Calcium/calmodulin-dependent myosin light chain kinase implicated in smooth muscle contraction via phosphorylation of myosin light chains (MLC). Also regulates actin-myosin interaction through a non-kinase activity. Phosphorylates PTK2B/PYK2 and myosin light-chains. Involved in the inflammatory response (e.g. Apoptosis, vascular permeability, leukocyte diapedesis), cell motility and morphology, airway hyperreactivity and other activities relevant to asthma. Required for tonic airway smooth muscle contraction that is necessary for physiological and asthmatic airway resistance. Necessary for gastrointestinal motility. Implicated in the regulation of endothelial as well as vascular permeability, probably via the regulation of cytoskeletal rearrangements. In the nervous system it has been shown to control the growth initiation of astrocytic processes in culture and to participate in transmitter release at synapses formed between cultured sympathetic ganglion cells. Critical participant in signaling sequences that result in fibroblast apoptosis. Plays a role in the regulation of epithelial cell survival. Required for epithelial wound healing, especially during actomyosin ring contraction during purse-string wound closure. Mediates RhoA-dependent membrane blebbing. Triggers TRPC5 channel activity in a calcium-dependent signaling, by inducing its subcellular localization at the plasma membrane. Promotes cell migration (including tumor cells) and tumor metastasis. PTK2B/PYK2 activation by phosphorylation mediates ITGB2 activation and is thus essential to trigger neutrophil transmigration during acute lung injury (ALI). May regulate optic nerve head astrocyte migration. Probably involved in mitotic cytoskeletal regulation. Regulates tight junction probably by modulating ZO-1 exchange in the perijunctional actomyosin ring. Mediates burn-induced microvascular barrier injury; triggers endothelial contraction in the development of microvascular hyperpermeability by phosphorylating MLC. Essential for intestinal barrier dysfunction. Mediates Giardia spp.-mediated reduced epithelial barrier function during giardiasis intestinal infection via reorganization of cytoskeletal F-actin and tight junctional ZO-1. Necessary for hypotonicity-induced Ca(2+) entry and subsequent activation of volume-sensitive organic osmolyte/anion channels (VSOAC) in cervical cancer cells. Responsible for high proliferative ability of breast cancer cells through anti-apoptosis.
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
The angiotensin II type 1 receptor (AT(1)R) is a G alpha(q/11)-coupled G protein-coupled receptor that is widely expressed in multiple tissues, including vascular smooth muscle cells, brain, and kidney. Activation of the AT(1)R in vascular smooth muscle cells leads to alterations in actin-based membrane protrusions such as lamellipodia, filopodia, and membrane blebs that ultimately lead to cell migration, which is important for the regulation of vascular tone. In the present study, we examine the role of small G proteins in mediating AT(1)R-induced alterations in membrane dynamics in human embryonic kidney 293 cells. We find that the activation of the AT(1)R with 100 nM angiotensin II results in the rapid formation of membrane blebs at early time points of agonist stimulation that cease within 40 min of agonist stimulation. AT(1)R-stimulated membrane bleb formation is independent of RalA, RalB, Rac1, cdc42, Arf6, and Ras, but it involves RhoA. Furthermore, membrane blebbing activated by the AT(1)R is attenuated in the presence of the beta-arrestin amino-terminal domain, Ral GDP dissociation stimulator (RalGDS) beta-arrestin binding domain, and short interfering RNA (siRNA) depletion of beta-arrestin2. However, siRNA depletion of RalGDS protein did not affect membrane blebbing in response to AT(1)R activation. The inhibition of the downstream RhoA effectors Rho kinase (ROCK) and myosin light chain kinase (MLCK) effectively attenuated AT(1)R-mediated membrane blebbing. Thus, we show that membrane blebbing in response to AT(1)R signaling is dependent on beta-arrestin2 and is mediated by a RhoA/ROCK/MLCK-dependent pathway.
We have shown previously that only the long myosin light chain kinase (MLCK), which is the predominant MLCK isoform expressed in nonmuscle cells, localizes to the cleavage furrow. To further examine the in vivo localization of the long MLCK in HeLa cells and the mechanisms responsible for kinase targeting during the cell cycle, we examined the distribution of the endogenous kinase and constructed green fluorescent protein (GFP) fusions of long HeLa MLCK truncations. A GFP fusion containing the N-terminal IgG domain and the five DXR motifs localized to stress fibers during interphase and the cleavage furrow during mitosis. Although individual fusions of the five DXRs and IgG domain both independently localized to stress fibers, only the five DXRs demonstrated a cortical localization in mitotic cells. Thus, robust targeting of the long MLCK to the cleavage furrow required the five DXRs and additional sequences from the IgG domain. Expression of the IgG domain alone or with five DXRs increased the number of multinucleate cells tenfold, whereas expression of the five DXRs or GFP had no effect. Furthermore, expression of the IgG domain alone or with five DXRs disrupted normal spindle morphology during mitosis. Extended astral microtubules and increased bundling of kinetochore microtubules, and spindle pole fragmentation were detected in mitotic cells. These microtubule defects were associated with abnormalities in metaphase chromosome alignment and a subsequent metaphase arrest caused by activation of the spindle assembly checkpoint at the kinetochores of mono-oriented chromosomes. Together, these results suggest that MLCK has an unexpected regulatory function during mitosis.
Hypotonicity-induced Ca2+ entry is a critical signal for the normal regulatory volume decrease in human cervical cancer cells. The aim of this study was to explore the role of myosin light chain kinase (MLCK) in the regulation of hypotonicity-induced Ca2+ signalling and Cl- channel activity. Blockade of MLCK activity by MLCK(11-19) amide, a substrate-specific peptide inhibitor, markedly attenuated hypotonicity-induced Ca2+ entry. A similar result was obtained with ML-7, a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK. More than 85% of the activity of the volume-regulated Cl- channel was suppressed when intracellular Ca2+ was buffered to near zero in the absence of extracellular Ca2+, suggesting that hypotonicity-induced Ca2+ signalling is important for the activation of the volume-regulated Cl- channel. Intracellular dialysis with MLCK(11-19) amide or ML-7 concentration-dependently reduced the amplitude and rate of activation of the volume-regulated Cl- channel. Swelling-activated taurine transport was also inhibited concentration dependently by ML-7 and MLCK(11-19) amide with IC(50) values of 6.4 and 2.0 microM, respectively. Hypotonicity induced MLC phosphorylation which was mediated totally by MLCK and depended on Ca2+ entry. However, phosphorylated MLC per se was not involved critically in the regulation of Ca2+ entry and activation of volume-sensitive organic osmolyte/anion channels (VSOAC). We propose that MLCK has a novel function in regulating the activation of VSOAC by mediating Ca2+ entry in response to hypotonicity. This function of MLCK on Ca2+ signalling does not correlate with MLC phosphorylation.
Myosin light-chain kinase (MLCK) plays a crucial role in the cell migration and tumor metastasis. Herein, we investigated the signaling pathways involved in MLCK using ML-7, a specific inhibitor of MLCK, in breast cancer cell proliferation and migration. Our data showed that reduction of MLCK in breast cancer cells mediated by 20 microM ML-7 was able to depress the cell proliferation and migration using two parallel cell lines (MCF-7 and LM-MCF/MDA-MB-231) with different metastatic abilities through reciprocal cross-talk with activated ERK1/2, in which both phosphorylated myosin light chain (p-MLC) and cascades of beta-catenin, cyclin D1, survivin, and c-Myc serve as essential downstream effectors.
BACKGROUND: The enhancement of cell motility is a critical event during tumor cell spreading. Since myosin light chain kinase (MLCK) regulates cell behavior, it is regarded as a promising target in terms of preventing tumor invasion and metastasis. Since MLCK was identified to be associated with human arrest defective-1 (hARD1) through yeast two-hybrid screening, we here tested the possibility that hARD1 acts as a regulator of MLCK and by so doing controls tumor cell motility. METHODOLOGY/PRINCIPAL FINDINGS: The physical interaction between MLCK and hARD1 was confirmed both in vivo and in vitro by immunoprecipitation assay and affinity chromatography. hARD1, which is known to have the activity of protein lysine epsilon-acetylation, bound to and acetylated MLCK activated by Ca(2+) signaling, and by so doing deactivated MLCK, which led to a reduction in the phosphorylation of MLC. Furthermore, hARD1 inhibited tumor cell migration and invasion MLCK-dependently. Our mutation study revealed that hARD1 associated with an IgG motif of MLCK and acetylated the Lys608 residue in this motif. The K608A-mutated MLCK was neither acetylated nor inactivated by hARD1, and its stimulatory effect on cell motility was not inhibited by hARD1. CONCLUSION/SIGNIFICANCE: These results indicate that hARD1 is a bona fide regulator of MLCK, and that hARD1 plays a crucial role in the balance between tumor cell migration and stasis. Thus, hARD1 could be a therapeutic target in the context of preventing tumor invasion and metastasis.
The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca(2+)] levels with comparable EC(50) [Ca(2+)] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60(Src)-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function.
BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.
The authors investigated the role of myosin light chain kinase (MYLK) and transforming growth factor beta (TGFbeta) receptor pathways in optic nerve head (ONH) astrocyte migration. They further investigated how the expression of these genes is altered by elevated hydrostatic pressure (HP).
Myosin II activation is essential for stress fiber and focal adhesion formation, and is implicated in integrin-mediated signaling events. In this study we investigated the role of acto-myosin contractility, and its main regulators, i.e. myosin light chain kinase (MLCK) and Rho-kinase (ROCK) in cell survival in normal and Ras-transformed MCF-10A epithelial cells. Treatment of cells with pharmacological inhibitors of MLCK (ML-7 and ML-9), or expression of dominant-negative MLCK, led to apoptosis in normal and transformed MCF-10A cells. By contrast, treatment of cells with a ROCK inhibitor (Y-27632) did not induce apoptosis in these cells. Apoptosis following inhibition of myosin II activation by MLCK is probably meditated through the death receptor pathway because expression of dominant-negative FADD blocked apoptosis. The apoptosis observed after MLCK inhibition is rescued by pre-treatment of cells with integrin-activating antibodies. In addition, this rescue of apoptosis is dependent on FAK activity, suggesting the participation of an integrin-dependent signaling pathway. These studies demonstrate a newly discovered role for MLCK in the generation of pro-survival signals in both untransformed and transformed epithelial cells and supports previous work suggesting distinct cellular roles for Rho-kinase- and MLCK-dependent regulation of myosin II.
Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of beta(2) integrins. Our results demonstrate that MYLK-mediated activation of beta(2) integrins through Pyk2 links beta(2) integrin signaling to the actin motile machinery of neutrophils.
Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.
Interacting selectively and non-covalently with calmodulin, a calcium-binding protein with many roles, both in the calcium-bound and calcium-free states.
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.
The process in which the structure of the smooth muscle tissue surrounding the aorta is generated and organized. An aorta is an artery that carries blood from the heart to other parts of the body.
Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.
The angiotensin II type 1 receptor (AT(1)R) is a G alpha(q/11)-coupled G protein-coupled receptor that is widely expressed in multiple tissues, including vascular smooth muscle cells, brain, and kidney. Activation of the AT(1)R in vascular smooth muscle cells leads to alterations in actin-based membrane protrusions such as lamellipodia, filopodia, and membrane blebs that ultimately lead to cell migration, which is important for the regulation of vascular tone. In the present study, we examine the role of small G proteins in mediating AT(1)R-induced alterations in membrane dynamics in human embryonic kidney 293 cells. We find that the activation of the AT(1)R with 100 nM angiotensin II results in the rapid formation of membrane blebs at early time points of agonist stimulation that cease within 40 min of agonist stimulation. AT(1)R-stimulated membrane bleb formation is independent of RalA, RalB, Rac1, cdc42, Arf6, and Ras, but it involves RhoA. Furthermore, membrane blebbing activated by the AT(1)R is attenuated in the presence of the beta-arrestin amino-terminal domain, Ral GDP dissociation stimulator (RalGDS) beta-arrestin binding domain, and short interfering RNA (siRNA) depletion of beta-arrestin2. However, siRNA depletion of RalGDS protein did not affect membrane blebbing in response to AT(1)R activation. The inhibition of the downstream RhoA effectors Rho kinase (ROCK) and myosin light chain kinase (MLCK) effectively attenuated AT(1)R-mediated membrane blebbing. Thus, we show that membrane blebbing in response to AT(1)R signaling is dependent on beta-arrestin2 and is mediated by a RhoA/ROCK/MLCK-dependent pathway.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of detection of, or exposure to, a hypotonic environment, i.e. an environment with a lower concentration of solutes than the organism or cell.
Hypotonicity-induced Ca2+ entry is a critical signal for the normal regulatory volume decrease in human cervical cancer cells. The aim of this study was to explore the role of myosin light chain kinase (MLCK) in the regulation of hypotonicity-induced Ca2+ signalling and Cl- channel activity. Blockade of MLCK activity by MLCK(11-19) amide, a substrate-specific peptide inhibitor, markedly attenuated hypotonicity-induced Ca2+ entry. A similar result was obtained with ML-7, a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK. More than 85% of the activity of the volume-regulated Cl- channel was suppressed when intracellular Ca2+ was buffered to near zero in the absence of extracellular Ca2+, suggesting that hypotonicity-induced Ca2+ signalling is important for the activation of the volume-regulated Cl- channel. Intracellular dialysis with MLCK(11-19) amide or ML-7 concentration-dependently reduced the amplitude and rate of activation of the volume-regulated Cl- channel. Swelling-activated taurine transport was also inhibited concentration dependently by ML-7 and MLCK(11-19) amide with IC(50) values of 6.4 and 2.0 microM, respectively. Hypotonicity induced MLC phosphorylation which was mediated totally by MLCK and depended on Ca2+ entry. However, phosphorylated MLC per se was not involved critically in the regulation of Ca2+ entry and activation of volume-sensitive organic osmolyte/anion channels (VSOAC). We propose that MLCK has a novel function in regulating the activation of VSOAC by mediating Ca2+ entry in response to hypotonicity. This function of MLCK on Ca2+ signalling does not correlate with MLC phosphorylation.
Any process that activates or increases the frequency, rate or extent of the directed movement of calcium ions into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
BACKGROUND: The enhancement of cell motility is a critical event during tumor cell spreading. Since myosin light chain kinase (MLCK) regulates cell behavior, it is regarded as a promising target in terms of preventing tumor invasion and metastasis. Since MLCK was identified to be associated with human arrest defective-1 (hARD1) through yeast two-hybrid screening, we here tested the possibility that hARD1 acts as a regulator of MLCK and by so doing controls tumor cell motility. METHODOLOGY/PRINCIPAL FINDINGS: The physical interaction between MLCK and hARD1 was confirmed both in vivo and in vitro by immunoprecipitation assay and affinity chromatography. hARD1, which is known to have the activity of protein lysine epsilon-acetylation, bound to and acetylated MLCK activated by Ca(2+) signaling, and by so doing deactivated MLCK, which led to a reduction in the phosphorylation of MLC. Furthermore, hARD1 inhibited tumor cell migration and invasion MLCK-dependently. Our mutation study revealed that hARD1 associated with an IgG motif of MLCK and acetylated the Lys608 residue in this motif. The K608A-mutated MLCK was neither acetylated nor inactivated by hARD1, and its stimulatory effect on cell motility was not inhibited by hARD1. CONCLUSION/SIGNIFICANCE: These results indicate that hARD1 is a bona fide regulator of MLCK, and that hARD1 plays a crucial role in the balance between tumor cell migration and stasis. Thus, hARD1 could be a therapeutic target in the context of preventing tumor invasion and metastasis.
BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.
Myosin light chain kinase (MLCK), a key enzyme in muscle contraction, has been shown by immunohistology to be present in neurons and glia. We describe here the cloning of the cDNA for human MLCK from hippocampus, encoding a protein sequence 95% similar to smooth muscle MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA clone detected two RNA transcripts in human frontal and entorhinal cortex, in hippocampus, and in jejunum, one corresponding to MLCK and the other probably to telokin, the carboxy-terminal 154 codons of MLCK expressed as an independent protein in smooth muscle. Levels of expression were lower in brain compared to smooth muscle. We show that within the protein sequence, a motif of 28 or 24 residues is repeated five times, the second repeat ending with the putative methionine start codon. These repeats overlap with a second previously reported module of 12 residues repeated five times in the human sequence. In addition, the acidic C-terminus of all MLCKs from both brain and smooth muscle resembles the C-terminus of tubulins. The chromosomal localization of the gene for human MLCK is shown to be at 3qcen-q21, as determined by PCR and Southern blotting using two somatic cell hybrid panels.
A process in which force is generated within smooth muscle tissue, resulting in a change in muscle geometry. Force generation involves a chemo-mechanical energy conversion step that is carried out by the actin/myosin complex activity, which generates force through ATP hydrolysis. Smooth muscle differs from striated muscle in the much higher actin/myosin ratio, the absence of conspicuous sarcomeres and the ability to contract to a much smaller fraction of its resting length.
A process in which force is generated within tonic smooth muscle tissue, resulting in a change in muscle geometry. Force generation involves a chemo-mechanical energy conversion step that is carried out by the actin/myosin complex activity, which generates force through ATP hydrolysis. In the tonic smooth muscle, the muscle contraction occurs without an ordered sarcomeric structure. Tonic smooth muscle contraction occurs as a sustained continuous contraction.
ISSOrtholog Curator
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
Isoform
Iso 1
Is activated by phosphorylation on Tyr-464 and Tyr-471. Isoforms which lack these tyrosine residues are not regulated in this way. All catalytically active isoforms require binding to calcium and calmodulin for activation. Repressed by organometallic pyridylnaphthalimide complexes, wortmannin, ML-7 (a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK) and ML-9.
Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. We previously reported the involvement of intracellular Ca(2+) in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.
Hypotonicity-induced Ca2+ entry is a critical signal for the normal regulatory volume decrease in human cervical cancer cells. The aim of this study was to explore the role of myosin light chain kinase (MLCK) in the regulation of hypotonicity-induced Ca2+ signalling and Cl- channel activity. Blockade of MLCK activity by MLCK(11-19) amide, a substrate-specific peptide inhibitor, markedly attenuated hypotonicity-induced Ca2+ entry. A similar result was obtained with ML-7, a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK. More than 85% of the activity of the volume-regulated Cl- channel was suppressed when intracellular Ca2+ was buffered to near zero in the absence of extracellular Ca2+, suggesting that hypotonicity-induced Ca2+ signalling is important for the activation of the volume-regulated Cl- channel. Intracellular dialysis with MLCK(11-19) amide or ML-7 concentration-dependently reduced the amplitude and rate of activation of the volume-regulated Cl- channel. Swelling-activated taurine transport was also inhibited concentration dependently by ML-7 and MLCK(11-19) amide with IC(50) values of 6.4 and 2.0 microM, respectively. Hypotonicity induced MLC phosphorylation which was mediated totally by MLCK and depended on Ca2+ entry. However, phosphorylated MLC per se was not involved critically in the regulation of Ca2+ entry and activation of volume-sensitive organic osmolyte/anion channels (VSOAC). We propose that MLCK has a novel function in regulating the activation of VSOAC by mediating Ca2+ entry in response to hypotonicity. This function of MLCK on Ca2+ signalling does not correlate with MLC phosphorylation.
Myosin II regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a fluorescent biosensor MLCK where Ca(2+)-dependent increases in kinase activity were coincident with decreases in fluorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca(2+)](i) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca(2+)](i). At saturating [Ca(2+)](i) MLCK was not fully activated probably due to limited availability of cellular Ca(2+)/CaM.
The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca(2+)] levels with comparable EC(50) [Ca(2+)] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60(Src)-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function.
Myosin II activation is essential for stress fiber and focal adhesion formation, and is implicated in integrin-mediated signaling events. In this study we investigated the role of acto-myosin contractility, and its main regulators, i.e. myosin light chain kinase (MLCK) and Rho-kinase (ROCK) in cell survival in normal and Ras-transformed MCF-10A epithelial cells. Treatment of cells with pharmacological inhibitors of MLCK (ML-7 and ML-9), or expression of dominant-negative MLCK, led to apoptosis in normal and transformed MCF-10A cells. By contrast, treatment of cells with a ROCK inhibitor (Y-27632) did not induce apoptosis in these cells. Apoptosis following inhibition of myosin II activation by MLCK is probably meditated through the death receptor pathway because expression of dominant-negative FADD blocked apoptosis. The apoptosis observed after MLCK inhibition is rescued by pre-treatment of cells with integrin-activating antibodies. In addition, this rescue of apoptosis is dependent on FAK activity, suggesting the participation of an integrin-dependent signaling pathway. These studies demonstrate a newly discovered role for MLCK in the generation of pro-survival signals in both untransformed and transformed epithelial cells and supports previous work suggesting distinct cellular roles for Rho-kinase- and MLCK-dependent regulation of myosin II.
A new metal-containing scaffold for the design of protein kinase inhibitors is introduced. Key feature is a 3-(2-pyridyl)-1,8-naphthalimide "pharmacophore chelate ligand" which is designed to form two hydrogen bonds with the hinge region of the ATP-binding site and is at the same time capable of serving as a stable bidentate ligand through C-H-activation at the 4-position of the electron-deficient naphthalene moiety. This C-H-activation leads to a reduced demand for coordinating heteroatoms and thus sets the basis for a very efficient three-step synthesis starting from 1,8-naphthalic anhydride. The versatility of this ligand is demonstrated with the discovery of a ruthenium complex that functions as a nanomolar inhibitor for myosin light-chain kinase (MYLK or MLCK).
In asthmatic patients, overexpression promotes actin filament propulsion, thus contributing to airway hyperresponsiveness. Some MYLK variants may contribute to acute lung injury (ALI) susceptibility. Potential therapeutic target in the treatment of burn edema.
CuratedUniProtKB
Biophysicochemical properties
Kinetic
parameters
KM
6.5 uM for MLC (isoform 1 at 22 degrees Celsius)
KM
7.2 uM for MLC (isoform 2 at 22 degrees Celsius)
Vmax
11.9 umol/min/mg enzyme (isoform 1 at 22 degrees Celsius)
Vmax
10.9 umol/min/mg enzyme (isoform 1 at 22 degrees Celsius)
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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