Reduced power output in skeletal muscles devoid of skMLCK: RLC phosphorylation contributes to peak performance

Regulatory light chain (RLC) phosphorylation in fast twitch muscle is catalyzed by skeletal myosin light chain kinase (skMLCK), a reaction known to increase muscle force, work, and power. The purpose of this study was to explore the contribution of RLC phosphorylation on the power of mouse fast muscle during high frequency (100 Hz) concentric contractions. To determine peak power shortening ramps (1.05 to 0.90 Lo) were applied to Wildtype (WT) and skMLCK knockout (skMLCK-/-) EDL muscles at a range of shortening velocities between 0.05-0.65 of maximal shortening velocity (Vmax), before and after a conditioning stimulus (CS). As a result, mean power was increased to 1.28 ± 0.05 and 1.11 ± .05 of pre-CS values, when collapsed for shortening velocity in WT and skMLCK-/-, respectively (n = 10). In addition, fitting each data set to a second order polynomial revealed that WT mice had significantly higher peak power output (27.67 ± 1.12 W/ kg-1) than skMLCK-/- (25.97 ± 1.02 W/ kg-1), (p < .05). No significant differences in optimal velocity for peak power were found between conditions and genotypes (p > .05). Analysis with Urea Glycerol PAGE determined that RLC phosphate content had been elevated in WT muscles from 8 to 63 % while minimal changes were observed in skMLCK-/- muscles: 3 and 8 %, respectively. Therefore, the lack of stimulation induced increase in RLC phosphate content resulted in a ~40 % smaller enhancement of mean power in skMLCK-/-. The increase in power output in WT mice suggests that RLC phosphorylation is a major potentiating component required for achieving peak muscle performance during brief high frequency concentric contractions.

The influence of myosin regulatory light chain phosphorylation on the contractile performance of fatigued mammalian skeletal muscle

ABSTRACT The myosm regulatory light chain (RLC) of type II fibres is phosphorylated by Ca2+ -calmodulin dependent myosin light chain kinase (skMLCK) during muscular activation. The purpose of this study was to explore the effect of skMLCK gene ablation on the fatigability of mouse skeletal muscles during repetitive stimulation. The absence of myosin RLC phosphorylation in skMLCK knockout muscles attenuated contractile performance without a significant metabolic cost. Twitch force was potentiated to a greater extent in wildtype muscles until peak force had diminished to ~60% of baseline (37.2 ± 0.05% vs. 14.3 ± 0.02%). Despite no difference in peak force (Po) and shortening velocity (Vo), rate of force development (+dP/dt) and shortening-induced deactivation (SID) were almost two-fold greater in WT muscles. The present results demonstrate that myosin RLC phosphorylation may improve contractile performance during fatigue; providing a contractile advantage to working muscles and protecting against progressive fatigue.

Myosin Regulatory Light Chain Phosphorylation and Its Effect on the Contractile Economy of Mouse Fast Muscle

Activated by elevations in myoplasmic calcium concentration, myosin light chain kinase (skMLCK) phosphorylates the regulatory light chains (RLCs) of fast muscle myosin. This covalent modification potentiates force production, but requires an investment of ATP. Our objective was to investigate the effect of RLC phosphorylation on the contractile economy (mechanical output:metabolic input) of fast twitch skeletal muscle. Extensor digitorum longus muscles isolated from Wildtype and skMLCK-/- mice mounted in vitro (25°C) were subjected to repetitive low-frequency stimulation (10Hz,15s) known to cause activation of skMLCK, and staircase potentiation of force. With a 3-fold increase in RLC phosphate content, Wildtype generated 44% more force than skMLCK-/- muscles over the stimulation period (P = .002), without an accompanied increase in energy cost (P = .449). Overall, the contractile economy of Wildtype muscles, with an intact RLC phosphorylation mechanism, was 73% greater than skMLCK /- muscles (P = .043), demonstrating an important physiological function of skMLCK during repetitive contractile activity.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Force potentiation as a modulator of contractile performance: Implications for control of skeletal muscle force and energetics of work

This thesis investigated the modulation of dynamic contractile function and energetics of work by posttetanic potentiation (PTP). Mechanical experiments were conducted in vitro using software-controlled protocols to stimulate/determine contractile function during ramp shortening, and muscles were frozen during parallel incubations for biochemical analysis. The central feature of this research was the comparison of fast hindlimb muscles from wildtype and skeletal myosin light chain kinase knockout (skMLCK-/-) mice that does not express the primary mechanism for PTP: myosin regulatory light chain (RLC) phosphorylation. In contrast to smooth/cardiac muscles where RLC phosphorylation is indispensable, its precise physiological role in skeletal muscle is unclear. It was initially determined that tetanic potentiation was shortening speed dependent, and this sensitivity of the PTP mechanism to muscle shortening extended the stimulation frequency domain over which PTP was manifest. Thus, the physiological utility of RLC phosphorylation to augment contractile function in vivo may be more extensive than previously considered. Subsequent experiments studied the contraction-type dependence for PTP and demonstrated that the enhancement of contractile function was dependent on force level. Surprisingly, in the absence of RLC phosphorylation, skMLCK-/- muscles exhibited significant concentric PTP; consequently, up to ~50% of the dynamic PTP response in wildtype muscle may be attributed to an alternate mechanism. When the interaction of PTP and the catchlike property (CLP) was examined, we determined that unlike the acute augmentation of peak force by the CLP, RLC phosphorylation produced a longer-lasting enhancement of force and work in the potentiated state. Nevertheless, despite the apparent interference between these mechanisms, both offer physiological utility and may be complementary in achieving optimal contractile function in vivo. Finally, when the energetic implications of PTP were explored, we determined that during a brief period of repetitive concentric activation, total work performed was ~60% greater in wildtype vs. skMLCK-/- muscles but there was no genotype difference in High-Energy Phosphate Consumption or Economy (i.e. HEPC: work). In summary, this thesis provides novel insight into the modulatory effects of PTP and RLC phosphorylation, and through the observation of alternative mechanisms for PTP we further develop our understanding of the history-dependence of fast skeletal muscle function.

Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

ATM mediated phosphorylation of CHD4 contributes to genome maintenance

Background: In order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling. Results: Here we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage. Conclusion: These results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

A crystallographic snapshot of tyrosine trans-phosphorylation in action

Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Phosphorylation of ATPase subunits of the 26S proteasome

Abstract The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2DPAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S oteasomes were immunoprecipitated as above and dissociated and Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.