Brain-derived neurotrophic factor regulates LYN kinase-mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons.

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

Molecular Insights into the MLCK Activation by CaM.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I.

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.

Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.

Calmodulin (CaM) mediates a wide range of biological responses to changes in intracellular Ca2+ concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca2+ ions to each of the two four-helix-bundle domains of CaM results in major conformational changes that create a potential binding site for the CaM binding domain of a target protein, which also undergoes major conformational changes to form the complex with CaM. Details of the molecular mechanism of complex formation are not well established, despite numerous structural, spectroscopic, thermodynamic, and kinetic studies. Here, we report a study of the process by which the 26-residue peptide M13, which represents the CaM binding domain of skeletal muscle myosin light chain kinase, forms a complex with CaM in the presence of excess Ca2+ on the millisecond time scale. Our experiments use a combination of selective 13C labeling of CaM and M13, rapid mixing of CaM solutions with M13/Ca2+ solutions, rapid freeze-quenching of the mixed solutions, and low-temperature solid state nuclear magnetic resonance (ssNMR) enhanced by dynamic nuclear polarization. From measurements of the dependence of 2D 13C-13C ssNMR spectra on the time between mixing and freezing, we find that the N-terminal portion of M13 converts from a conformationally disordered state to an α-helix and develops contacts with the C-terminal domain of CaM in about 2 ms. The C-terminal portion of M13 becomes α-helical and develops contacts with the N-terminal domain of CaM more slowly, in about 8 ms. The level of structural order in the CaM/M13/Ca2+ complexes, indicated by 13C ssNMR line widths, continues to increase beyond 27 ms.

Charge-dependent modulation of specific and nonspecific protein-metal ion interactions in nanoelectrospray ionization mass spectrometry.

RATIONALE: Previous studies found that charge state could affect both specific and nonspecific binding of protein-metal ion interactions in nanoelectrospray ionization mass spectrometry (nESI-MS). However, the two kinds of interactions have been studied individually in spite of the problem that they often coexist in the same system. Thus, it is necessary to study the effects of charge state on specific and nonspecific protein-metal ion interactions in one system to reveal more accurate binding state. METHODS: The HIV-1 nucleocapsid protein (NCp7(31-55)) which can bind specifically and nonspecifically to Zn2+ served as the model to show the charge-dependent protein-metal ion interactions. Hydrogen/deuterium exchange (HDX) and photodissociation (PD) were used to demonstrate that specific binding state was correlated with protein structure. In addition to NCp7(31-55), three other model proteins were used to investigate the reason for the charge-dependent nonspecific binding. RESULTS: For specific binding, we proposed that protein ions with different charge states had different conformations. The HDX results showed that labile protons in the NCp7(31-55)-Zn complex were exchanged in a charge-state-dependent way. The PD experiments revealed differential fragment yields for different charge states. For nonspecific binding, higher charge states had more Zn2+ additions, but less SO4 2- additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO4 2- . These results could reveal that the nonspecific binding was caused by electrostatic interaction. CONCLUSIONS: For specific binding, NCp7(31-55) with lower charge states have folding and undenatured structures. The binding states of lower charge states can better reflect more native binding states. For nonspecific binding, when multiple metal ions adduct to proteins, the proteins have more net positive charges, which tend to generate higher charge ions during electrospray.

Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation.

To investigate the cyanylated cysteine vibrational probe group's ability to report on binding-induced changes along a protein-protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.

Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. SIGNIFICANCE OF THE STUDY: Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Myosin Light Chain Kinase MYLK1: Anatomy, Interactions, Functions, and Regulation.

This review discusses and summarizes the results of molecular and cellular investigations of myosin light chain kinase (MLCK, MYLK1), the key regulator of cell motility. The structure and regulation of a complex mylk1 gene and the domain organization of its products is presented. The interactions of the mylk1 gene protein products with other proteins and posttranslational modifications of the mylk1 gene protein products are reviewed, which altogether might determine the role and place of MLCK in physiological and pathological reactions of cells and entire organisms. Translational potential of MLCK as a drug target is evaluated.

Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle.

The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

KEY POINTS: Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. ABSTRACT: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

Galectin 3 regulates HCC cell invasion by RhoA and MLCK activation.

Hepatocellular carcinoma (HCC) carries a poor prognosis with no effective treatment available other than liver transplantation for selected patients. Vascular invasion of HCC is one of the most important negative predictor of survival. As the regulation of invasion of HCC cells is not well understood, our aim was to study the mechanisms by which galectin 3, a ß-galactosidase-binding lectin mediates HCC cell migration. HCC was induced by N-diethylnitrosamine in wild-type and galectin 3(-/-) mice, and tumor formation, histology, and tumor cell invasion were assessed. The galectin 3(-/-) mice developed significantly smaller tumor burden with a less invasive phenotype than the wild-type animals. Galectin 3 was upregulated in the wild-type HCC tumor tissue, but not in the surrounding parenchyma. Galectin 3 expression in HCC was induced by NF-κB transactivation as determined by chromatin immunoprecipitation assays. In vitro studies assessed the pro-migratory effects of galectin 3. The migration of hepatoma cells was significantly decreased after transfection by the galectin 3 siRNA and also after using the Rho kinase inhibitor Y-27632. The reorganization of the actin cytoskeleton, RhoA GTPase activity and the phosphorylation of MLC2 (myosin light chain 2) were decreased in the galectin 3 siRNA-transfected cells. In addition, in vitro and in vivo evidence showed that galectin 3 deficiency reduced hepatoma cell proliferation and increased their apoptosis rate. In conclusion, galectin 3 is an important lectin that is induced in HCC cells, and promotes hepatoma cell motility and invasion by an autocrine pathway. Targeting galectin 3 therefore could be an important novel treatment strategy to halt disease progression.

Myosin regulatory light chain phosphorylation enhances cardiac ß-myosin in vitro motility under load.

Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac ß-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level.

Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

Myosin phosphatase isoforms as determinants of smooth muscle contractile function and calcium sensitivity of force production.

The dephosphorylation of myosin by the MP causes smooth muscle relaxation. MP is also a key target of signals that regulate vascular tone and thus blood flow and pressure. Here, we review studies from the past two decades that support the hypothesis that the regulated expression of MP subunits is a critical determinant of smooth muscle responses to constrictor and dilator signals. In particular, the highly regulated splicing of the regulatory subunit Mypt1 Exon 24 is proposed to tune sensitivity to NO/cGMP-mediated relaxation. The regulated transcription of the MP inhibitory subunit CPI-17 is proposed to determine sensitivity to agonist-mediated constriction. The expression of these subunits is specific in the microcirculation and varies in developmental and disease contexts. To date, the relationship between MP subunit expression and vascular function in these different contexts is correlative; confirmation of the hypothesis will require the generation of genetically engineered mice to test the role of MP subunits and their isoforms in the specificity of vascular smooth muscle responses to constrictor and dilator signals.

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.

Myosin light chain kinase signaling in endothelial barrier dysfunction.

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.

Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²âºCaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

Importance of the interaction protein-protein of the CaM-PDE1A and CaM-MLCK complexes in the development of new anti-CaM drugs.

Protein-protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein-protein-ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII-spectrin peptide (αII-spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C-mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM-protein complex under analysis. For the Ca(2+) -CaM, Ca(2+) -CaM-PDE1A, and Ca(2+) -CaM-MLCK complexes, CPZ apparent dissociation constants (Kds ) were 1.11, 0.28, and 0.55 µM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 µM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII-spec) to Ca(2+) -hCaM M124C-mBBr quenched the fluorescence (Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 µM) did not affect the fluorescent signal. Instead, the additions of αII-spec to a preformed Ca(2+) -hCaM M124C-mBBr-MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca(2+) -CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca(2+) -CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands.