RESUMO
BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.
Assuntos
Mucosa Gástrica/metabolismo , Gastroparesia/metabolismo , Músculo Liso/metabolismo , Adulto , Anoctamina-1/metabolismo , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Heme Oxigenase-1/metabolismo , Humanos , Células Intersticiais de Cajal/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
For many years the simple view was held that contractile force in smooth muscle was proportional to cytosolic Ca2+ concentrations ([Ca2+]i). With the discovery that phosphorylation of myosin light chain by Ca2+/calmodulin-dependent myosin light chain kinase initiated contraction, regulation of the contractile elements developed more complex properties. Molecular and biochemical investigations have identified important domains of myosin light chain kinase: light chain binding sites, catalytic core, pseudosubstrate prototope, and calmodulin-binding domain. New protein phosphatase inhibitors such as okadaic acid and calyculin A should help in the identification of the physiologically important phosphatase and potential modes of regulation. The proposal of an attached, dephosphorylated myosin cross bridge (latch bridge) that can maintain force has evoked considerable controversy about the detailed functions of the myosin phosphorylation system. The latch bridge has been defined by a model based on physiological properties but has not been identified biochemically. Thin-filament proteins have been proposed as secondary sites of regulation of contractile elements, but additional studies are needed to establish physiological roles. Changes in the Ca2+ sensitivity of smooth muscle contractile elements with different modes of cellular stimulation may be related to inactivation of myosin light chain kinase or activation of protein phosphatase activities. Thus, contractile elements in smooth muscle cells are not dependent solely on [Ca2+]i but use additional regulatory mechanisms. The immediate challenge is to define their relative importance and to describe molecular-biochemical properties that provide insights into proposed physiological functions.
Assuntos
Contração Muscular , Proteínas Musculares/fisiologia , Músculo Liso Vascular/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
Cyclic nucleotides can relax smooth muscle without a change in [Ca2+]i, a phenomenon termed Ca2+ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca2+ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an approximately 30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca2+ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca2+ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally approximately 5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca2+ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 microM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.
Assuntos
Cálcio/farmacologia , GMP Cíclico/farmacologia , Relaxamento Muscular , Músculo Liso/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/fisiologia , Peptídeos/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Aorta/efeitos dos fármacos , Íleo/efeitos dos fármacos , Camundongos , Camundongos Knockout , Relaxamento Muscular/genética , Relaxamento Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/ultraestrutura , Quinase de Cadeia Leve de Miosina/deficiência , Quinase de Cadeia Leve de Miosina/genética , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fragmentos de Peptídeos , Peptídeos/deficiência , Peptídeos/genéticaRESUMO
Several allosterically modulated protein kinases have been shown to be regulated by an autoinhibitory domain located within the kinase molecules. The inhibitory domain has been proposed to act as a "pseudosubstrate" inhibitor binding to the substrate binding site of the kinase, thereby blocking the binding of the enzyme's true substrate. In this report, site-directed mutagenesis has been used to further investigate the mechanism of activation of the inhibitory domain of rabbit skeletal muscle myosin light chain kinase. Basic residues within the pseudosubstrate domain (572-573, 577-579, 580-581), which are analogous to the important substrate determinants of the myosin light chain, were found not to be required in order to maintain the kinase in an inhibited state. Two groups of these residues (577-579 and 581-582) were, however, found to be important for high affinity calmodulin binding to the kinase. These data suggest that the autoinhibitory domain of myosin light chain kinase may not function by directly mimicking the light chain substrate.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Músculos/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biotina/química , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Mutação , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Coelhos , Especificidade por SubstratoRESUMO
It has been proposed that the carboxyl terminus of the smooth muscle myosin light chain kinase is expressed as an independent protein. This protein has been purified from tissues and named telokin (Ito, M., Dabrowska, R., Guerriero, V., Jr., and Hartshorne, D. J. (1989) J. Biol. Chem. 264, 13971-13974). In this study we have isolated and characterized cDNA and genomic clones encoding telokin. Analysis of a genomic DNA clone suggests that the mRNA encoding telokin arises from a promoter which appears to be located within an intron of the smooth muscle myosin light chain kinase (MLCK) gene. This intron interrupts exons encoding the calmodulin binding domain of the kinase. The amino acid sequence deduced from the cDNA predicts that telokin is identical to the carboxyl-terminal 155 residues of the smooth muscle MLCK. Unlike the smooth muscle MLCK which is expressed in both smooth and non-muscle tissues, telokin is expressed in some smooth muscle tissues but has not been detected in aortic smooth muscle or in any non-muscle tissues.
Assuntos
Proteínas Musculares/genética , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/genética , Sequência de Aminoácidos , Animais , Anticorpos , Sequência de Bases , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Biblioteca Genômica , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/isolamento & purificação , Fragmentos de Peptídeos , Peptídeos , Coelhos , Mapeamento por Restrição , Útero/enzimologiaRESUMO
Telokin transcription is initiated from a smooth muscle-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene. We have previously identified a 310-base pair fragment of the promoter that mediates A10 smooth muscle cell-specific expression of telokin. In the current study, telokin-luciferase reporter gene assays in A10 cells and REF52 nonmuscle cells revealed that the promoter region between -81 and +80 contains the regulatory elements required to mediate the in vitro cell specificity of the promoter. Several positive-acting elements, including an E box, myocyte enhancer factor 2 (MEF2)-TATA box, and CArG-serum response element, were identified within this region. Telokin transcription in A10 smooth muscle cells requires all three transcription initiation sites and an AT-rich sequence between -71 and -62 that includes a TATA box. MEF2 interacts with the AT-rich region with low affinity; however, MEF2 binding is not required for transcriptional activity in A10 cells. Binding of serum response factor (SRF) to a CArG element proximal to the TATA sequence is also critical for high levels of transcription in A10 cells. Together these data suggest that an AT-rich motif, acting in concert with SRF and an unusual transcription initiation mechanism, is required for the cell-specific expression of the telokin promoter in A10 smooth muscle cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Proteínas Musculares/genética , Músculo Liso Vascular/fisiologia , Proteínas Nucleares/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Fatores de Regulação Miogênica , Quinase de Cadeia Leve de Miosina , Fragmentos de Peptídeos , Peptídeos , Regiões Promotoras Genéticas , Ratos , Fator de Resposta Sérica , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The carboxy terminus of the smooth muscle myosin light chain kinase (smMLCK) is expressed as an independent protein, telokin. Western and Northern blotting analyses demonstrated that telokin protein and mRNA are expressed at high levels only in adult and embryonic smooth muscle tissues and cells. In vitro transfection assays in A10 smooth muscle cells identified a functional promoter located in an intron in the 3' region of the smMLCK gene that directs the smooth muscle cell-specific transcription of telokin. To test the cell specificity of the telokin promoter in vivo, transgenic mice were generated in which the telokin promoter was used to drive expression of SV40 large T-antigen. Expression of T-antigen in the transgenic mice paralleled that of the endogenous telokin gene. High levels of T-antigen expression were observed in smooth muscle tissues of the digestive, urinary, and reproductive tracts, with lower levels of expression in airway and vascular smooth muscle. Expression was restricted to smooth muscle cells, with no expression detected in any other cell type.
Assuntos
Expressão Gênica , Proteínas Musculares/genética , Músculo Liso/citologia , Músculo Liso/fisiologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , Embrião de Mamíferos/metabolismo , Genoma , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Fragmentos de Peptídeos , Peptídeos , Coelhos , Ratos , Transcrição GênicaRESUMO
The rate of exchange of phosphate bound to ventricular myosin light chain-2 (LC2-P) was measured in rat hearts perfused with [32P]Pi at various levels of perfusate Ca2+. Computer simulations of the light-chain labelling suggested the presence of two isotopically distinct pools of LC2-P, one large pool comprising 90% of the total and a small pool consisting of the remaining 10%. At control levels of perfusate Ca2+ the phosphate of the large pool turned over very slowly (t 1/2 congruent to 250 min), whereas that of the small pool turned over much more rapidly (t 1/2 congruent to 1 min). At high levels of perfusate free Ca2+ (5mM) the turnover of the phosphate of the small pool decreased markedly, whereas that of the large pool remained little changed. Conversely, at low perfusate free Ca2+ (0.2 mM), the turnover of the large pool decreased, whereas that of the small pool remained unchanged. The possible identity of these two pools is discussed. The total myosin-light-chain kinase activity of rat ventricle was found to be only 2-3-fold higher than the kinase activity expressed in the heart under control conditions. This, coupled with the very low turnover of most of the LC2-bound phosphate, implies that, in heart, there is insufficient myosin-light-chain kinase activity to cause a rapid rise in the overall level of light-chain phosphorylation, even under conditions of increased cytoplasmic Ca2+.
Assuntos
Miocárdio/metabolismo , Miosinas/metabolismo , Fosfatos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Modelos Biológicos , Perfusão , Fosforilação , Ratos , Ratos EndogâmicosRESUMO
Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.
Assuntos
Quinase de Cadeia Leve de Miosina , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA/genética , Análise Mutacional de DNA , Epitopos , Biblioteca Gênica , Dados de Sequência Molecular , Peso Molecular , Quinase de Cadeia Leve de Miosina/genética , Mapeamento de Peptídeos , Conformação Proteica , Coelhos , Mapeamento por RestriçãoRESUMO
Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.
Assuntos
Músculo Liso/enzimologia , Músculos/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Galinhas , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TransfecçãoRESUMO
The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.
Assuntos
Músculo Esquelético/enzimologia , Músculo Liso/enzimologia , Miocárdio/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos/genética , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Clonagem Molecular , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Liso/citologia , Músculo Liso/embriologia , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Músculos Papilares/enzimologiaRESUMO
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.
Assuntos
Embrião de Mamíferos/enzimologia , Quinase de Cadeia Leve de Miosina/análise , Animais , Cálcio/fisiologia , Células Cultivadas , Feminino , Camundongos , Peso Molecular , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/imunologia , Fosforilação , GravidezRESUMO
CC(A/T)(6)GG or serum response elements represent a common regulatory motif important for regulating the expression of many smooth muscle-specific genes. They are multifunctional elements that bind serum response factor (SRF) and are important for serum induction of genes, expression of muscle-specific genes, and differentiation of vascular smooth muscle cells. In the current study, a yeast two-hybrid screen was used to identify proteins from mouse intestine that interact with SRF. A novel homeodomain-containing transcription factor, called Barx2b, was identified that specifically interacts with SRF and promotes the DNA binding activity of SRF. Northern blotting, RNase protection analysis, and Western blotting revealed that Barx2b mRNA and protein are expressed in several smooth muscle-containing tissues, as well as in skeletal muscle and brain. In vitro binding studies using bacterial fusion proteins revealed that the DNA-binding domain of SRF interacts with a region of Barx2b located amino-terminal of the homeobox domain. The results of these studies support the hypothesis that interaction of SRF with different homeodomain-containing proteins may play a critical role in determining the cell-specific functions of SRF.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Diferenciação Celular , Linhagem Celular , DNA/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Genes Reporter , Proteínas de Homeodomínio/biossíntese , Mucosa Intestinal/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleases/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Resposta Sérica , Fatores de Tempo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
Site-directed and chimeric mutations of myosin regulatory light chains were used to identify residues important for phosphorylation of Ser19 by smooth muscle myosin light chain kinase. Arg16 and hydrophobic residues C-terminal of Ser19 in smooth muscle light chain were important substrate determinants in the intact protein. However, changes in the kinetic properties with mutations in the light chain were substantially smaller than results reported with structurally similar synthetic peptide substrates. These results together with the low Vmax value for short peptide substrates containing the consensus phosphorylation sequence suggest that there may be additional sites of interactions between the kinase and protein substrate. Chimeras of skeletal and smooth muscle light chains were constructed with exchanges at the N terminus and subdomains I, II, III, and IV. Analysis of results obtained on the kinetic properties for phosphorylation showed that subdomains I and II contribute to high Vmax values. Thus, a region distant from the consensus phosphorylation sequence in smooth muscle light chain is also an important substrate determinant for myosin light chain kinase.
Assuntos
Músculo Liso/química , Miosinas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Músculo Esquelético/química , Mutação , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Fosforilação , CoelhosRESUMO
A 1.85-kilobase (kb) cDNA has been isolated that encodes the catalytic and calmodulin binding domains of rat skeletal muscle myosin light chain kinase. The cDNA hybridized to a 3.3-kb RNA present in fast- and slow-twitch skeletal muscles. The reported enzymatic activity (3-fold greater in fast- than slow-twitch skeletal muscles) reflects the relative abundance of this RNA in the two types of skeletal muscle. No hybridization of the cDNA was detected to RNA isolated from smooth or nonmuscle tissues. The clone cross hybridized to a 2.2-kb RNA present in cardiac tissue. Ribonuclease protection analysis of skeletal and cardiac muscle RNA revealed major differences in the two hybridizing RNAs. Thus rat skeletal muscle contains a single myosin light chain kinase isoform, which is distinct from the cardiac, smooth, and nonmuscle forms.
Assuntos
Músculos/enzimologia , Quinase de Cadeia Leve de Miosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Recombinante/metabolismo , Genes , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA/genética , RNA Antissenso , RNA Mensageiro/antagonistas & inibidores , RatosRESUMO
It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.
Assuntos
Calmodulina/metabolismo , Músculo Liso/enzimologia , Mutagênese Sítio-Dirigida , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/farmacologia , Calmodulina/farmacologia , Ativação Enzimática , Immunoblotting , Cinética , Dados de Sequência Molecular , CoelhosRESUMO
A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.
Assuntos
Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/genética , Útero/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Galinhas , DNA/genética , DNA/isolamento & purificação , Feminino , Biblioteca Gênica , Moela das Aves/enzimologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Quinase de Cadeia Leve de Miosina/metabolismo , Oligodesoxirribonucleotídeos , Coelhos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.
Assuntos
Subfragmentos de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cinética , Dados de Sequência Molecular , Músculos/enzimologia , Mutação , Quinase de Cadeia Leve de Miosina/isolamento & purificação , Quinase de Cadeia Leve de Miosina/metabolismo , Sondas de Oligonucleotídeos , CoelhosRESUMO
Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Musculares/genética , Músculo Liso/embriologia , Sequência de Aminoácidos/fisiologia , Animais , Sequência de Bases/fisiologia , Clonagem Molecular/métodos , DNA Complementar/genética , Feminino , Genitália Feminina/citologia , Genitália Feminina/crescimento & desenvolvimento , Genitália Feminina/metabolismo , Genitália Masculina/citologia , Genitália Masculina/crescimento & desenvolvimento , Genitália Masculina/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Liso/citologia , Músculo Liso/crescimento & desenvolvimento , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina , Fragmentos de Peptídeos , Peptídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.