Regulation Of Myosin Light Chain Kinase Research Articles

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

A technique for simultaneous measurement of Ca2+, FRET fluorescence and force in intact mouse small arteries

FRET (Forster resonance energy transfer)-based biosensor molecules are powerful tools to reveal specific molecular interactions in cells. Typically however, they are used in cultured cells that (inevitably) express different genes than their counterparts in intact organisms. In such cells it may be impossible to administer physiological stimuli and measure physiological outputs. Here, through the use of transgenic mice that express a FRET-based myosin light chain kinase (MLCK) biosensor molecule, we report a technique for dynamically observing activation and regulation of MLCK within the smooth muscle cells of intact, functioning small arteries, together with measurement of arterial force production and intracellular [Ca(2+)].

A Mathematical Model of Airway and Pulmonary Arteriole Smooth Muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca 2+), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca 2+ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca 2+ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca 2+ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca 2+ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca 2+-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.

Signal transduction and regulation in smooth muscle

Smooth muscle cells in the walls of many organs are vital for most bodily functions, and their abnormalities contribute to a range of diseases. Although based on a sliding-filament mechanism similar to that of striated muscles, contraction of smooth muscle is regulated by pharmacomechanical as well as by electromechanical coupling mechanisms. Recent studies have revealed previously unrecognized contractile regulatory processes, such as G-protein-coupled inhibition of myosin light-chain phosphatase, regulation of myosin light-chain kinase by other kinases, and the functional effects of smooth muscle myosin isoforms. Abnormalities of these regulatory mechanisms and isoform variations may contribute to diseases of smooth muscle, and the G-protein-coupled inhibition of protein phosphatase is also likely to be important in regulating non-muscle cell functions mediated by cytoplasmic myosin II.

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility.

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.