RESUMO
Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas de Fusão Oncogênica/fisiologia , Fatores de Transcrição/fisiologia , Animais , Medula Óssea/patologia , Autorrenovação Celular , Feminino , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Células Mieloides/patologia , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Fenótipo , RNA Interferente Pequeno/genética , Quimera por Radiação , Trombopoese/genética , Fatores de Transcrição/genéticaRESUMO
Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFß1 mRNA, and addition of exogenous TGFß1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFßR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFß signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.
Assuntos
Neoplasias Pulmonares/genética , Proteína Quinase C-alfa/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Bronquíolos/metabolismo , Bronquíolos/patologia , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Células-Tronco/patologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have shown that ILK expression is vital during both embryonic development and tissue homeostasis. At the cellular and tissue levels, ILK regulates signaling pathways for cell adhesion-mediated cell survival (anoikis), apoptosis, proliferation and mitosis, migration, invasion, and vascularization and tumor angiogenesis. ILK also has central roles in cardiac and smooth-muscle contractility, and ILK dysregulation causes cardiomyopathies in humans. ILK protein levels are increased in several human cancers and often the expression level predicts poor patient outcome. Abundant evidence has accumulated suggesting that, of the diverse functions of ILK, some may require kinase activity whereas others depend on protein-protein interactions and are, therefore, independent of kinase activity. However, the past several years have seen an ongoing debate about whether ILK indeed functions as a protein serine/threonine kinase. This debate centers on the atypical protein kinase domain of ILK, which lacks some amino-acid residues thought to be essential for phosphotransferase activity. However, similar deficiencies are present in the catalytic domains of other kinases now known to possess protein kinase activity. Numerous studies have shown that ILK phosphorylates peptide substrates in vitro, corresponding to ILK-mediated phosphorylations in intact cells, and a recent report characterizing in vitro phosphotransferase activity of highly purified, full-length ILK, accompanied by detailed enzyme kinetic analyses, shows that, at least in vitro, ILK is a bona fide protein kinase. However, several genetic studies suggest that, not all biological functions of ILK require kinase activity, and that it can function as an adaptor/scaffold protein. Here, we review evidence for and against ILK being an active kinase, and provide a framework for strategies to further analyze the kinase and adaptor functions of ILK in different cellular contexts.
Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Domínio Catalítico , Humanos , Técnicas In Vitro , Mutação , Neoplasias/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genéticaRESUMO
PbSeTe-based quantum dot superlattice structures grown by molecular beam epitaxy have been investigated for applications in thermoelectrics. We demonstrate improved cooling values relative to the conventional bulk (Bi,Sb)2(Se,Te)3 thermoelectric materials using a n-type film in a one-leg thermoelectric device test setup, which cooled the cold junction 43.7 K below the room temperature hot junction temperature of 299.7 K. The typical device consists of a substrate-free, bulk-like (typically 0.1 millimeter in thickness, 10 millimeters in width, and 5 millimeters in length) slab of nanostructured PbSeTe/PbTe as the n-type leg and a metal wire as the p-type leg.
RESUMO
Voltage-gated K(+) channels (Kv) play a critical role in regulating arterial tone by modulating the membrane potential of vascular smooth muscle cells. Our previous work demonstrated that the dominant 4-aminopyridine (4-AP)-sensitive, delayed rectifier Kv current of rabbit portal vein (RPV) myocytes demonstrates similar 4-AP sensitivity and biophysical properties to Kv1alpha-containing channels. To identify the molecular constituents underlying the 4-AP-sensitive Kv current of vascular myocytes, we characterized the expression pattern of Kv1alpha subunits and their modulatory Kvbeta subunits in RPV. The mRNAs encoding pore-forming subunits Kv1.2, Kv1.4, and Kv1.5 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Kv1.1, Kv1.3, and Kv1.6 transcripts were undetectable. Kvbeta1.1, beta1.2, beta1.3, beta2.1, and beta2.2 messages were expressed, whereas Kvbeta3.1 and beta4 mRNAs were undetected by RT-PCR. Kv1.2, Kv1.4, Kv1.5, Kvbeta1.2, beta1.3, and beta2.1 proteins were detected in RPV by Western blotting and/or immunocytochemistry of freshly isolated myocytes. We provide the first evidence, from coimmunoprecipitation studies, for the formation of heteromultimeric Kv channel complexes composed of Kv1.2, Kv1.5, and Kvbeta1.2 subunits in vascular smooth muscle.
Assuntos
4-Aminopiridina/farmacologia , Músculo Liso Vascular/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio/química , Animais , Western Blotting , Imuno-Histoquímica , Canal de Potássio Kv1.2 , Canal de Potássio Kv1.4 , Canal de Potássio Kv1.5 , Veia Porta/metabolismo , Canais de Potássio/genética , Canais de Potássio/imunologia , Canais de Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Testes de Precipitina , Subunidades Proteicas , RNA Mensageiro/biossíntese , CoelhosRESUMO
The molecular identity of vascular delayed rectifier K(+) channels (K(DR)) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of K(DR) of rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of K(DR) was pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of K(DR). Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2](2) channels. K(DR) currents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular K(DR) are inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular K(DR) channel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.
Assuntos
4-Aminopiridina/farmacologia , Músculo Liso Vascular/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/química , 4-Aminopiridina/metabolismo , Animais , Células Cultivadas , Charibdotoxina/farmacologia , Canais de Potássio de Retificação Tardia , Condutividade Elétrica , Cinética , Canal de Potássio Kv1.2 , Canal de Potássio Kv1.5 , Técnicas de Patch-Clamp , Veia Porta/citologia , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/fisiologia , Subunidades Proteicas , Coelhos , TransfecçãoRESUMO
The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.
Assuntos
Actinas/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Carbocianinas , Separação Celular , Células Cultivadas , Dendritos/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes , Hipocampo , Camundongos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/farmacologia , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
The effects of PKC activation on a transient (It) and a sustained (Iss) cardiac K+ current and the subcellular distribution of the epsilon isoform of PKC (PKC(epsilon)) were compared in epicardial and endocardial regions of the rat ventricle. Activation of PKC(epsilon) with a diacylglycerol analogue (di-octanoyl-glycerol (DiC8), 20 (mu)M) leads to differential effects in epicardial and endocardial cells. In epicardial cells (n = 20) It and Iss are attenuated by 17.7 +/- 2.1 % and 11.9 +/- 3.1 %, respectively (means +/- S.E.M.). In endocardial cells It attenuation was significantly smaller (4.6 +/- 1.6 %, n = 14, P < 0.0005). Iss attenuation was similar to that in epicardial cells (10.5 +/- 3.8 %). PKC[epsilon] expression was measured by Western blotting. Calculated endocardial/epicardial ratios showed no regional differences in total protein extracts (1.04 +/- 0.11, mean +/- S.E.M, n = 4), but PKC[epsilon] distribution in the cytosolic fraction showed a marked difference, with significantly (P < 0.05) higher levels in endocardial extracts. The cytosolic endocardial/epicardial PKC[epsilon] ratio was 2.64 +/- 0.24 (n = 4), indicating a reduced amount of PKC[epsilon] in the membrane fraction of the endocardium. This could account for the reduced effect of DiC8 on It in endocardial myocytes. Under both hypothyroid and streptozotocin-induced diabetic conditions the difference in endocardial and epicardial cytosolic PKC[epsilon] levels was absent (ratios of 0.86 +/- 0.21 (n = 4) and 1.09 +/- 0.16 (n = 3), respectively; means +/- S.E.M.). Ratios in the total protein extracts were not significantly different from those in control conditions. The results show transmural differences in the functional effects of PKC(epsilon) activation on a cardiac K+ current, and in the subcellular distribution of PKC(epsilon). These differences are absent in diabetic and hypothyroid conditions.
Assuntos
Isoenzimas/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Miocárdio/citologia , Canais de Potássio/fisiologia , Potássio/metabolismo , Proteína Quinase C/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diglicerídeos/farmacologia , Endocárdio/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ventrículos do Coração/citologia , Hipotireoidismo/metabolismo , Isoenzimas/análise , Técnicas de Patch-Clamp , Proteína Quinase C/análise , Proteína Quinase C-épsilon , RatosRESUMO
Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-akt , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca(2+)](i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Galinhas , Inibidores Enzimáticos/farmacologia , Moela das Aves , Humanos , Cinética , Microcistinas , Modelos Biológicos , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/enzimologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Proteínas de Ligação ao Cálcio/genética , Camundongos Knockout/genética , Camundongos Knockout/fisiologia , Contração Muscular/fisiologia , Proteínas Musculares/genética , Músculo Liso/fisiologia , Animais , Masculino , Camundongos , Proteínas dos Microfilamentos , Bexiga Urinária/fisiologia , Ducto Deferente/fisiologia , CalponinasRESUMO
Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20 kDa light chains of myosin (LC(20)). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC(20) phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC(20) phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC(20) phosphorylation in intact RTA is mediated by MLCK; (ii) alpha(1)-adrenoceptor stimulation increases LC(20) phosphorylation via MLCK and PKC; (iii) Ca(2+)-induced LC(20) phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs alpha and beta following detergent treatment; (iv) very little LC(20) diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC(20) phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension-LC(20) phosphorylation relationship is markedly different between the two preparations such that the basal level of LC(20) phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.
Assuntos
Músculo Liso Vascular/metabolismo , Miosinas/metabolismo , Proteína Quinase C/metabolismo , Cauda/irrigação sanguínea , Animais , Masculino , Músculo Liso Vascular/enzimologia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
The effect of phosphorylation in the N-terminal region of myosin phosphatase target subunit 1 (MYPT1) on the interactions with protein phosphatase 1 catalytic subunit (PP1c) and with phosphorylated 20 kDa myosin light chain (P-MLC20) was studied. Protein kinase C (PKC) phosphorylated threonine-34 (1 mol/mol), the residue preceding the consensus PP1c-binding motif ((35)KVKF(38)) in MYPT1(1-38), but this did not affect binding of the peptide to PP1c. PKC incorporated 2 mol P(i) into MYPT1(1-296) suggesting a second site of phosphorylation within the ankyrin repeats (residues 40-296). This phosphorylation diminished the stimulatory effect of MYPT1(1-296) on the P-MLC20 phosphatase activity of PP1c. Binding of PP1c or P-MLC20 to phosphorylated MYPT1(1-296) was also attenuated. It is concluded that phosphorylation of MYPT1 by PKC may therefore result in altered dephosphorylation of myosin.
Assuntos
Cadeias Leves de Miosina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Animais , Repetição de Anquirina/fisiologia , Domínio Catalítico , Peso Molecular , Fosfatase de Miosina-de-Cadeia-Leve , Peptídeos/metabolismo , Fosforilação , Proteína Fosfatase 1 , CoelhosRESUMO
Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Plantas/enzimologia , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , ATPases Transportadoras de Cálcio/metabolismo , Ativação EnzimáticaRESUMO
Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation.
Assuntos
Proteínas de Ligação ao Cálcio/química , Serina/química , Actinas/metabolismo , Alanina/química , Sítio Alostérico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Ligação Competitiva , Encéfalo/enzimologia , Galinhas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epitopos , Escherichia coli/metabolismo , Moela das Aves/metabolismo , Cinética , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Músculo Liso/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tropomiosina/química , CalponinasRESUMO
ATP-sensitive K(+) channels (K(ATP)) contribute to the regulation of tone in vascular smooth muscle cells. We determined the effects of protein kinase C (PKC) activation on the nucleoside diphosphate-activated (K(NDP)) subtype of vascular smooth muscle K(ATP) channel. Phorbol 12,13-dibutyrate (PdBu) and angiotensin II inhibited K(NDP) activity of C-A patches of rabbit portal vein (PV) myocytes, but an inactive phorbol ester was without effect, and pretreatment with PKC inhibitor prevented the actions of PdBu. Constitutively active PKC inhibited K(NDP) in I-O patches but was without effect in the presence of a specific peptide inhibitor of PKC. PdBu increased the duration of a long-lived interburst closed state but was without effect on burst duration or intraburst kinetics. PdBu treatment inhibited K(NDP), but not a 70-pS K(ATP) channel of rat PV. The results indicate that the K(NDP) subtype of vascular smooth muscle K(ATP) channel is inhibited by activation of PKC. Control of K(NDP) activity by intracellular signaling cascades involving PKC may, therefore, contribute to control of tone and arterial diameter by vasoconstrictors.
Assuntos
Músculo Liso Vascular/fisiologia , Veia Porta/fisiologia , Canais de Potássio/fisiologia , Proteína Quinase C/metabolismo , Trifosfato de Adenosina/farmacologia , Angiotensina II/farmacologia , Animais , Células Cultivadas , Desoxiglucose/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Dibutirato de 12,13-Forbol/farmacologia , Bloqueadores dos Canais de Potássio , Coelhos , RatosRESUMO
Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca(2+) release channels, but has additional effects such as the induction of Ca(2+) sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca(2+)] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC(50) 23 microM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca(2+)]. Ca(2+)-independent contraction was induced by RuR (EC(50) 843 microM) and by microcystin (EC(50) 59 nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300 microM RuR enhanced the contraction induced by 30 nM microcystin but markedly decreased that induced by 1 microM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca(2+) sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Músculo Liso/enzimologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Rutênio Vermelho/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Microcistinas , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve , Peptídeos Cíclicos/farmacologia , FosforilaçãoRESUMO
Potassium channels that are inhibited by internal ATP (K(ATP) channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K(ATP) channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K(ATP) channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K(ATP) channel isoforms suggests that this pathway may have wide significance for the physiological regulation of K(ATP) channels in various tissues and organelles.
Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Humanos , Dados de Sequência Molecular , Fosforilação , Canais de Potássio/química , CoelhosRESUMO
This study reports an attempt to confirm a published and well-defined biological effect of magnetic fields. The biological model investigated was the phosphorylation of myosin light chain in a cell free system. The rate of phosphorylation has been reported to be affected in an approximately linear manner by static magnetic field strengths in the range 0-200 microT. We performed three series of experiments, two to test the general hypothesis and a third that was a direct replication of published work. We found no effect of static magnetic field strength on the rate of phosphorylation. Hence, we were unable to confirm that weak static magnetic fields affect the binding of calcium to calmodulin. In view of the difficulty we and other authors have had making independent verifications of claimed biological effects of magnetic fields, we would urge caution in the interpretation of published data until they have been independently confirmed. There are still few well defined biological effects of low level magnetic fields that have been successfully transferred to an independent laboratory.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Magnetismo , Cadeias Leves de Miosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Sistema Livre de Células , Corantes Fluorescentes , Fura-2 , Quinase de Cadeia Leve de Miosina/metabolismo , Radioisótopos de Fósforo , Fosforilação , Compostos Radiofarmacêuticos , Processamento de Sinais Assistido por Computador , TemperaturaRESUMO
The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML-9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1(191-210), an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.