RESUMO
Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.
Assuntos
Lipoilação , Ácidos Nucleicos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteína Quinase C/genéticaRESUMO
The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.
RESUMO
The most frequent ERK2 (MAPK1) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, we found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. Here, we catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.
Assuntos
Ácido Aspártico , Proteínas de Drosophila , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno , Animais , Drosophila , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação , Fosforilação , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteínas de Drosophila/genética , Multimerização ProteicaRESUMO
Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.
Assuntos
Células Endoteliais , Junções Intercelulares , Neovascularização Fisiológica , Fator de Crescimento Transformador beta , Proteína Quinase 1 Deficiente de Lisina WNK , Animais , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Camundongos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Receptor Tirosina Quinase AxlRESUMO
The with no lysine (K) 1 (WNK1) protein kinase maintains cellular ion homeostasis in many tissues through actions on ion cotransporters and channels. Increased accumulation of WNK1 protein leads to pseudohypoaldosteronism type II (PHAII), a form of familial hypertension. WNK1 can be degraded via its adaptor-dependent recruitment to the Cullin3-RBX1 E3 ligase complex by the ubiquitin-proteasome system. Disruption of this process also leads to disease. To determine if this is the primary mechanism of WNK1 turnover, we examined WNK1 protein stability and degradation by measuring its rate of decay after blockade of translation. Here, we show that WNK1 protein degradation exhibits atypical kinetics in HeLa cells. Consistent with this apparent complexity, we found that multiple degradative pathways can modulate cellular WNK1 protein amount. WNK1 protein is degraded by not only the proteasome but also the lysosome. Non-lysosomal cysteine proteases calpain and caspases also influence WNK1 degradation, as inhibitors of these proteases modestly increased WNK1 protein expression. Importantly, we discovered that the E3 ubiquitin ligase UBR5 interacts with WNK1 and its deficiency results in increased WNK1 protein. Our results further demonstrate that increased WNK1 in UBR5-depleted cells is attributable to reduced lysosomal degradation of WNK1 protein. Taken together, our findings provide insights into the multiplicity of degradative pathways involved in WNK1 turnover and uncover UBR5 as a previously unknown regulator of WNK1 protein stability that leads to lysosomal degradation of WNK1 protein.
Assuntos
Proteínas Serina-Treonina Quinases , Pseudo-Hipoaldosteronismo , Células HeLa , Humanos , Antígenos de Histocompatibilidade Menor/genética , Complexo de Endopeptidases do Proteassoma , Proteínas Serina-Treonina Quinases/genética , Pseudo-Hipoaldosteronismo/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismoRESUMO
Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Pirrolidinas/farmacologia , Células Tumorais Cultivadas , Proteína Quinase 1 Deficiente de Lisina WNK/antagonistas & inibidores , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Mutação/genética , Animais , Animais Geneticamente Modificados , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Ativação Enzimática , Estabilidade Enzimática , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismoRESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating amino acids is dominated by l-leucine but also includes l-methionine, l-isoleucine, and l-valine. l-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation.
Assuntos
Aminoácidos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/genética , Animais , Células HeLa , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais Epiteliais de Sódio/genética , Células HeLa , Humanos , Proteínas Imediatamente Precoces/genética , Immunoblotting , Imunoprecipitação , Camundongos , Antígenos de Histocompatibilidade Menor , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Ratos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.
Assuntos
Doença , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Diabetes Mellitus/enzimologia , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias/enzimologia , Doenças Renais Policísticas/enzimologiaRESUMO
Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.
Assuntos
Dano ao DNA , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Biologia Computacional , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Citometria de Fluxo , Humanos , Hidroxiureia/farmacologia , Immunoblotting , MAP Quinase Quinase Quinases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Espectrometria de Massas em Tandem , Proteínas Supressoras de Tumor/metabolismo , Raios UltravioletaRESUMO
Cells integrate signals to select the appropriate response from an array of possible outcomes. Signal integration causes the reorganization of signaling pathways by undescribed events. To analyze the molecular changes in signaling pathways that elicit different responses, we focused on the interaction between cyclic AMP (cAMP) and growth factors. We show that the activation of extracellular signal-regulated kinase 5 (ERK5), but not ERK1/2, by growth factors is disrupted by cAMP through cAMP-dependent protein kinase (PKA). Activation of MEKK2, a mitogen-activated protein (MAP) kinase kinase kinase upstream of ERK5 that is required for growth factor activation of ERK5, is also disrupted by PKA. Transcription of c-Jun is induced by ERK5, and like ERK5, c-Jun induction is also blocked by cAMP. Transcription from the serum response element, like activation of ERK1/2, is not blocked by cAMP. Collectively, these results support a model in which cAMP shapes the growth factor-induced cellular response through PKA-dependent uncoupling of selected MAP kinase cascades from activating signals.
Assuntos
AMP Cíclico/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-jun/metabolismo , Elementos de Resposta/genética , Transcrição GênicaRESUMO
WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.
Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/patologia , Humanos , Hipertensão/etiologia , Soluções Hipotônicas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos de Histocompatibilidade Menor , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Estresse Fisiológico/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
TAO2 is a mitogen-activated protein kinase kinase kinase (MAP3K) that doubly phosphorylates and activates the MAP kinase kinases (MAP2Ks) MEK3 and MEK6. The structure of the kinase domain of TAO2 (1-320) has been solved in its phosphorylated active conformation. The structure, together with structure-based mutagenic analysis, reveals that positively charged residues in the substrate binding groove mediate the first step in the dual phosphorylation of MEK6, on the threonine residue in the motif DS*VAKT*I (*denotes phosphorylation site) of MEK6. TAO2 is a Ste20p homolog, and the structure of active TAO2, in comparison with that of low-activity p21-activated protein kinase (PAK1), a Ste20p-related MAP4K, reveals how this group of kinases is activated by phosphorylation. Finally, active TAO2 displays unusual interactions with ATP, involving, in part, a subgroup-specific C-terminal extension of TAO2. The observed interactions may be useful in making specific inhibitors of TAO kinases.
Assuntos
MAP Quinase Quinase Quinases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalografia , Cristalografia por Raios X , Ativação Enzimática , MAP Quinase Quinase 6/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Ratos , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Phosphatidylinositol 4,5-bisphosphate (PIP2) affects profoundly several cardiac ion channels and transporters, and studies of PIP2-sensitive currents in excised patches suggest that PIP2 can be synthesized and broken down within 30 s. To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and PIP(2) levels actually change in intact heart, we used a new, nonradioactive HPLC method to quantify anionic phospholipids. Total PIP and PIP2 levels (10-30 micromol/kg wet weight) do not change, or even increase, with activation of Galpha(q)/phospholipase C (PLC)-dependent pathways by carbachol (50 microM), phenylephrine (50 microM), and endothelin-1 (0.3 microM). Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1microM) both cause 30% reduction of PIP2 in ventricles, suggesting that diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac PIP2. PIP2, but not PIP, increases reversibly by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig left atria; the increase is blocked by nickel (2 mM). Both PIP and PIP2 increase within 3 min in hypertonic solutions, roughly in proportion to osmolarity, and similar effects occur in multiple cell lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism might be sensitive to membrane tension, per se.
Assuntos
Hormônios/fisiologia , Miocárdio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Adenosina/farmacologia , Animais , Células Cultivadas , Diglicerídeos/farmacologia , Condutividade Elétrica , Estimulação Elétrica , Cobaias , Coração/efeitos dos fármacos , Coração/fisiologia , Átrios do Coração , Ventrículos do Coração , Soluções Hipertônicas/farmacologia , Miocárdio/citologia , Fosfatidilinositol 4,5-Difosfato/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Sarcolema/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.