Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

The On-Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca²⁺ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca²⁺. Our results indicate that loss of Ca²⁺ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.     

Different Head Environments in Tarantula Thick Filaments Support a Cooperative Activation Process

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.     

Different Head Environments in Tarantula Thick Filaments Support a?Cooperative Activation Process

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.     

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.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

Theoretical model of the interactions between Ca2+, calmodulin and myosin light chain kinase

Active Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays an important role in the process of MLC phosphorylation and consecutive smooth muscle contraction. Here, we propose a mathematical model of a detailed kinetic scheme describing interactions among Ca²⁺, CaM and MLCK and taking into account eight different aggregates. The main model result is the prediction of the Ca²⁺ dependent active form of MLCK, which is in the model taken as proportional to the concentration of Ca₄CaM · MLCK complex. Wegscheider’s condition is additionally applied as a constraint enabling the prediction of some parameter values that have not yet been obtained by experiments.     

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

Characterization of Tightly Associated Smooth Muscle Myosin-Myosin Light-Chain Kinase-Calmodulin Complexes

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ∼ 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ∼ 1 nM free [Ca²⁺]. There were two MLCK pools that bound unphosphorylated SMM with K_d ∼ 10 and 0.2 μM and phosphorylated SMM with K_d ∼ 20 and 0.2 μM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca²⁺ and phosphorylation of SMM occurred at a pCa₅₀ of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.     

Protein kinase C βII and δ/θ play critical roles in bone morphogenic protein-4-stimulated osteoblastic differentiation of MC3T3-E1 cells

Multiple drug resistance protein 1 (MDR1) is composed of two homologous halves separated by an intracellular linker region. The linker has been reported to bind myosin regulatory light chain (RLC), but it is not clear how this can occur in the context of a myosin II complex. We characterized MDR1-RLC interactions and determined that binding occurs via the amino terminal of the RLC, a domain that typically binds myosin heavy chain. MDR1-RLC interactions were sensitive to the phosphorylation state of the light chain in that phosphorylation by myosin light chain kinase (MLCK) resulted in a loss of binding in vitro. We used ML-7, a specific inhibitor of MLCK, to study the functional consequences of disrupting RLC phosphorylation in intact cells. Pretreatment of polarized Madin-Darby canine kidney cells stably expressing MDR1 with ML-7 produced a significant increase in apical to basal permeability and a corresponding decrease in the efflux ratio (threefold; p < 0.01) of [³H]-digoxin, a classic MDR1 substrate. Together these data show that MDR1-mediated transport of [³H]-digoxin can be modulated by pharmacological manipulation of myosin RLC, but direct MDR1-RLC interactions are atypical and not explained by the structure of the myosin II holoenzyme.     

Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca²⁺. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0–2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca²⁺-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0–2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8–4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the V_max of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphrylation site B (S⁸²⁸).     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca²⁺, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca²⁺-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca²⁺-induced changes in cTnI-cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC-cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca²⁺ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca²⁺ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.     

Role of the tail in the regulated state of myosin 2

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.     

Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.     

Protein phosphatase activity is necessary for myofibrillogenesis

During myofibrillogenesis, myosin light-chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin II, enabling patterned assembly of myosin thick filaments. A protein phosphatase (PP) has been shown to mediate RLC dephosphorylation in adult smooth and striated muscle. A role for PP activity in regulating myofibrillogenesis during embryonic development, however, has not been investigated. Tautomycin (TM) was used to inhibit both PP1 and PP2A activities, whereas okadaic acid (OA) and fostriecin (FOS) were used to inhibit PP2A. TM affected both actin and myosin assembly at 5nM; the IC50 value was 20 and 8.5nM, respectively. In contrast, OA applied at 10 times above its reported Ki for PP2A caused no significant disruption. There was also no disruption when FOS was applied at a concentration 30 times above its reported Ki for PP2A. Thus, our results suggest a primary role for PP1 isoforms during myofibrillogenesis. Although rho kinase (RK) regulates PP activity in embryonic smooth and cardiac muscle, application of the RK inhibitor Y27632 did not affect actin or myosin assembly in skeletal myocytes. Collectively, our pharmacological results suggest that PP1 is involved in dynamic regulation of RLC phosphorylation. To specifically test involvement of the myosin-targeted isoform (PP1M), we used a morpholino antisense approach to knock down the myosin targeting (M) subunit of PP1. Embryos injected with morpholino targeted to the 110-kDa M targeting subunit had fewer somites, and myosin organization was significantly perturbed. The combined pharmacological and molecular results suggest a dynamic equilibrium between MLCK and PP1M activities is required for proper myofibrillogenesis.     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.