Myosin Light Chain Kinase In Cells Research Articles

Endothelial cell myosin light chain kinase (MLCK) regulates TNF?-induced NF?B activity

Tumor necrosis factor (TNFalpha-) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa-B (NFkappaB) activation, a regulator of anti-apoptotic genes. We previously demonstrated that increased EC contractility, rearrangement of the actin cytoskeleton, and increased myosin light chain (MLC) phosphorylation occurs as a consequence of TNFalpha-induced activation of EC MLC kinase (EC MLCK) and is required for bovine lung EC apoptosis. As the association between MLCK and pro-survival signals such as NFkappaB activation is unknown, we studied the role of MLCK in the regulation of NFkappaB-dependent transactivation in bovine pulmonary artery EC. Both TNFalpha-induced increase in NFkappaB dependent transactivation measured by NFkappaB luciferase reporter assay (approximately fivefold) and nuclear translocation of NFkappaB were significantly inhibited by MLCK-selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease). Furthermore, our data revealed that inhibition of MLCK attenuated the TNFalpha-induced IkappaB phosphorylation, translocation of p65, NFkappaB-DNA binding, and NFkappaB transcriptional activity. Molecular approaches to either reduce EC MLCK expression (AdV EC MLCK antisense construct) or to reduce kinase activity (kinase-dead EC MLCK ATPdel mutant) produced similar attenuation of the TNFalpha-induced NFkappaB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFalpha-induced luciferase activity. Together, these novel observations indicate that TNFalpha-induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFalpha-dependent NFkappaB activation and amplification of pro-survival signals.

P38 MAP kinase-dependent regulation of endothelial cell permeability.

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.

Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction.

We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.

Novel interaction of cortactin with endothelial cell myosin light chain kinase

Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca 2+/calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60 src and 80 kDa cortactin, an actin-binding protein and known p60 src target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y 464, Y 471) or cortactin (Y 421, Y 466, and Y 482) by p60 src significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972–979 and 1019–1025 as sites of cortactin interaction. EC MLCK–cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin–EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement.

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

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.

Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src).

Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60(src). Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60(src)-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.

Relaxin treatment alters the kinetic properties of myosin light chain kinase activity in rat myometrial cells in culture.

Relaxin treatment altered the kinetic properties of rat myometrial cell myosin light chain kinase (MLCK) by increasing the K50 of the enzyme for calmodulin (CaM) from 1.1 +/- 0.1 to 38 +/- 14 nM. When MLCK was assayed in the presence of 7 nM CaM to maximize the effect of the decreased affinity for CaM between control and relaxin-treated groups, a rapid concentration-dependent effect of the hormone was observed. Relaxin decreased MLCK activity significantly within 1 min. The ED50 of the effect was 0.4 microgram/ml. In addition to its effect on Ca2+-CaM-dependent activity, relaxin also decreased Ca2+-CaM-independent MLCK activity. This decrease was not attributable to a decrease in the affinity of the enzyme for myosin. There was a temporal association between the effects of relaxin on mean cell length, elevation of cAMP levels in the presence of 0.4 microM forskolin previously shown in other studies, and the alteration of MLCK activity. All three parameters were changed significantly within 1 min after exposure to relaxin. The ED50 of relaxin for cell shape changes, cAMP elevation, and effects on MLCK activity were all approximately 0.4 microgram/ml. Relaxin may act in part by a cAMP-mediated phosphorylation of MLCK, thereby decreasing its affinity for CaM. The effect on MLCK may be linked to a decrease in the phosphorylation of myosin light chains and the promotion of uterine relaxation.