Smooth Muscle Myosin Light Chain Kinase Research Articles

Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

Notch signaling regulates arterial vasoreactivity through opposing functions of Jagged1 and Dll4 in the vessel wall.

Functional interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) in the arterial wall are necessary for controlling vasoreactivity that underlies vascular resistance and tone. Key signaling pathways converge on the phosphorylation of myosin light chain (p-MLC), the molecular signature of force production in SMCs, through coordinating the relative activities of myosin light chain kinase (MLCK) and myosin phosphatase (MP). Notch signaling in the vessel wall serves critical roles in arterial formation and maturation and has been implicated in arterial vasoregulation. In this report, we hypothesized that Notch signaling through ligands Jagged1 (in SMCs) and delta-like protein-4 (Dll4; in ECs) regulates vasoreactivity via homotypic (SMC-SMC) and heterotypic (EC-SMC) cell interactions. Using ligand induction assays, we demonstrated that Jagged1 selectively induced smooth muscle MLCK gene expression and p-MLC content while inhibiting MP function (i.e., increased Ca2+ sensitization) in a Rho kinase II-dependent manner. Likewise, selective deficiency of smooth muscle Jagged1 in mice resulted in MLCK and p-MLC loss, reduced Ca2+ sensitization, and impaired arterial force generation measured by myography. In contrast, smooth muscle Notch signaling triggered by Dll4 increased expression of MP-targeting subunit 1 (MYPT1; the MP regulatory subunit), whereas arteries from endothelial Dll4-deficient mice featured reduced MYPT1 levels, enhanced force production, and impaired relaxation independent of endothelium-derived nitric oxide signaling. Taken together, this study identifies novel opposing vasoregulatory functions for ligand-specific Notch signaling in the vessel wall, underscoring instructional signaling between ECs and SMCs and suggesting that Notch signals might behave as a "rheostat" in arterial tone control. NEW & NOTEWORTHY The present study unveils novel roles for ligand-specific Notch signaling in arterial function. Smooth muscle Jagged1 and endothelial cell delta-like protein-4 ligands exhibit selective regulation of myosin light chain kinase and myosin phosphatase-targeting subunit 1/myosin phosphatase, respectively, providing a mechanistic link through which Notch signals modulate contractile activities in vascular smooth muscle. These findings may inform vascular derangements observed in human syndromes of Notch signaling deficiency while offering fundamental molecular insights into arterial physiological function.

Berberine Depresses Contraction of Smooth Muscle via Inhibiting Myosin Light-chain Kinase.

Background:Berberine is a natural isoquinoline alkaloid possessing various pharmacological effects, particularly apparent in the treatment of diarrhea, but the underlying mechanism remains unclear. Smooth muscle myosin light-chain kinase (MLCK) plays a crucial role in the smooth muscle relaxation-contraction events, and it is well known that berberine can effectively depress the contraction of smooth muscle. Hence, whether berberine could inhibit MLCK and then depress the smooth muscle contractility might be researched.Objective:The purpose of this study is to investigate the effects of berberine on MLCK. Based on this, the contractility of gastro-intestine, catalysis activity of MLCK, and molecular docking are going to be evaluated.Materials and Methods:The experiment of smooth muscle contraction was directly monitored the contractions of the isolated gastrointestine by frequency and amplitude at different concentration of berberine. The effects of berberine on MLCK were measured in the presence of Ca2+-calmodulin, using the activities of 20 kDa myosin light chain (MLC20) phosphorylation, and myosin Mg2+-ATPase induced by MLCK. The docking study was conducted with expert software in the meantime.Results:The phosphorylation of myosin and the Mg2+-ATPase activity is reduced in the presence of berberine. Moreover, berberine could inhibit the contractibility of isolated gastric intestine smooth muscle. Berberine could bind to the ATP binding site of MLCK through hydrophobic effect and hydrogen bonding according to the docking study.Conclusion:The present work gives a deep insight into the molecular mechanism for the treatment of diarrhea with berberine, i.e., berberine could suppress the contractility of smooth muscle through binding to MLCK and depressing the catalysis activity of MLCK.SUMMARY Berberine significantly reduced the amplitude of contraction in isolated duodenum and gastric strips in ratsBerberine inhibited the phosphorylated extents of MLC20 and Mg2+-ATPase activity of phosphorylated myosin induced by MLCKBerberine binds to the ATP binding site of MLCK by hydrophobic effect and hydrogen bondingBerberine may modulate contraction of smooth muscle by inhibiting MLCK. Abbreviations used: MLCK: Myosin light chain kinase; MLC20: 20 KDa regulating myosin light chain; CaM: Calmodulin.

Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling

Smooth muscle myosin light chain kinase (SM-MLCK) is the key enzyme responsible for phosphorylation of regulatory myosin light chain (MLC20), resulting in actin-myosin cross-bridging and force generation in vascular smooth muscle required for physiological vasoreactivity and blood pressure control. In this study, we investigated the combinatorial role of myocardin/serum response factor (SRF) and Notch signaling in the transcriptional regulation of MLCK gene expression. Promoter reporter analyses in rat A10 smooth muscle cells revealed a bimodal pattern of MLCK promoter activity and gene expression upon stimulation with constitutively active Notch1 in presence of myocardin or by Jagged1 ligand stimulation. An initial Notch1-induced increase in MLCK transcription was followed by loss in promoter sensitivity, which could be restored with further Notch1 dose escalation. Real-time PCR analyses revealed that endogenous levels of Hairy Related Transcription (HRT) factor 2 (HRT2) peaked concurrently with inhibitory concentrations of Notch1. Forced expression of HRT2 demonstrated simultaneous repression of both myocardin- and Notch1-induced MLCK promoter activity. HRT2-mediated repression was further confirmed by HRT2 truncations and siHRT2 treatments that rescued MLCK promoter activity and gene expression. Chromatin immunoprecipitation studies revealed both Jagged1 ligand- and Notch1-enhanced myocardin/SRF complex formation at the promoter CArG element. In contrast, heightened levels of HRT2 concomitantly disrupted myocardin/SRF and Notch transcription complex formation at respective CArG and CSL binding elements. Taken together, SM-MLCK promoter activity appears highly sensitive to the relative levels of Notch1 signaling, HRT2, and myocardin. These findings identify a novel Notch-dependent HRT2 autoregulatory circuit coordinating transcriptional regulation of SM-MLCK.

Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

An intronic MYLK variant associated with inflammatory lung disease regulates promoter activity of the smooth muscle myosin light chain kinase isoform

Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.

Moving beyond the “Allegory of the Cave” in the assessment of pulmonary arterial hypertension

PULMONARY ARTERIAL HYPERTENSION (PAH) remains a clinically vexing problem considering the high morbidity and mortality that plagues this entity despite indication of improved survival after the introduction of more targeted therapy. While this syndrome is well described from a pathological standpoint the mechanisms involved in the pulmonary vascular remodeling are less clear but are thought to involve endothelial dysfunction and proliferation of various cell types that comprise the wall of distal pulmonary vessels (3). The specific mechanisms leading to distal vessel obliteration and increased pulmonary vascular resistance have been particularly elusive for several decades (14), and although vasoconstriction as a primary event was the focus of research and targeted therapy in early studies, the PAH research community has witnessed a paradigm shift in recent years with a strong tendency to now view the PAH lesion as a quasi-cancerous process (12). In this issue of the Journal of Applied Physiology, Schwenke et al. (8) bring a new tool for assessment of the distal pulmonary vasculature and shake an old specter, vasoconstriction. The investigators employ synchrotron radiation microangiography (SRA), a fairly novel and powerful technique, to more directly evaluate vascular tone in a classic animal model of PAH, monocrotaline (MCT)-challenged rats, providing new insight into regional specific responses of the pulmonary circulation in PAH. SRA allows for both the measurement and assessment of control of blood flow in vivo and collection of data on small arteries and arterioles in situ over time (10). Thus it can supply superior information on kinetic events relative to ex vivo techniques. Since SRA has no focal plane, it can visualize both surface and penetrating vessels simultaneously unlike intravital microscopy, which is limited to assessment of vessel caliber at a single vascular plan. In dynamic organs such as the lung and heart, which are subject to motion, temporal

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

Identification of Cardiac-Specific Myosin Light Chain Kinase

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 Ca(2+)/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.

Abstract 1102: Cardiac-Specific Myosin Light Chain Kinase (MLCK), a Third Member of MLCK Family, Potentiates Sarcomere Formation and Cardiac Contraction

Background: Two myosin light chain kinase (MLCK) proteins, skeletal (encoded by mylk2 gene) and smooth muscle MLCK (encoded by mylk1 gene) have been shown to be expressed in mammals. Human mylk2 has been mapped as a disease locus for familial cardiac hypertrophy (OMIM 606566 ), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. While phosphorylation of the putative substrate of skeletal MLCK, myosin light chain 2 (MLC2), is recognized as a key regulator of cardiac contraction, the abundance of skeletal MLCK in the heart is controversial, suggesting the existence of an additional MLCK that is preferentially expressed in cardiac muscle. Methods and Results: We characterized a new kinase named cardiac MLCK that is encoded by a gene homologous to mylk1 and 2 and is specifically expressed in the heart in both atrium and ventricle. Expression of cardiac MLCK was highly regulated by the cardiac homeobox transcription factor, Nkx2.5, in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK including putative catalytic and adjacent Ca2+/calmodulin binding domains at the carboxyl-terminus. The amino-terminus is unique without significant homology to other known proteins. Cardiac MLCK phosphorylated MLC2v with a catalytic value of Km=4.3 micro M (Lineweaver-Burk analysis) indicating high affinity of cardiac MLCK to MLC2v, similar to the affinity of skeletal muscle MLCK to skeletal muscle MLC2 and smooth muscle MLCK to smooth muscle MLC2. Adenoviral-mediated overexpression of cardiac MLCK and knockdown of cardiac MLCK using RNAi in cultured cardiomyocytes revealed that cardiac MLCK regulates MLC2v phosphorylation, sarcomere organization and cardiac myocyte contraction. Expression of cardiac MLCK protein was significantly decreased in severe heart failure in vivo (post-myocardial infarction heart failure mouse model). Conclusion: Cardiac MLCK is a new key regulator of cardiac contraction and sarcomere organization. Reduction of cardiac MLCK function leading to decreased phosphorylation of MLC2v may contribute to compromised contractile function in the failing heart.

Abstract 1084: Myosin Light Chain Kinase Signaling Regulates Ectopic Atrial Activity

Objective : Determine whether calmodulin (CaM) acts through novel signaling pathways to elicit left atrial ectopic activity. Methods : Rat left atria (n=6 –9 per group) were isolated and superfused at 30°C. Mechanical function was recorded using a Grass recorder. Left atrial proteins were analyzed using Western analysis. Findings : Our rat left atria are quiescent in the absence of external pacing. However, in the presence of 2-aminoethoxydiphenyl borate (20μM; 2APB), a novel sarcoplasmic reticulum (SR) calcium leak inducer, left atria contract spontaneously at 67±12 contractions (min) − 1 in the absence of pacing. Pre-treating left atria with two irreversible CaM inhibitors, phenoxybenzamine and fluphenazine chloroethane (90μM each), decreases spontaneous contraction frequency to 5±3 and 2±1 (min) − 1 , respectively, indicating CaM signaling is activated and required for this ectopic activity. Pre-treating left atria with 20μM KN62 did not decrease the frequency of 2APB-induced ectopic activity (72±8 contractions (min) − 1 ) indicating calmodulin-dependent protein kinase II (CAMKII) is not a downstream target here. In contrast, 20μM ML7, a specific inhibitor of myosin light chain kinase (MLCK), a second cardiac CaM-regulated kinase, decreases 2APB-induced spontaneous contractions to 7±3 min −1 . Western analysis shows left atria contain smooth muscle MLCK. This kinase preferentially phosphorylates non-muscle myosin light chain 2 (nmMLC2). Western analyses also show left atria contain nmMLC2 which is not phosphorylated in untreated muscles. However, nmMLC2 phosphorylation increases significantly in left atria exposed to 2APB. Conclusions : CaM and MLCK inhibitors prevent the ectopic atrial activity induced by 2APB. This suggests a unique SR calcium leak occurs under this condition which activates CaM signaling whose specific downstream target is MLCK, a major myocardial CaM-dependent kinase. MLCK signaling via its sole substrate, nmMLC2, is required for this novel ectopic atrial activity. Hyperphosphorylation of SR ryanodine receptors by CAMKII is known to induce an SR calcium leak that underlies triggered ectopic activity. Our data suggest multiple calcium leaks and CaM signaling pathways regulate the stability of intact atrial muscle.

Salt Enhances Calmodulin-Target Interaction

Calmodulin (CaM) operates as a Ca 2+ sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca 2+-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.

Endogenously Bound Calmodulin Is Essential for the Function of the Inositol 1,4,5-Trisphosphate Receptor

Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.

Characterization of Recombinant Full-Length, Smooth-Muscle Myosin Light Chain Kinase.

Background : Myosin light chain kinase (MLCK), a regulatory protein of smooth muscle contraction, not only catalyzes the phosphorylation of the myosin regulatory light chain (MLc 20) but also binds to actin. Full-length MLCK has not yet been successfully expressed, in spite of using various expression systems. Material and Methods : cDNA of the bovine stomach MLCK (BsMLCK) was expressed using an E. coli expression system. Result : Recombinant BsMLCK was expressed in a soluble form and phosphorylated MLc20. It also demonstrated actin-binding and actin-bundling activities. Conclusion : Recombinant BsMLCK, with the properties known for MLCK, was quantifiably expressed and is the first step in analyzing the structure and function of MLCK.

Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse.

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.

Relative Affinity Constants by Electrospray Ionization and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Calmodulin Binding to Peptide Analogs of Myosin Light Chain Kinase

Synthetic RS20 peptide and a set of its point-mutated peptide analogs have been used to analyze the interactions between calmodulin (CaM) and the CaM-binding sequence of smooth-muscle myosin light chain kinase both in the presence and the absence of Ca 2+. Particular peptides, which were expected to have different binding strengths, were chosen to address the effects of electrostatic and bulky mutations on the binding affinity of the RS20 sequence. Relative affinity constants for protein/ligand interactions have been determined using electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry. The results evidence the importance of electrostatic forces in interactions between CaM and targets, particularly in the presence of Ca 2+, and the role of hydrophobic forces in contributing additional stability to the complexes both in the presence and the absence of Ca 2+.

Low lung volume alters contractile properties of airway smooth muscle in sheep.

Breathing at volumes lower than functional residual capacity (FRC) can induce changes in nonasthmatic airways consistent with the behaviour of asthmatic airways. This study investigated the chronic effect of breathing at volumes lower than FRC on the contractility of airway smooth muscle and myosin light chain kinase (MLCK) content and activity. Sheep of three age groups (neonate, adolescent and adult) had their FRC reduced by approximately 25%, for 4 weeks using a leather corset. Contractile responses to carbachol were then recorded in isolated tracheal strips and bronchial rings. MLCK content and activity were assessed by immunoblotting. The rate of stress generation increased in the bronchial smooth muscle of both adult and adolescent but not neonatal corseted sheep: adolescent corseted versus control, 65.0 +/- 4.1 versus 103.4 +/- 7.0 s (to reach 50% maximum stress), respectively; and adult corseted versus control, 57.0 +/- 6.4 versus 93.4 +/- 8.2 s, respectively. This was not due to increases in either bronchial or tracheal smooth muscle amount or MLCK content and activity. The present results indicate that chronic breathing at low lung volumes increases the rate of stress generation in airway smooth muscle.

Unique Features in the C-terminal Domain Provide Caltractin with Target Specificity

Caltractin (centrin) is a member of the calmodulin (CaM) superfamily of EF-hand calcium-binding proteins. It is an essential component of the centrosomal structures in a wide range of organisms. Caltractin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity. In an effort to understand the structural basis for such differences, the high-resolution three-dimensional solution structure of the complex between the Ca 2+-activated C-terminal domain of Chlamydomonas reinhardtii caltractin (CRC-C) and a 19 residue peptide fragment comprising the putative cdc31p-binding region of Kar1p (K 19) has been determined by multi-dimensional heteronuclear NMR spectroscopy. Formation of the complex is calcium-dependent and is stabilized by extensive interactions between CRC-C and three key hydrophobic anchors (Trp10, Leu13 and Leu14) in the peptide as well as favorable electrostatic interactions at the protein–peptide interface. In-depth comparisons have been made to the structure of the complex of Ca 2+-activated calmodulin and R 20, the CaM-binding domain of smooth muscle myosin light-chain kinase. Although the overall structures of CRC and CaM domains in their respective complexes are very similar, differences in critical regions in the sequences of these proteins and their targets lead to clear differences in the complementarity of their respective binding surfaces. These subtle differences reveal the structural basis for the Ca 2+-dependent regulation of distinct cellular signaling events by CRC and CaM.