Phosphorylation of the regulatory light chains of myosin affects Ca2+ sensitivity of skeletal muscle contraction.

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.     

Acceleration of stretch activation in murine myocardium due to phosphorylation of myosin regulatory light chain

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca(2+) sensitivity of isometric force, reduced the steepness of the force-pCa relationship, and increased both Ca(2+)-activated and Ca(2+)-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.     

Influence of Ca2+ on force redevelopment kinetics in skinned rat myocardium.

The influence of Ca2+ on isometric force kinetics was studied in skinned rat ventricular trabeculae by measuring the kinetics of force redevelopment after a transient decrease in force. Two protocols were employed to rapidly detach cycling myosin cross-bridges: a large-amplitude muscle length ramp followed by a restretch back to the original length or a 4% segment length step. During the recovery of force, the length of the central region of the muscle was controlled by using a segment marker technique and software feedback control. Tension redevelopment was fit by a rising exponential governed by the rate constant ktr for the ramp/restretch protocol and kstep for the step protocol. ktr and kstep averaged 7.06 s-1 and 15.7 s-1, respectively, at 15 degrees C; neither ktr nor kstep increased with the level of Ca2+ activation. Similar results were found at submaximum Ca2+ levels when sarcomere length control by laser diffraction was used. The lack of activation dependence of ktr contrasts with results from fast skeletal fibers, in which ktr varies 10-fold from low to high activation levels, and suggests that Ca2+ does not modulate the kinetics of cross-bridge attachment or detachment in mammalian cardiac muscle.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

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.     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation.

The Ca2+ sensitivities of tonic (pulmonary and femoral artery) and phasic (portal vein and ileum) smooth muscles and the effects of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) and norepinephrine on Ca2+ sensitivity of force development and myosin light chain (MLC20) phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenous calmodulin. The Ca2+ sensitivity of force was higher (approximately 3-fold) in the tonic than in the phasic smooth muscles. The nucleotide specificity of Ca2+ sensitization was: GTP gamma S much greater than GTP greater than ITP much greater than CTP = UTP. Baseline phosphorylation (7% at pCa greater than 8) and maximal phosphorylation (58% at pCa 5.0) were both lower in portal vein than in femoral artery (20 and 97%). Norepinephrine and GTP gamma S increased phosphorylation at constant [Ca2+] (pCa 7.0-6.5). MLC20 phosphorylation induced by norepinephrine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S). In portal vein at pCa 5, GTP gamma S increased phosphorylation from 58%, the maximal Ca2(+)-activated value, to 75%, and at pCa greater than 8, from 7 to 13%. In femoral artery at pCa 5, neither phosphorylation (97%) nor force was affected by GTP gamma S, while at pCa greater than 8, GTP gamma S caused an increase in force (16% of maximum) with a borderline increase in MLC20 phosphorylation (from 20 to 27%). MLC20 phosphorylation (up to 100%) was positively correlated with force. The major results support the hypothesis that the G-protein coupled Ca2(+)-sensitizing effect of agonists on force development is secondary to increased MLC20 phosphorylation.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

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.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

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.     

The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts

Phosphorylation of the myosin regulatory light chain (RLC) by Ca(2+)-calmodulin-activated myosin light chain kinase (MLCK) is known to be essential for the inotropic function of the heart. In this study, we have examined the effects of MLCK-phosphorylation of transgenic (Tg) mouse cardiac muscle preparations expressing the D166V (aspartic acid to valine)-RLC mutation, identified to cause familial hypertrophic cardiomyopathy with malignant outcomes. Our previous work with Tg-D166V mice demonstrated a large increase in the Ca(2+) sensitivity of contraction, reduced maximal ATPase and force and a decreased level of endogenous RLC phosphorylation. Based on studies demonstrating the beneficial and/or protective effects of cardiac myosin phosphorylation for heart function, we hypothesized that an ex vivo phosphorylation of Tg-D166V cardiac muscle may rescue the detrimental contractile phenotypes observed earlier at the level of single myosin molecules and in Tg-D166V papillary muscle fibres. We showed that MLCK-induced phosphorylation of Tg-D166V cardiac myofibrils and muscle fibres was able to increase the reduced myofibrillar ATPase and reverse an abnormally increased Ca(2+) sensitivity of force to the level observed for Tg-wild-type (WT) muscle. However, in contrast to Tg-WT, which displayed a phosphorylation-induced increase in steady-state force, the maximal tension in Tg-D166V papillary muscle fibres decreased upon phosphorylation. With the exception of force generation data, our results support the notion that RLC phosphorylation works as a rescue mechanism alleviating detrimental functional effects of a disease causing mutation. Further studies are necessary to elucidate the mechanism of this unexpected phosphorylation-induced decrease in maximal tension in Tg-D166V-skinned muscle fibres.     

Characterization of myosin light-chain kinase from bovine adrenal medulla

Partially purified bovine adrenal medullary myosin light-chain kinase (MLCK) possesses a Stoke's radius of 79 A and a sedimentation coefficient of 3.95 +/- 0.45 S, yielding a native molecular weight of 150,000 +/- 17,000 g/mol and a frictional ratio of 2.24. It catalyzes the phosphorylation of the isolated light chain of skeletal muscle myosin and the light chain of intact adrenal medullary myosin, but not phosphorylase b or histone. The activation of MLCK by calmodulin is specific and dose dependent, yielding a K0.5 value of 9.0 nM; the dose response curve with respect to free Ca2+ is biphasic, exhibiting a stimulatory phase at low free Ca2+ concentrations (K0.5 = 0.17 microM) and an inhibitory phase at higher free Ca2+ concentrations (400-3000 microM). Michaelis-Menten kinetics are observed for ATP, yielding a Km for ATP of 25 microM and a Vmax of 23.2 nmol/min/mg. However, positive cooperative kinetics are observed for the skeletal muscle myosin light chain, yielding a Hill coefficient of 3.57, a K0.5 for light chain of 27 microM and a Vmax of 16.6 nmol/min/mg. A stoichiometry of phosphorylation of approximately 1 mol of phosphate/mol of skeletal muscle myosin light chain was observed. Therefore, adrenal medullary MLCK is similar in most, but not all, of its physical and kinetics properties to MLCKs isolated from other sources and may serve to regulate actin-myosin contractile activity in the adrenal medulla.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.     

Ca2+-induced conformational changes of spin-labeled g2 chain bound to myosin and the effect of phosphorylation.

One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiC1. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.1.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated on the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.