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1.
Nature ; 474(7351): 403-6, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21572435

RESUMO

The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/deficiência , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Biochem J ; 441(1): 325-37, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22032326

RESUMO

Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Fosforilação , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de Soluto , Proteína Quinase 1 Deficiente de Lisina WNK
3.
J Cell Biol ; 176(1): 89-100, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17190791

RESUMO

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sorbitol/farmacologia , Sequência de Aminoácidos , Animais , Domínio Catalítico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clatrina/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Pressão Osmótica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK
4.
J Immunol ; 185(7): 4385-92, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20802146

RESUMO

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1ß and IL-18. We show, however, that mice lacking both IL-1ß and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1ß and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.


Assuntos
Endotoxemia/metabolismo , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Animais , Western Blotting , Caspase 1/imunologia , Caspase 1/metabolismo , Endotoxemia/imunologia , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Proteína HMGB1/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo
5.
Biochem J ; 397(1): 223-31, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16669787

RESUMO

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Humanos , Hiperpotassemia/genética , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular , Rim/citologia , Antígenos de Histocompatibilidade Menor , Mutação , Concentração Osmolar , Fosforilação , Homologia de Sequência de Aminoácidos , Serina , Simportadores de Cloreto de Sódio-Potássio , Membro 2 da Família 12 de Carreador de Soluto , Especificidade por Substrato , Treonina , Proteína Quinase 1 Deficiente de Lisina WNK
6.
Biochem J ; 391(Pt 1): 17-24, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16083423

RESUMO

Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed 'STE20/SPS1-related proline/alanine-rich kinase' (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed 'oxidative stress response kinase-1' (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.


Assuntos
Hipertensão/enzimologia , Mutação/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Hiperpotassemia/genética , Hiperpotassemia/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Masculino , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Síndrome , Testículo/enzimologia , Proteína Quinase 1 Deficiente de Lisina WNK
7.
Science ; 351(6278): 1208-13, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26912361

RESUMO

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Desoxiadenosinas/metabolismo , Deleção de Genes , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Purina-Núcleosídeo Fosforilase/genética , RNA Interferente Pequeno/genética , Tionucleosídeos/metabolismo
8.
Biochem J ; 378(Pt 1): 257-68, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14611643

RESUMO

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Especificidade de Anticorpos , Catálise , Linhagem Celular , Humanos , Hipertensão/genética , Fator de Crescimento Insulin-Like I/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos de Histocompatibilidade Menor , Mutação , Fosforilação , Fosfotreonina/análise , Fosfotreonina/imunologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , Pseudo-Hipoaldosteronismo/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Síndrome , Proteína Quinase 1 Deficiente de Lisina WNK
9.
J Cell Biol ; 187(1): 61-70, 2009 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-19805629

RESUMO

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1beta (IL-1beta) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)-sensitive K(+) (K(ATP)) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K(ATP) subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1beta secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome-associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1beta secretion.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Glibureto/imunologia , Hipoglicemiantes/imunologia , Inflamação/metabolismo , Adenosina Trifosfatases/imunologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Proteínas de Transporte/genética , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Glibureto/química , Glibureto/metabolismo , Glibureto/farmacologia , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Inflamação/enzimologia , Inflamação/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Mutação , Proteína 3 que Contém Domínio de Pirina da Família NLR , Distribuição Aleatória , Salmonella typhimurium/classificação , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia
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