RESUMO
BACKGROUND: Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) govern myosin light chain (LC20) phosphorylation and smooth muscle contraction. Rho kinase (ROK) inhibits MLCP, resulting in greater LC20 phosphorylation and force generation at a given [Ca(2+) ](i) . Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles. METHODS: Protein and phosphorylation levels were determined by western blotting. The effects of Y27632, nicardipine, and GF109203X on phosphorylation levels and contraction were measured. KEY RESULTS: γ-Actin expression is similar in all three smooth muscles. LC20 and pS19 are highest, but ROK1 and ROK2 are lowest, in antrum and proximal colon smooth muscles. LZ +/- myosin phosphatase targeting subunit 1 (MYPT1), CPI-17, and pT696, pT853, and pT38 are highest in fundus and proximal colon smooth muscles. Myosin phosphatase-rho interacting protein (M-RIP) expression is lowest in fundus, and highest in antrum and proximal colon smooth muscles. Y27632 reduced pT853 in each smooth muscle, but reduced pT696 only in fundus smooth muscles. Nicardipine had no effect on pT38 in each smooth muscle, while GF109203X reduced pT38 in proximal colon and fundus smooth muscles. Y27632 or nicardipine reduced pS19 in proximal colon and fundus smooth muscles. Y27632 or nicardipine inhibited antrum and proximal colon smooth muscle spontaneous contractions, but only Y27632 reduced fundus smooth muscle tone. Zero external Ca(2+) relaxed each smooth muscle and abolished LC20 phosphorylation. CONCLUSIONS & INFERENCES: Organ-specific mechanisms involving the MLCP interacting proteins LZ +/- MYPT1, M-RIP, and CPI-17 are critical to regulating basal LC20 phosphorylation in gastrointestinal smooth muscles.
Assuntos
Trato Gastrointestinal/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfoproteínas/metabolismo , Miosinas de Músculo Liso/metabolismo , Animais , Western Blotting , Colo/metabolismo , Eletroforese em Gel de Poliacrilamida , Fundo Gástrico/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosforilação , Antro Pilórico/metabolismoRESUMO
BACKGROUND: Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca(2+) signalling contributes to the impaired contractility of colon circular smooth muscles. METHODS: Acute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days. KEY RESULTS: Spontaneous and acetylcholine-evoked contractions, caffeine-evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca(2+) waves were increased compared with controls. Caffeine abolished intracellular Ca(2+) waves in control smooth muscle cells, but not in smooth muscle cells from DSS-treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and I kappaB alpha levels were decreased in distal colon smooth muscles from DSS-treated mice. CONCLUSIONS & INFERENCES: These results suggest that disruptions in intracellular Ca(2+) mobilization due to down-regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF-kappaB activity.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Colite/fisiopatologia , Colo/metabolismo , Motilidade Gastrointestinal/fisiologia , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Western Blotting , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colo/efeitos dos fármacos , Colo/fisiopatologia , Sulfato de Dextrana/toxicidade , Regulação para Baixo , Eletrofisiologia , Motilidade Gastrointestinal/efeitos dos fármacos , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Vascular ATP-sensitive potassium (K(ATP)) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca(2+)-dependent phosphatase, inhibits K(ATP) channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. EXPERIMENTAL APPROACH: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K(ATP) channel (K(IR)6.1/SUR2B). The effect of intracellular Ca(2+) and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. KEY RESULTS: Constitutively active calcineurin A alpha but not A beta significantly attenuated K(ATP) currents activated by low intracellular Ca(2+), whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K(ATP) currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca(2+) (equivalent to increasing calcineurin activity), almost completely inhibited K(ATP) channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. CONCLUSIONS AND IMPLICATIONS: Calcineurin A alpha regulates K(IR)6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the K(ATP) channel.
Assuntos
Calcineurina/fisiologia , Cálcio/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Músculo Liso Vascular/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Inibidores de Calcineurina , Linhagem Celular , Clonagem Molecular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Humanos , Isoenzimas/síntese química , Isoenzimas/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Biológicos , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidoresRESUMO
Intestinal obstructions lead to a variety of motility disorders. Small intestine smooth muscles undergo dramatic phenotypic changes in response to obstruction, but the underlying molecular mechanisms are unknown. Using RT-PCR, ChIP, Re-ChIP, and Western blots, we examined the effect of small bowel mechanical obstruction on smooth muscle gene expression. Obstruction caused a transient hyperplasia, followed by a prolonged hypertrophic response of small intestine smooth muscle cells. Smooth muscle myosin heavy chain (MHC), alpha-actin, and gamma-actin expression decreased initially, and then increased as hypertrophy developed. Myocardin expression decreased initially and then increased, while kruppel-like factors (KLF)4 and KLF5 expression increased initially, and then decreased. Serum response factor (SRF) expression decreased initially, and then recovered to sham-operated levels as hypertrophy developed. SRF binding to smooth muscle MHC and alpha-actin promoters decreased initially, but then increased above sham-operated levels as hypertrophy developed. Elk-1 binding to smooth muscle myosin heavy chain and alpha-actin promoters increased initially, and then decreased to sham-operated levels as hypertrophy developed. c-fos expression increased initially, which was associated with increased SRF/Elk-1 binding to the c-fos promoter. The Elk-1 phosphorylation inhibitor U-0126 inhibited the increase in c-fos expression. These findings indicate a dynamic response of small intestine smooth muscles to bowel obstruction involving switching between differentiated, proliferative, and hypertrophic phenotypes. These results suggest that changes in the expression and interactions between SRF, myocardin, Elk-1, and c-fos play key roles in the phenotypic switching of small intestine smooth muscles in response to mechanical obstruction.
Assuntos
Obstrução Intestinal , Intestino Delgado/patologia , Intestino Delgado/fisiologia , Fator de Resposta Sérica/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Biomarcadores/metabolismo , Proliferação de Células , Intestino Delgado/anatomia & histologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Músculo Liso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
A rapidly inactivating K(+) current (A-type current) participates in the regulation of colonic muscle excitability. We found 19-pS K(+) channels in cell-attached patches of murine colonic myocytes that activated and inactivated with kinetics similar to the A-type current. The A-type current in colonic myocytes is regulated by Ca(2+)/calmodulin-dependent protein kinase II. Therefore, we studied regulation of the 19-pS K(+) channels by Ca(2+)-dependent phosphorylation/dephosphorylation. The rates of inactivation of ensemble-averaged currents resulting from 19-pS K(+) channels were increased by the calmodulin antagonist W-7. Inhibitors of calcineurin, cyclosporin A and FK-506, slowed the inactivation of the 19-pS K(+) channels. Okadaic acid, an inhibitor of the calcineurin/inhibitor-1/protein phosphatase 1 cascade, also slowed inactivation of the 19-pS K(+) channels. Polymerase chain reaction detected transcripts encoding calcineurin A in isolated colonic smooth muscle cells, and immunohistochemical studies demonstrated specific expression of calcineurin A-like immunoreactivity in colonic muscle tissues and in colonic myocytes. These data, when considered with previous findings, suggest that Ca(2+)-dependent phosphorylation/dephosphorylation regulates the A-type current in murine colonic smooth muscle cells.
Assuntos
Cálcio/metabolismo , Colo/citologia , Músculo Liso/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Calcineurina/química , Calcineurina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Colo/metabolismo , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Ácido Okadáico/farmacologia , Técnicas de Patch-Clamp , Fosforilação , Proteína Fosfatase 1 , Sulfonamidas/farmacologia , Tacrolimo/farmacologiaRESUMO
We investigated the regulation of ATP-sensitive K(+) (K(ATP)) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K(+) (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha-Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K(ATP) channels was increased by pinacidil, and PDBu suppressed openings of K(ATP) channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon-isoform (PKCepsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K(ATP) were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K(ATP) in colonic muscle cells and that the effects of ACh on K(ATP) are largely mediated by PKC. PKCepsilon appears to be the major isozyme that regulates K(ATP) in murine colonic myocytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Colo/fisiologia , Isoenzimas/metabolismo , Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Proteína Quinase C/metabolismo , Acetilcolina/farmacologia , Alcaloides , Animais , Benzofenantridinas , Colo/efeitos dos fármacos , Colo/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Glibureto/farmacologia , Técnicas In Vitro , Cinética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Naftalenos/farmacologia , Técnicas de Patch-Clamp , Fenantridinas/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Pinacidil/farmacologia , Canais de Potássio/efeitos dos fármacos , Proteína Quinase C-épsilonRESUMO
It has been proposed that N-terminal myristoylation of calcineurin B is necessary for the membrane association of calcineurin. We tested the effects of Ca(2+) and myristoylation on the binding of calcineurin B alone or heterodimeric calcineurin to phosphatidylserine or phosphatidylcholine vesicles. In the presence of excess phosphatidylserine, 50-60% of total calcineurin associated with phosphatidylserine in a Ca(2+)-sensitive manner. Calcineurin did not associate with phosphatidylcholine. Calcineurin containing both the alpha and beta catalytic subunit isoforms bound to phosphatidylserine. Calmodulin interfered with the association of calcineurin with phosphatidylserine. In the presence of Ca(2+), myristoylated calcineurin B alone did not bind to phosphatidylcholine but did bind to phosphatidylserine, although to a lesser extent than the calcineurin heterodimer. Non-myristoylated calcineurin B alone, or calcineurin containing non-myristoylated calcineurin B did not associate with phosphatidylserine in the presence of Ca(2+). These results indicate: (i) Both isoforms of calcineurin bind to phosphatidylserine. (ii) A phospholipid binding site is located on the calcineurin B subunit. (iii) Calcineurin B myristoylation is required for the Ca(2+)-sensitive binding of calcineurin to phosphatidylserine vesicles in vitro.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Ácido Mirístico/química , Fosfatidilserinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Calcineurina/química , Cálcio/química , Calmodulina/química , Calmodulina/metabolismo , Domínio Catalítico , Bovinos , Fosfatidilcolinas/química , Fosfatidilserinas/química , Fosfolipídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Interactions between phospholipid membranes and the acyl chain and specific amino acid residues of myristoylated proteins are necessary for membrane association. In the present study we tested the effects of mutations of calcineurin B subunit amino acid residues K(20)K(21), K(24)R(25), K(27)K(28) to Glu on the interactions between calcineurin and phosphatidylserine vesicles. Calcineurin-phosphatidylserine interactions were measured using binding assays and assays of phosphatidylserine-stimulated calcineurin phosphatase activity. The reverse-charge calcineurin B subunit mutant had a slower mobility in SDS-PAGE relative to wild-type calcineurin B. In addition, the myristoylated calcineurin B reverse-charge mutant had a slower mobility in SDS-PAGE compared to the non-myristoylated form, in contrast to the faster mobility of myristoylated wild-type calcineurin B relative to non-myristoylated calcineurin B. The reverse-charge mutations had no apparent effect on N-terminal myristoylation, Ca(2+)-binding, or calcineurin heterodimer formation and stimulation of Ca(2+)/calmodulin-dependent phosphatase activity. However, in contrast to the results obtained using native calcineurin, phosphatidylserine vesicles did not bind to or activate the phosphatase activity of calcineurin containing the calcineurin B reverse-charge mutant. These results indicate that calcineurin B contains an amino terminal basic residue cluster that is involved in the binding of calcineurin to acidic phospholipids.
Assuntos
Aminoácidos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Mutação/genética , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Aminoácidos/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Calcineurina/química , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Bovinos , Técnicas In Vitro , Mutação/fisiologia , Ácidos Mirísticos/química , Fosfatidilserinas/química , Fosfolipídeos/química , Monoéster Fosfórico Hidrolases/metabolismo , Subunidades Proteicas , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/metabolismo , Bovinos , Inibidores Enzimáticos/química , Expressão Gênica , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
1. The kinetics of inactivation of delayed rectifier K+ current in murine colonic myocytes differed in amphotericin-permeabilized patch and conventional patch clamp. The difference was accounted for by Ca2+ buffering. 2. Calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitors increased the rate of inactivation and slowed recovery from inactivation of the outward current. This was seen in single steps and in the envelope of the current tails. The effect was largely on the TEA-insensitive component of current. 3. Dialysis of myocytes with autothiophosphorylated CaMKII slowed inactivation. This effect was reversed by addition of CaMKII inhibitor. 4. Antibodies revealed CaMKII-like immunoreactivity in murine colonic myocytes and other cells. Immunoblots identified a small protein with CaMKII-like immunoreactivity in homogenates of colonic muscle. 5. We conclude that CaMKII regulates delayed rectifier K+ currents in murine colonic myocytes. The changes in the delayed rectifier current may participate in the Ca2+-dependent regulation of gastrointestinal motility.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Colo/metabolismo , Potássio/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Benzilaminas/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Motilidade Gastrointestinal/fisiologia , Imuno-Histoquímica , Técnicas In Vitro , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Sulfonamidas/farmacologiaRESUMO
cAMP and cyclosporin A exert antiproliferative effects in many different cell types. In cultured pituitary cells, the antiproliferative effects of both agents correlate with the inhibition of a serine/threonine protein phosphatase activity. This inhibition is mediated by the heat-stable protein, inhibitor-1. The increase of cAMP levels, through the activation of the protein kinase A, induces inhibitor-1 phosphorylation and activation. On the other hand, cyclosporin A, inhibiting the calcium-dependent serine/threonine phosphatase calcineurin, prevents the dephosphorylation and inactivation of inhibitor-1. This dual regulation by cAMP and calcium on the inhibitor-1 activity parallel the effects of these agents on DNA synthesis and serine/threonine phosphatase activity. Because inhibitor-1 is the main regulator of protein phosphatase 1 activity, these results suggest that protein phosphatase 1 may be the common target of cAMP and cyclosporin A in regulating cell proliferation. We propose that protein-phosphatase 1 stimulates growth in these cells and that cAMP and cyclosporin A block this effect through their actions on inhibitor-1.
Assuntos
AMP Cíclico/farmacologia , Ciclosporina/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Hipófise/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Dopamina/farmacologia , Inibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/fisiologia , Hipófise/citologia , Hipófise/efeitos dos fármacos , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/metabolismoRESUMO
The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Fosfoproteínas Fosfatases/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Calcineurina , Proteínas de Ligação a DNA/genética , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFATC , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Transcrição/genéticaRESUMO
NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Fosfoproteínas Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Calcineurina , Cálcio/metabolismo , Células Clonais , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fosforilação , Ligação Proteica , Linfócitos T/metabolismoRESUMO
The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B.A., Chang, B.H., & Soderling, T.R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B.A., Ng, L.Y., & Soderling, T.R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calcineurina , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Bovinos , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Ratos , Homologia de Sequência de AminoácidosRESUMO
Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Química Encefálica , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas/metabolismo , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calcineurina , Proteínas de Ligação a Calmodulina/análise , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Transporte/análise , Bovinos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/análise , Hipocampo/química , Dados de Sequência Molecular , Neuritos/química , Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Proteínas/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Tacrolimo/farmacologiaRESUMO
Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Sítios de Ligação , Encéfalo/enzimologia , Calcineurina , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/química , Células Cultivadas , Ativação Enzimática , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Ratos , Spodoptera , Especificidade por SubstratoRESUMO
Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit. A purified fusion protein containing residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC50 = 0.1 microM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val349-Phe350 and Phe356-Val357) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn2+/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A subunit bind B subunit and that the mutated hydrophobic residues are essential.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Calcineurina , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Células Cultivadas , Clonagem Molecular , Insetos , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Ligação Proteica , RatosRESUMO
The nuclear factor of activated T cells (NF-AT) is essential for transcription of the interleukin-2 gene upon T cell activation. Here we use a technique involving elution and renaturation of proteins from SDS-acrylamide gels to identify a DNA-binding component of NF-AT (NF-ATp) that is present in hypotonic extracts of T cells prior to activation and appears in nuclear extracts when T cells are activated. NF-ATp is present in resting T cells predominantly in a form migrating with an apparent molecular weight of 110,000-140,000, while NF-ATp from nuclear extracts of activated T cells migrates with a lower apparent molecular weight (90,000-125,000). This difference is likely to reflect dephosphorylation of NF-ATp, since treatment of NF-ATp with calf intestinal phosphatase or the calcium- and calmodulin-dependent phosphatase calcineurin in vitro results in a similar decrease in its apparent molecular weight. We show that NF-ATp is dephosphorylated in cell lysates by a calcium-dependent process that is blocked by inclusion of EGTA or a specific peptide inhibitor of calcineurin in the cell lysis buffer. Moreover, dephosphorylation of NF-ATp in cell extracts is inhibited by prior treatment of T cells with the immunosuppressive drugs cyclosporin A or FK506, which inhibit the phosphatase activity of calcineurin when complexed with their specific binding proteins, cyclophilin and FK506-binding protein. This work identifies NF-ATp as a DNA-binding phosphoprotein and a target for the drug/immunophilin/calcineurin complexes thought to mediate the inhibition of interleukin-2 gene induction by cyclosporin A and FK506.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunossupressores/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Linfócitos T/metabolismo , Isomerases de Aminoácido/metabolismo , Animais , Calcineurina , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Células Clonais , Ciclosporina/metabolismo , Ciclosporina/farmacologia , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Camundongos , Peptidilprolil Isomerase , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/isolamento & purificação , Tacrolimo/metabolismo , Tacrolimo/farmacologia , Proteínas de Ligação a TacrolimoRESUMO
Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.