RESUMO
Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.
Assuntos
Aterosclerose/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Integrina alfa5beta1/metabolismo , Proteína Fosfatase 2/metabolismo , Sistemas do Segundo Mensageiro , Animais , Aterosclerose/genética , Aterosclerose/patologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Integrina alfa5beta1/genética , Camundongos , Camundongos Mutantes , Proteína Fosfatase 2/genéticaRESUMO
Medulloblastomas, the most common malignant pediatric brain tumors, have been genetically defined into four subclasses, namely WNT-activated, Sonic Hedgehog (SHH)-activated, Group 3, and Group 4. Approximately 30% of medulloblastomas have aberrant SHH signaling and thus are referred to as SHH-activated medulloblastoma. The tumor suppressor gene TP53 has been recently recognized as a prognostic marker for patients with SHH-activated medulloblastoma; patients with mutant TP53 have a significantly worse outcome than those with wild-type TP53. It remains unknown whether p53 activity is impaired in SHH-activated, wild-type TP53 medulloblastoma, which is about 80% of the SHH-activated medulloblastomas. Utilizing the homozygous NeuroD2:SmoA1 mouse model with wild-type Trp53, which recapitulates human SHH-activated medulloblastoma, it was discovered that the endogenous Inhibitor 2 of Protein Phosphatase 2A (SET/I2PP2A) suppresses p53 function by promoting accumulation of phospho-MDM2 (S166), an active form of MDM2 that negatively regulates p53. Knockdown of I2PP2A in SmoA1 primary medulloblastoma cells reduced viability and proliferation in a p53-dependent manner, indicating the oncogenic role of I2PP2A. Importantly, this mechanism is conserved in the human medulloblastoma cell line ONS76 with wild-type TP53. Taken together, these findings indicate that p53 activity is inhibited by I2PP2A upstream of PP2A in SHH-activated and TP53-wildtype medulloblastomas. IMPLICATIONS: This study suggests that I2PP2A represents a novel therapeutic option and its targeting could improve the effectiveness of current therapeutic regimens for SHH-activated or other subclasses of medulloblastoma with wild-type TP53.
Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Chaperonas de Histonas/metabolismo , Meduloblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Peptídeos/farmacologia , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
Protein phosphatase 2A (PP2A) is a heterotrimer composed of single catalytic and scaffolding subunits and one of several possible regulatory subunits. We identified PPTR-2, a regulatory subunit of PP2A, as a binding partner for the giant muscle protein UNC-89 (obscurin) in Caenorhabditis elegans. PPTR-2 is required for sarcomere organization when its paralogue, PPTR-1, is deficient. PPTR-2 localizes to the sarcomere at dense bodies and M-lines, colocalizing with UNC-89 at M-lines. PP2A components in C. elegans include one catalytic subunit LET-92, one scaffolding subunit (PAA-1), and five regulatory subunits (SUR-6, PPTR-1, PPTR-2, RSA-1, and CASH-1). In adult muscle, loss of function in any of these subunits results in sarcomere disorganization. rsa-1 mutants show an interesting phenotype: one of the two myosin heavy chains, MHC A, localizes as closely spaced double lines rather than single lines. This "double line" phenotype is found in rare missense mutants of the head domain of MHC B myosin, such as unc-54(s74). Analysis of phosphoproteins in the unc-54(s74) mutant revealed two additional phosphoserines in the nonhelical tailpiece of MHC A. Antibodies localize PPTR-1, PAA-1, and SUR-6 to I-bands and RSA-1 to M-lines and I-bands. Therefore, PP2A localizes to sarcomeres and functions in the assembly or maintenance of sarcomeres.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Músculo Estriado/enzimologia , Proteína Fosfatase 2/metabolismo , Sarcômeros/metabolismo , Animais , Mutação de Sentido Incorreto/genética , Fenótipo , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Leucine carboxyl methyltransferase-1 (LCMT-1) methylates the C-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In this study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down â¼30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and usually death by embryonic day 16.5. Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit+Lin-Sca1+ cells. The percent cycling cells and mitotic indices of WT and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, noncompetitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multilineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis.
Assuntos
Embrião de Mamíferos/patologia , Feto/patologia , Hematopoese , Fígado/patologia , Proteína O-Metiltransferase/fisiologia , Animais , Apoptose , Proliferação de Células , Metilação de DNA , Embrião de Mamíferos/enzimologia , Feto/enzimologia , Fígado/enzimologia , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/metabolismoRESUMO
The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.
Assuntos
Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteína O-Metiltransferase/metabolismo , Animais , Células Cultivadas , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , Metilação , Camundongos , Camundongos Knockout , Fosfoproteínas Fosfatases/genética , Fosforilação/genética , Proteína O-Metiltransferase/genéticaRESUMO
The mammalian striatin family consists of three proteins, striatin, S/G2 nuclear autoantigen, and zinedin. Striatin family members have no intrinsic catalytic activity, but rather function as scaffolding proteins. Remarkably, they organize multiple diverse, large signaling complexes that participate in a variety of cellular processes. Moreover, they appear to be regulatory/targeting subunits for the major eukaryotic serine/threonine protein phosphatase 2A. In addition, striatin family members associate with germinal center kinase III kinases as well as other novel components, earning these assemblies the name striatin-interacting phosphatase and kinase (STRIPAK) complexes. Recently, there has been a great increase in functional and mechanistic studies aimed at identifying and understanding the roles of STRIPAK and STRIPAK-like complexes in cellular processes of multiple organisms. These studies have identified novel STRIPAK and STRIPAK-like complexes and have explored their roles in specific signaling pathways. Together, the results of these studies have sparked increased interest in striatin family complexes because they have revealed roles in signaling, cell cycle control, apoptosis, vesicular trafficking, Golgi assembly, cell polarity, cell migration, neural and vascular development, and cardiac function. Moreover, STRIPAK complexes have been connected to clinical conditions, including cardiac disease, diabetes, autism, and cerebral cavernous malformation. In this review, we discuss the expression, localization, and protein domain structure of striatin family members. Then we consider the diverse complexes these proteins and their homologs form in various organisms, emphasizing what is known regarding function and regulation. Finally, we explore possible roles of striatin family complexes in disease, especially cerebral cavernous malformation.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a Calmodulina/genética , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Transdução de SinaisRESUMO
Heterotrimeric protein phosphatase 2A (PP2A) consists of catalytic C (PP2Ac), structural A, and regulatory B-type subunits, and its dysfunction has been linked to cancer. Reversible methylation of PP2Ac by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) differentially regulates B-type subunit binding and thus PP2A function. Polyomavirus middle (PyMT) and small (PyST) tumor antigens and SV40 small tumor antigen (SVST) are oncoproteins that block PP2A function by replacing certain B-type subunits, resulting in cellular transformation. Whereas the B-type subunits replaced by these oncoproteins seem to exhibit a binding preference for methylated PP2Ac, PyMT does not. We hypothesize that circumventing the normal cellular control of PP2A by PP2Ac methylation is a general strategy for ST- and MT-mediated transformation. Two predictions of this hypothesis are (1) that PyST and SVST also bind PP2A in a methylation-insensitive manner and (2) that down-regulation of PP2Ac methylation will activate progrowth and prosurvival signaling and promote transformation. We found that SVST and PyST, like PyMT, indeed form PP2A heterotrimers independently of PP2Ac methylation. In addition, reducing PP2Ac methylation through LCMT-1 knockdown or PME-1 overexpression enhanced transformation by activating the Akt and p70/p85 S6 kinase (S6K) pathways, pathways also activated by MT and ST oncoproteins. These results support the hypothesis that MT and ST oncoproteins circumvent cellular control of PP2A by methylation to promote transformation. They also implicate LCMT-1 as a negative regulator of Akt and p70/p85 S6K. Therefore, disruption of PP2Ac methylation may contribute to cancer, and modulation of this methylation may serve as an anticancer target.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular , Proliferação de Células , Tamanho Celular , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Ativação Enzimática/genética , Regulação da Expressão Gênica , Humanos , Metilação , Multimerização Proteica , Proteína O-Metiltransferase/genética , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
BACKGROUND: Striatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. RESULTS: To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. CONCLUSIONS: Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between striatin's calmodulin-binding and WD-domains and recruits the PP2A A/C heterodimer to its coiled-coil/oligomerization domain. Residues outside the previously reported coiled-coil domain of striatin are necessary for its oligomerization. Striatin-associated PP2A is critical for Mst3 dephosphorylation and inactivation. Upon inhibition of PP2A, Mst3 activation appears to involve autophosphorylation of multiple activation loop phosphorylation sites. Mob3 can associate with striatin sequences C-terminal to the Mst3 binding site but also with sequences proximal to striatin-associated PP2A, consistent with a possible role for Mob 3 in the regulation of Mst3 by PP2A.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Sistema Nervoso Central/patologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a Calmodulina/genética , Sistema Nervoso Central/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Especificidade por Substrato , Ativação TranscricionalRESUMO
Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the B alpha subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. B alpha is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that B alpha-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70(S6K), were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I(1)(PP2A) enhanced E4orf4-induced cell killing and G(2)/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with B alpha-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.
Assuntos
Adenovírus Humanos/patogenicidade , Ciclo Celular , Morte Celular , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Virais/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Ligação Proteica , Proteína Fosfatase 2/metabolismoRESUMO
The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2A(BAC)), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2A(BAC) holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2A(BAC) heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2A(BAC) heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.
Assuntos
Domínio Catalítico/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Metiltransferases/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas Virais/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Metiltransferases/metabolismo , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína Fosfatase 2/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Imunoprecipitação/métodos , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fosforilação , Proteína Fosfatase 2/genética , Piridinas/farmacologia , Ratos , Transdução de Sinais , Fatores de Tempo , Transfecção/métodosRESUMO
Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.
Assuntos
Apoptose , Fragmentação do DNA , Metiltransferases/metabolismo , Mitose , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Inibidores de Caspase , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fragmentação do DNA/efeitos dos fármacos , Perda do Embrião/enzimologia , Perda do Embrião/genética , Perda do Embrião/patologia , Células HeLa , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/genética , Camundongos , Camundongos Knockout , Mitose/efeitos dos fármacos , Mitose/genética , Nocodazol/farmacologia , Inibidores de Proteases/farmacologia , Proteína O-Metiltransferase/genética , Proteína O-Metiltransferase/metabolismo , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Timidina/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Safingol [(2S,3S)-2-amino-1,3-octadecanediol] is an unnatural l-threo-stereoisomer of sphinganine that is cytotoxic for cancer cells in culture and is being tested in phase 1 human clinical trials. To determine if safingol can be absorbed orally and if it affects prostate cancer in a mouse strain used in prostate cancer studies, safingol was fed to TRAMP (transgenic adenocarcinoma of mouse prostate) mice for 2 weeks at 0.0125% to 0.1% w/w of the diet. Analysis of safingol and safingol metabolites in blood and tissues by liquid chromatography electrospray ionization tandem mass spectrometry revealed uptake in tissue and extensive conversion of safingol to N-acyl species (comparable to natural "ceramides") and mono-, di-, and tri-N-methyl metabolites that have not been observed previously. Safingol caused significant hepatotoxicity at all dosages, as reflected in elevated liver alanine aminotransferase, and at the highest dose (0.1 %) caused changes in liver histology (appearance of autophagosomal vacuoles) and renal toxicity (based on elevation of blood urea nitrogen) and decreases in packed blood cell volume and body weight. Safingol did not inhibit the prostate pre-neoplastic lesion (prostate intraepithelial neoplasia) in TRAMP mice; however, additional studies at lower dosages for longer time were not pursued due to host toxicity. Safingol and its N-methyl metabolites were cytotoxic to both a human prostate cell line (DU145) and mouse BALB 3T3 cells; therefore, the host and potential antitumor toxicity may be due to multiple molecular species of safingol.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Fígado/efeitos dos fármacos , Neoplasia Prostática Intraepitelial/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Esfingosina/análogos & derivados , Acilação , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Células 3T3 BALB , Biotransformação , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hematócrito , Humanos , Nefropatias/induzido quimicamente , Fígado/patologia , Hepatopatias/patologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/administração & dosagem , Esfingosina/farmacocinética , Esfingosina/uso terapêutico , Esfingosina/toxicidade , Distribuição TecidualRESUMO
Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B''-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.
Assuntos
Regulação da Expressão Gênica/genética , Microtúbulos/metabolismo , Mutação/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Acetilação , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Neuroblastoma , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2 , Interferência de RNA/fisiologia , Transfecção/métodos , Tirosina/metabolismoRESUMO
Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. The enzyme is, in fact, largely a collection of varied heterotrimeric species composed of a catalytic (C) subunit and regulatory (B-type) subunit bound together by a structural (A) subunit. One important feature of the C subunit is that its carboxy-terminus can be modified by phosphorylation and methylation. The mechanisms that trigger such posttranslational modifications, as well as their consequences, are still under investigation. However, data collected thus far indicate that these modifications alter the binding to B subunits for an AC dimer, thereby affecting the makeup of the PP2A species in the cell. In this chapter, we describe an in vivo assay for assessing stable PP2A heterotrimer formation that is based on specific subcellular localizations of PP2A heterotrimers. This assay can be used to study the impact of a wide variety of alterations (such as mutations and covalent modifications) on PP2A heterotrimer formation. We specifically describe the use of this assay to quantify the effects of methylation on the stable formation of PP2ARts1p and PP2ACdc55p heterotrimers.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Domínio Catalítico , Dimerização , Metilação , Fosfoproteínas Fosfatases/química , Fosforilação , Proteína Fosfatase 2 , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , Leveduras/enzimologia , Leveduras/metabolismoRESUMO
[structure: see text] A family of cyclic 1-deoxysphingolipid derivatives of structure 4 has been designed and synthesized, which may serve as tumorigenesis suppressors for various cancers. Compound 4 is a second-generation analogue developed from sphingosine (1), in which a hydroxyl substituent is moved from C1 to C5 and a methylene is added for conformational rigidity between the C2-nitrogen substituent and C4. The synthetic chemistry for pyrrolidine ring closure at C3-C4 features ring-closing metathesis followed by hydroboration-oxidation.
Assuntos
Pirrolidinas/química , Pirrolidinas/síntese química , Esfingosina/análogos & derivados , Ciclização , Estrutura Molecular , Esfingosina/síntese química , Esfingosina/química , EstereoisomerismoRESUMO
Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.
Assuntos
Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/química , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinetocoros/metabolismo , Metilação , Modelos Biológicos , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 2 , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate gene expression. SHRs also mediate rapid, nongenomic cellular activation by steroids. In vascular endothelial cells, the SHR for estrogen, estrogen receptor (ER) alpha, is targeted by unknown mechanisms to a functional signaling module in membrane caveolae that enables estrogen to rapidly activate the mitogen-activated protein kinase and phosphatidylinositol 3-Akt kinase pathways, and endothelial NO synthase (eNOS). Here we identify the 110-kDa caveolin-binding protein striatin as the molecular anchor that localizes ERalpha to the membrane and organizes the ERalpha-eNOS membrane signaling complex. Striatin directly binds to amino acids 183-253 of ERalpha, targets ERalpha to the cell membrane, and serves as a scaffold for the formation of an ERalpha-Galphai complex. Disruption of complex formation between ERalpha and striatin blocks estrogen-induced rapid activation mitogen-activated protein kinase, Akt kinase, and eNOS, but has no effect on ER-dependent regulation of an estrogen response element-driven reporter plasmid. These findings identify striatin as a molecular scaffold required for rapid, nongenomic estrogen-mediated activation of downstream signaling pathways. Furthermore, by demonstrating independent regulation of nongenomic vs. genomic ER-dependent signaling, these findings provide conceptual support for the potential development of "pathway-specific" selective ER modulators.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Receptor alfa de Estrogênio/fisiologia , Substâncias Macromoleculares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Transdução de Sinais , Proteínas de Ligação a Calmodulina/fisiologia , Cavéolas/metabolismo , Linhagem Celular , Endotélio Vascular/citologia , Ativação Enzimática , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Humanos , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Óxido Nítrico Sintase Tipo III , Ligação ProteicaRESUMO
One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Transformação Celular Neoplásica/genética , Regulação da Expressão Gênica/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Western Blotting , Adesão Celular/genética , Divisão Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Genes cdc , Humanos , Integrinas/genética , Integrinas/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Vírus 40 dos Símios/imunologia , Transcrição Gênica/genética , Quinases da Família src/genética , Quinases da Família src/fisiologiaRESUMO
Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G(1) cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G(1) tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G(1) tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G(1) tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G(1) tetraploidy checkpoint.