Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.     

Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction

Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.     

AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain

The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.     

Two isoforms of regulatory light chain of abalone smooth muscle myosin

Abalone myosin contains two kinds of light chain, regulatory light chain (LC2) and essential light chain (LC1) according to SDS-PAGE. Three distinct light chain bands were observed on polyacrylamide gel electrophoresis of purified abalone myosin in the presence of urea (urea-PAGE). The slower two components showed had mobility on SDS-PAGE and they also showed regulatory activity as the regulatory light chain. They were termed LC2-a and LC2-b in order of increasing mobility on urea-PAGE and isolated by DE-32 ion exchange column chromatography in the presence 8 M urea. The ratio of LC2-a and LC2-b in the central portion of adductor muscle of abalone (LC2-a: LC2-b = 7:3) was different from that (1:1) in the peripheral portion. These results suggest that the two light chains are isoforms of the regulatory light chain. The amino acid compositions of LC2-a and LC2-b were very similar to each other except for the Cys content. The UV absorption spectra were also quite similar, as were the UV difference absorption spectra induced by Ca2+. Phosphorylation was not detectable with the myosin light chain kinase of chicken gizzard. The Ca2+ concentration dependencies of Mg-ATPase activity of LC2-a or LC2-b hybridized abalone myosin (a-myosin, b-myosin) were similar to each other in the absence of rabbit F-actin, but differed in the presence of actin. The b-myosin had a higher maximum value of actomyosin ATPase activity and a lower apparent binding constant of actin and myosin than a-myosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The regulatory light chain is required for folding of smooth muscle myosin

Light chain phosphorylation causes the folded monomeric form of myosin to extend and assemble into filaments. This observation established the involvement of the 20-kDa regulatory light chain (LC20) in conformational transitions of smooth muscle myosin. To further assess the role of this subunit in the intramolecular folding of myosin, LC20 was removed from turkey gizzard myosin at elevated temperatures in the presence of EDTA through the use of an antibody affinity column. Metal-shadowed images showed that LC20-deficient myosin had a tendency to aggregate through the neck region. When MgATP was added to filaments formed from this myosin, less than 10% of the myosin was solubilized, indicating that myosin could not fold in the absence of light chain. Readdition of native regulatory light chain restored the myosin to its original solubility properties, thus establishing reversibility. Addition of foreign light chains from skeletal muscle myosin or a chymotryptic-cleaved gizzard light chain produced the same amount of monomeric myosin in high salt that was obtained by recombination with the homologous light chain. However, the ability of the hybrid myosins to assume the folded conformation was impaired, and only a partially folded species was obtained. Single-headed myosin, like rod and light chain-deficient myosin, remained filamentous in the presence of MgATP. These results are consistent with the hypothesis that the regulatory light chain in the neck region of myosin contributes to a binding site for the myosin tail.     

Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).     

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

We tested the hypothesis that increases in force at a given cytosolic Ca(2+) concentration (i.e., Ca(2+) sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in alpha-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 microM Ca(2+) in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 microM) plus GTP (1 microM) significantly shifted the Ca(2+)-force relationship curve to the left (EC(50): 0.39 +/- 0.06 to 0.078 +/- 0.006 microM Ca(2+)) and significantly increased the maximum force (104.4 +/- 4.8 to 120.2 +/- 2.8%; n = 6 observations). The Ca(2+)-rMLC phosphorylation relationship curve was also shifted to the left (EC(50): 1.26 +/- 0.57 to 0.13 +/- 0.04 microM Ca(2+)) and upward (maximum rMLC phosphorylation: 70.9 +/- 7.9 to 88.5 +/- 5. 1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca(2+) concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca(2+) sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.     

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.     

Signaling to myosin regulatory light chain in sarcomeres

Myosin regulatory light chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca(2+)-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca(2+)-dependent myosin light chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin light chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.     

Monitoring the orientation of myosin bridges on two-dimensional maps of birefringence in a single muscle fiber

Two-dimensional maps of birefringence in sarcomers of a single fiber of rabbit m.psoas were obtained by an automated interference microscope developed at our laboratory. The changes in birefringence of muscle fibers reflect the movement of myosin cross-bridges. The orientation of cross-bridges was modified by varying the pH (pH 7.0, 6.0, 8.0) and ionic strength (mu = 0.115, 0.085, 0.235) of the bathing rigor solution. The maximum value of total birefringence in the rigor state was observed at neutral pH. Total birefringence markedly decreased (by 40%) as pH was changed from 7.0 to both 8.0 and 6.0. No significant changes in light phase shifts were found at a 1.5 reduction of ionic strength in the rigor solution. The calculated birefringence values were 45% higher in rigor solutions of a high (mu = 0.235) ionic strength. The results observed are discussed in terms of changes in the orientation of cross-bridges due to the movement of the alpha-helical subfragment-2 away from the filament shaft (pH 8) or coming closer to it (mu = 0.235). The available data do not allow one to explain the results obtained at pH 6.0.     

Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain

A cDNA probe was constructed from a chicken skeletal muscle regulatory light chain cDNA and was used to screen a chicken gizzard cDNA library. A clone containing the entire coding region of the chicken gizzard regulatory light chain was isolated and sequenced. The deduced protein sequence is identical to the most recently reported chemical sequence of the chicken smooth muscle regulatory light chain, and has homologies with other troponin C-like calcium-binding proteins.     

Amino acid sequence of the regulatory light chain of squid mantle muscle myosin

The amino acid sequence of the regulatory light chain of mantle muscle myosin from squid (Todarodes pacificus) was determined by conventional methods. It was: xA-E-E-A-P-R-R-V-K-L-S-Q-R-Q-M-Q-E-L-K-E-A-F-T-M-I-D-Q-D-R-D-G-F-I-G-M- E-D-L-K-D-M-F-S-S-L-G-R-V-P-P-D-D-E-L-N-A-M-L-K-E-C-P-G-Q-L-N-F-T- A-F-L-T-L-F-G-E-K-V-S-G-T-D-P-E-D-A-L-R-N-A-F-S-M-F-D-E-D-G-Q-G-F-I-P- E-D-Y-L-K-D-L-L-E-N-M-G-D-N-F-S-K-E-E-I-K-N-V-W-K-D-A-P-L-K-N-K-Q-F- N-Y-N-K-M-V-D-I-K-G-K-A-E-D-E-D. The alpha-amino group of this light chain was blocked, and a typical calcium-binding structure was recognized at the sequence of residue 26 to residue 37, like those in other myosin regulatory light chains.     

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.     

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells

These experiments were performed totest the hypotheses that myosin light chain 17 (MLC₁₇) aand b isoform expression varies between individual vascular smoothmuscle (SM) cells and that their expression correlates with cellunloaded shortening velocity. Single SM cells isolated from rabbitaorta and carotid arteries were used to measure unloaded shorteningvelocity and subsequently were analyzed via RT-PCR forMLC₁₇ a and b mRNA ratio. The MLC_17b/a mRNA andprotein ratios from adjacent tissue sections correlate very well(R² = 0.68), allowing use of the mRNA ratio topredict the protein ratio. The rabbit MLC₁₇ isoform proteinsequence was found to be similar to, but unique from, the swine, mouse,and chicken sequences. Isolated single SM cells from the aorta andcarotid have resting lengths of 70-280 µm and shorten to33-88 µm after contraction. Isolated cell maximum unloadedshortening velocity is highly variable (0.5-7.5 µm/s) butbecomes more uniform when normalized to initial cell length(0.01-0.05 cell lengths/s). Carotid cells activated in thepresence of okadaic acid (1 µm) have mean maximal unloaded shorteningvelocities not significantly different from carotid cells activatedwithout okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting celllength before activation is significantly correlated with final celllength after unloaded shortening. Neither initial cell length, finalcell length, total cell length change, nor maximum unloaded shorteningvelocity (absolute or normalized) was significantly correlated withsingle-cell MLC_17b/a mRNA ratio. These studies wereperformed in isolated single SM cells where unloaded shorteningvelocity and MLC_17b/a mRNA ratios were measured in the samecell. In this preparation, the three-dimensional organization andmilieu of the cell is kept intact, but without the intercellularheterogeneity concerns of multicellular preparations. These resultssuggest the MLC_17b/a ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloadedshortening velocity in single SM cells.

     

Bifunctional rhodamine probes of Myosin regulatory light chain orientation in relaxed skeletal muscle fibers

The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30 degrees C; approximately 60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution-the maximum entropy distribution-consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the "lever" axis, linking Cys707 and Lys843 of the myosin heavy chain, was at 70-80 degrees to the fiber axis, and the "hook" helix (Pro830-Lys843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.     

Orientation of the N-terminal lobe of the myosin regulatory light chain in skeletal muscle fibers

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state. The peak angle between the lever axis of the myosin head and the fiber or actin filament axis was 100—110° in relaxation, isometric contraction, and rigor. In each state the hook helix was at an angle of ～40° to the lever/filament plane. The in situ orientation of the RLC D and E helices, and by implication of its N- and C-lobes, was similar in smooth and skeletal RLC isoforms. The angle between these two RLC lobes in rigor fibers was different from that in the crystal structure. These results extend previous crystallographic evidence for bending between the two lobes of the RLC to actin-attached myosin heads in muscle fibers, and suggest that such bending may have functional significance in contraction and regulation of vertebrate striated muscle.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.