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1.  A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability* 
The Journal of biological chemistry  2004;279(53):55506-55513.
Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.
PMCID: PMC1237105  PMID: 15507455
2.  Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo 
Journal of Clinical Investigation  2005;115(10):2702-2715.
Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell–mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.
PMCID: PMC1224297  PMID: 16184195
3.  Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model 
PLoS ONE  2013;8(10):e77523.
Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.
Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO2 cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.
Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.
These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.
PMCID: PMC3798300  PMID: 24147016
4.  Loss of Guanylyl Cyclase C (GCC) Signaling Leads to Dysfunctional Intestinal Barrier 
PLoS ONE  2011;6(1):e16139.
Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.
Methodology/Principal Findings
Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.
GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.
PMCID: PMC3031533  PMID: 21305056
5.  LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms 
Gastroenterology  2007;132(7):2383-2394.
LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a TNF core family member that regulates T cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT was capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling.
The effects of LIGHT and interferon-γ (IFN-γ) on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers.
LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. IFN-γ pretreatment, which induced lymphotoxin β receptor (LTβR) expression, was required for these effects and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LTβR-knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin co-localized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis.
T cell-derived LIGHT activates intestinal epithelial LTβR to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.
PMCID: PMC2709832  PMID: 17570213
tight junction; interferon-γ; tumor necrosis factor; LIGHT; lymphotoxin; endocytosis; cytoskeleton; myosin; inflammatory bowel disease
6.  Role of Non-muscle Myosin Light Chain Kinase in Neutrophil-mediated Intestinal Barrier Dysfunction During Thermal Injury 
Shock (Augusta, Ga.)  2012;38(4):436-443.
Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared to their wild-type counterparts, nmMLCK-/- mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels post-burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of FITC-dextran (4 kD), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.
PMCID: PMC3716260  PMID: 22814287
Myosin light chain kinase; neutrophil; intestinal barrier dysfunction; signal transduction
7.  Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function 
Molecular Biology of the Cell  2010;21(22):4042-4056.
This study identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy and examined their influence on nmMLCK function and human lung endothelial barrier regulation. The data indicate an essential role for Abl kinase in vascular barrier regulation via phosphorylation of nmMLCK and the actin-binding protein cortactin.
Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.
PMCID: PMC2982111  PMID: 20861316
8.  Myosin light chain kinase in microvascular endothelial barrier function 
Cardiovascular Research  2010;87(2):272-280.
Microvascular barrier dysfunction is implicated in the initiation and progression of inflammation, posttraumatic complications, sepsis, ischaemia–reperfusion injury, atherosclerosis, and diabetes. Under physiological conditions, a precise equilibrium between endothelial cell–cell adhesion and actin–myosin-based centripetal tension tightly controls the semi-permeability of microvascular barriers. Myosin light chain kinase (MLCK) plays an important role in maintaining the equilibrium by phosphorylating myosin light chain (MLC), thereby inducing actomyosin contractility and weakening endothelial cell–cell adhesion. MLCK is activated by numerous physiological factors and inflammatory or angiogenic mediators, causing vascular hyperpermeability. In this review, we discuss experimental evidence supporting the crucial role of MLCK in the hyperpermeability response to key cell signalling events during inflammation. At the cellular level, in vitro studies of cultured endothelial monolayers treated with MLCK inhibitors or transfected with specific inhibiting peptides have demonstrated that induction of endothelial MLCK activity is necessary for hyperpermeability. Ex vivo studies of live microvessels, enabled by development of the isolated, perfused venule method, support the importance of MLCK in endothelial permeability regulation in an environment that more closely resembles in vivo tissues. Finally, the role of MLCK in vascular hyperpermeability has been confirmed with in vivo studies of animal disease models and the use of transgenic MLCK210 knockout mice. These approaches provide a more complete view of the role of MLCK in vascular barrier dysfunction.
PMCID: PMC2895546  PMID: 20479130
MLCK; MLC; Contractile cytoskeleton; Endothelial barrier function; Microvascular permeability
9.  Helicobacter pylori Dysregulation of Gastric Epithelial Tight Junctions by Urease-Mediated Myosin II Activation 
Gastroenterology  2008;136(1):236-246.
Background & Aims
Helicobacter pylori-induced gastritis predisposes to the development of gastric cancer. Increased epithelial tight junctions permeability and alterations in apical-junctional complexes are also associated with an increased risk of carcinogenesis. Phosphorylation of myosin regulatory light chain (MLC) by MLC kinase (MLCK) regulates tight junction function. We determined whether MLCK was activated by H. pylori and defined the mechanisms through which such activation dysregulates gastric epithelial barrier function.
MKN28 gastric epithelial cells were co-cultured with the H. pylori cag+ strain 60190 or cagA-, cagE-, ureB-, or vacA- mutants. MLC phosphorylation and barrier integrity were determined by immunoblot analysis and transepithelial electrical resistance measurements, respectively. Localization of the tight junction protein occludin was determined by immunocytochemistry in MKN28 cells and INS-GAS mice.
H. pylori induced a progressive loss of barrier function that was attenuated by inactivation of ureB, but not cagA, cagE, or vacA. Reductions in transepithelial electrical resistance were also dependent on functional urease activity. H. pylori increased MLC phosphorylation in epithelial monolayers; this was significantly decreased by inhibition of MLCK or Rho kinase or by loss of UreB. H. pylori infection of either cultured monolayers or hypergastrinemic INS-GAS mice induced occludin endocytosis, reflecting cytoskeletally-mediated disruption of tight junctions.
H. pylori increases MLC phosphorylation, occludin internalization and barrier dysfunction in gastric epithelial cells. This process requires functional urease activity and is independent of the cag pathogenicity island or VacA. These data provide new insights into the mechanisms by which H. pylori disrupts gastric barrier function.
PMCID: PMC2678540  PMID: 18996125
10.  Modulation of smooth muscle tonus in the lower urinary tract: Interplay of MLCK and MLCP 
BJU international  2010;108(2 Pt 2):E66-E70.
Smooth muscle tonus in the bladder and urethra plays a critical role in the normal function of the lower urinary tract. The phosphorylation status of the myosin light chain (MLC) is a key regulator of smooth muscle tonus. MLC phosphorylation is modulated by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We investigated expression and activity of MLCK and MLCP in the rat bladder and urethra.
Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague Dawley rats. Real-time PCR and Western blotting were used to assess expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1), an inhibitor of MLCP. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity.
MLCK mRNA expression was higher in the bladder than the urethra but this difference was not statistically significant (0.26 ± 0.17 vs. 0.14 ± 0.12, p = 0.09). MYPT1 mRNA expression was significantly higher in the bladder than the urethra (2.31 ± 1.04 vs. 0.56 ± 0.36, p = 0.001). Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared to the urethra (1.63 ± 0.25 vs. 0.91 ± 0.29 and 0.97 ± 0.10 vs. 0.37 ± 0.29, respectively, p <0.001 for both). Functional enzymatic assay identified significantly greater MLCK activity in the bladder compared to the urethra. MLCP activity was lower in the bladder compared to the urethra but the difference was statistically significant only at 1 minute after initiation of assay.
In healthy young female rats, MLCK activity is higher and MLCP activity lower in the bladder relative to the urethra. These differences likely play a role in modulating the functional differences between bladder and urethral smooth muscle tone.
PMCID: PMC3158139  PMID: 21070574
bladder; urethra; myosin light chain kinase; myosin light chain phosphatase
11.  IFN-γ-induced TNFR2 Upregulation is Required for TNF-dependent Intestinal Epithelial Barrier Dysfunction 
Gastroenterology  2006;131(4):1153-1163.
Background & Aims:
Tumor necrosis factor (TNF) plays critical roles in intestinal disease. In intestinal epithelia, TNF causes tight junction disruption and epithelial barrier loss by upregulating myosin light chain kinase (MLCK) activity and expression. The aim of this study was to determine the signaling pathways by which TNF causes intestinal epithelial barrier loss.
Caco-2 cells that were either non-transfected or stably-transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiological, morphological, and biochemical analyses.
Colitis induced in vivo by adoptive transfer of CD4+CD45RBhi T cells was associated with increased epithelial myosin light chain kinase (MLCK) expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphological tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after IFN-γ pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-γ and TNF synergize to cause intestinal epithelial barrier loss. IFN-γ priming increased TNFR1 and TNFR2 expression and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-γ-independent TNF responses.
IFN-γ primes intestinal epithelia to respond to TNF by inducing TNFR2 upregulation, which in turn mediates the TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.
PMCID: PMC1693969  PMID: 17030185
tight junction; interferon-γ; tumor necrosis factor; cytoskeleton; myosin; inflammatory bowel disease
12.  Localization and Activity of Myosin Light Chain Kinase Isoforms during the Cell Cycle 
The Journal of Cell Biology  2000;151(3):697-708.
Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH2-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH2-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735–744) act cooperatively to regulate the level of Ser 19–phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.
PMCID: PMC2185581  PMID: 11062269
myosin; phosphorylation; myosin light chain kinase; cell division; cytokinesis
13.  Decreased myosin phosphatase target subunit 1(MYPT1) phosphorylation via attenuated rho kinase and zipper-interacting kinase activities in edematous intestinal smooth muscle 
Neurogastroenterology and Motility  2012;24(3):257-e109.
Intestinal edema development after trauma resuscitation inhibits intestinal motility which results in ileus, preventing enteral feeding and compromising patient outcome. We have shown previously that decreased intestinal motility is associated with decreased smooth muscle myosin light chain (MLC) phosphorylation. The purpose of the present study was to investigate the mechanism of edema-induced decreases in MLC in a rodent model of intestinal edema.
Intestinal edema was induced by a combination of resuscitation fluid administration and mesenteric venous hypertension. Sham operated animals served as controls. Contractile activity and alterations in the regulation of MLC including the regulation of MLC kinase (MLCK) and MLC phosphatase (MLCP) were measured.
Key Results
Contraction amplitude and basal tone were significantly decreased in edematous intestinal smooth muscle compared to non-edematous tissue. Calcium sensitivity was also decreased in edematous tissue compared to non-edematous intestinal smooth muscle. Although inhibition of MLCK decreased contractile activity significantly less in edematous tissue compared to non-edematous tissue, MLCK activity in tissue lysates was not significantly different. Phosphorylation of MYPT was significantly lower in edematous tissue compared to non-edematous tissue. In addition, activities of both rho kinase and zipper-interacting kinase were significantly lower in edematous tissue.
Conclusions and Inferences
We conclude from these data that interstitial intestinal edema inhibits MLC phosphorylation predominantly by decreasing inhibitory phosphorylation of the MLC targeting subunit (MYPT1) of MLC phosphatase via decreased ROCK and ZIPK activities, resulting in more MLC phosphatase activity.
PMCID: PMC3321580  PMID: 22235829
intestinal smooth muscle; myosin light chain; MYPT1; edema; intestinal motility; rho kinase
14.  Identification of Cardiac-Specific Myosin Light Chain Kinase 
Circulation research  2008;102(5):571-580.
Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca2+/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.
PMCID: PMC2504503  PMID: 18202317
kinase; transcription; contraction
Journal of neuroscience research  2011;89(6):852-861.
Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton leading to disruption of vascular barrier integrity in multiple organs including the blood brain barrier (BBB) after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase in MLCK in astrocytes and the signaling pathways involved in this increase. Following midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MCLK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK or rho kinase also prevented this increase. These results are further evidence of a role of MCLK in the mechanisms of BBB compromise following TBI, and identify astrocytes as a cell type, in addition to endothelium in the BBB which express MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB.
PMCID: PMC3079319  PMID: 21360574
myosin light chain kinase; blood brain barrier; astrocyte; traumatic brain injury; transforming growth factor
16.  Crystallization and preliminary X-ray analysis of the human long myosin light-chain kinase 1-specific domain IgCAM3 
To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized.
Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0 Å resolution and were consistent with the primitive trigonal space group P212121.
PMCID: PMC3034612  PMID: 21301090
IgCAM3 domain; myosin light-chain kinase 1
17.  Regulation of Cell Motility by Mitogen-activated Protein Kinase 
The Journal of Cell Biology  1997;137(2):481-492.
Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.
PMCID: PMC2139771  PMID: 9128257
18.  Rho-Kinase–Mediated Contraction of Isolated Stress Fibers 
The Journal of Cell Biology  2001;153(3):569-584.
It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca2+. However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca2+. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca2+-dependent MLCK and the Rho-kinase systems. We propose that Ca2+ is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.
PMCID: PMC2190572  PMID: 11331307
Rho; Rho-kinase; stress fiber; contraction; myosin regulatory light chain
19.  Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo 
Molecular and Cellular Biology  2005;25(14):6259-6266.
Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.
PMCID: PMC1168802  PMID: 15988034
20.  Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation 
EMBO Molecular Medicine  2012;4(2):109-124.
Epithelial myosin light chain kinase (MLCK)-dependent barrier dysfunction contributes to the pathogenesis of inflammatory bowel diseases (IBD). We reported that epithelial GM-CSF–STAT5 signalling is essential for intestinal homeostatic response to gut injury. However, mechanism, redundancy by STAT5 or cell types involved remained foggy. We here generated intestinal epithelial cell (IEC)-specific STAT5 knockout mice, these mice exhibited a delayed mucosal wound healing and dysfunctional intestinal barrier characterized by elevated levels of NF-κB activation and MLCK, and a reduction of zonula occludens expression in IECs. Deletion of MLCK restored intestinal barrier function in STAT5 knockout mice, and facilitated mucosal wound healing. Consistently, knockdown of stat5 in IEC monolayers led to increased NF-κB DNA binding to MLCK promoter, myosin light chain phosphorylation and tight junction (TJ) permeability, which were potentiated by administration of tumour necrosis factor-α (TNF-α), and prevented by concurrent NF-κB knockdown. Collectively, enterocyte STAT5 signalling protects against TJ barrier dysfunction and promotes intestinal mucosal wound healing via an interaction with NF-κB to suppress MLCK. Targeting IEC STAT5 signalling may be a novel therapeutic approach for treating intestinal barrier dysfunction in IBD.
PMCID: PMC3306555  PMID: 22228679
inflammatory bowel disease (IBD); myosin light chain kinase (MLCK); nuclear factor-κB (NF-κB); signals transducers and activators of transcription (STAT) 5; tight junction (TJ)
21.  Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation 
EMBO Molecular Medicine  2012;4(2):109-124.
Epithelial myosin light chain kinase (MLCK)-dependent barrier dysfunction contributes to the pathogenesis of inflammatory bowel diseases (IBD). We reported that epithelial GM-CSF–STAT5 signalling is essential for intestinal homeostatic response to gut injury. However, mechanism, redundancy by STAT5 or cell types involved remained foggy. We here generated intestinal epithelial cell (IEC)-specific STAT5 knockout mice, these mice exhibited a delayed mucosal wound healing and dysfunctional intestinal barrier characterized by elevated levels of NF-κB activation and MLCK, and a reduction of zonula occludens expression in IECs. Deletion of MLCK restored intestinal barrier function in STAT5 knockout mice, and facilitated mucosal wound healing. Consistently, knockdown of stat5 in IEC monolayers led to increased NF-κB DNA binding to MLCK promoter, myosin light chain phosphorylation and tight junction (TJ) permeability, which were potentiated by administration of tumour necrosis factor-α (TNF-α), and prevented by concurrent NF-κB knockdown. Collectively, enterocyte STAT5 signalling protects against TJ barrier dysfunction and promotes intestinal mucosal wound healing via an interaction with NF-κB to suppress MLCK. Targeting IEC STAT5 signalling may be a novel therapeutic approach for treating intestinal barrier dysfunction in IBD.
PMCID: PMC3306555  PMID: 22228679
inflammatory bowel disease (IBD); myosin light chain kinase (MLCK); nuclear factor-κB (NF-κB); signals transducers and activators of transcription (STAT) 5; tight junction (TJ)
22.  Expression of a Novel Myosin Light Chain Kinase in Embryonic Tissues and Cultured Cells* 
The Journal of biological chemistry  1995;270(49):29090-29095.
A novel, 208-kDa myosin light chain kinase (MLCK) distinct from smooth muscle and non-muscle MLCK has been identified by cross-reaction to two antibodies raised against smooth muscle MLCK. Additional antibodies directed against the amino and carboxyl termini of the smooth muscle MLCK do not react with the 208-kDa MLCK, suggesting these regions are distinct. 208-kDa MLCK phosphorylates 20-kDa myosin light chains in a Ca2+/calmodulin-dependent manner, consistent with it being a member of the MLCK family. Expression of 208-kDa MLCK and smooth muscle MLCK appears to be inversely regulated, with 208-kDa MLCK being most abundant during early development and declining at birth. In contrast, expression of smooth muscle MLCK is relatively low early during development and increases to become the predominant MLCK detected in all adult smooth and non-muscle tissues. The developmental expression pattern of the 208-kDa MLCK suggests this form be named, embryonic MLCK.
PMCID: PMC2836784  PMID: 7493932
23.  The Hormone Ghrelin Prevents Traumatic Brain Injury Induced Intestinal Dysfunction 
Journal of Neurotrauma  2010;27(12):2255-2260.
Intestinal barrier breakdown following traumatic brain injury (TBI) is characterized by increased intestinal permeability, leading to bacterial translocation, and inflammation. The hormone ghrelin may prevent intestinal injury and have anti-inflammatory properties. We hypothesized that exogenous ghrelin prevents intestinal injury following TBI. A weight-drop model created severe TBI in three groups of anesthetized Balb/c mice. Group TBI: animals underwent TBI only; Group TBI/ghrelin: animals were given 10 μg of ghrelin intraperitoneally prior and 1 h following TBI; Group sham: no TBI or ghrelin injection. Intestinal permeability was measured 6 h following TBI by detecting serum levels of FITC-Dextran after injection into the intact ileum. The terminal ileum was harvested for histology, expression of the tight junction protein MLCK and inflammatory cytokine TNF-α. Permeability increased in the TBI group compared to the sham group (109.7 ± 21.8 μg/mL vs. 32.2 ± 10.1 μg/mL; p < 0.002). Ghrelin prevented TBI-induced permeability (28.3 ± 4.2 μg/mL vs. 109.7 ± 21.8 μg/mL; p < 0.001). The intestines of the TBI group showed blunting and necrosis of villi compared to the sham group, while ghrelin injection preserved intestinal architecture. Intestinal MLCK increased 73% compared to the sham group (p < 0.03). Ghrelin prevented TBI-induced MLCK expression to sham levels. Intestinal TNF-α increased following TBI compared to the sham group (46.2 ± 7.1 pg/mL vs. 24.4 ± 2.2 pg/mL p < 0.001). Ghrelin reduced TNF-α to sham levels (29.2 ± 5.0 pg/mL; p = NS). We therefore conclude that ghrelin prevents TBI-induced injury, as determined by intestinal permeability, histology, and intestinal levels of TNF-α. The mechanism for ghrelin mediating intestinal protection is likely multifactorial, and further studies are needed to delineate these possibilities.
PMCID: PMC3304249  PMID: 20858122
ghrelin; intestinal permeability; tight junctions; traumatic brain injury
24.  Biochemistry of Smooth Muscle Myosin Light Chain Kinase 
The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca2+-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.
PMCID: PMC3382066  PMID: 21565153
25.  Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2 
The Febs Journal  2012;279(13):2444-2454.
Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.
PMCID: PMC3377847  PMID: 22564771
myosin light chain; phosphorylation; matrix metalloproteinase; ischemia-reperfusion; ML-7; doxycycline

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