation of myosin light chain in aortic intima smooth muscle. II. Effects of regulatory proteins on the actin-myosin interaction

Contractile and regulatory proteins were prepared from bovine aortic intima, and actin from bovine stomach smooth and rabbit skeletal muscles. In the desensitized and reconstituted actomyosin system, the superprecipitation activity was measured by the turbidity method. Superprecipitation of each system was not exhibited even in the presence of Ca ions, but was observable only in the presence of tropomyosin and Ca ions, while 20,000-dalton light chain of myosin remained dephosphorylated during the reaction. Addition of tropomyosin to the reconstituted acto-myosin digest system (trypsin-digested myosin was devoid of 20,000-dalton light chain) also restored the Ca2+-sensitivity. These results indicate that the phosphorylation of myosin light chain is not a crucial step in the contraction of aortic intima smooth muscle. For full activation of the actin-myosin-ATP interaction, additional factors other than the myosin light chain kinase are required, although some contribution of the kinase to the full activation cannot be ruled out.     

In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis

Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.