RESUMO
Scratch-induced disruption of cultured monolayers induces polarity in front row cells that can be visualized by spatially localized polymerization of actin at the front of the cell and reorientation of the centrosome/Golgi to face the leading edge. We previously reported that centrosomal reorientation and microtubule polarization depend on a Cdc42-regulated signal transduction pathway involving activation of the Par6/aPKC complex followed by inhibition of GSK-3beta and accumulation of the adenomatous polyposis coli (APC) protein at the plus ends of leading-edge microtubules. Using monolayers of primary rodent embryo fibroblasts, we show here that dishevelled (Dvl) and axin, two major components of the Wnt signaling pathway are required for centrosome reorientation and that Wnt5a is required for activation of this pathway. We conclude that disruption of cell-cell contacts leads to the activation of a noncanonical Wnt/dishevelled signal transduction pathway that cooperates with Cdc42/Par6/aPKC to promote polarized reorganization of the microtubule cytoskeleton.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polaridade Celular , Centrossomo/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteína Axina , Comunicação Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Proteínas Desgrenhadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Complexo de Golgi/metabolismo , Camundongos , Microtúbulos/metabolismo , Fosfoproteínas/genética , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Repressoras/genética , Proteínas Wnt/genética , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU)-induced mutation in Suppressor of Zeste 12 (Suz12), a core component of Polycomb Repressive Complex 2 (PRC2), we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC) activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are sufficient to ameliorate the stem cell defect and thrombocytopenia present in mice that lack the thrombopoietin receptor (c-Mpl). To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in global patterns of gene expression in cells deficient in Suz12. We identified a distinct set of genes that are regulated by Suz12 in hematopoietic cells, including eight genes that appear to be highly responsive to PRC2 function within this compartment. These data suggest that PRC2 is required to maintain a specific gene expression pattern in hematopoiesis that is indispensable to normal stem cell function.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/metabolismo , Alelos , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , RNA Mensageiro/metabolismo , Proteínas Repressoras/genéticaRESUMO
RA is an autoimmune disease characterized by sustained imbalance between pro- and antiinflammatory immune mechanisms. The SOCS proteins are negative regulators of cytokine signaling, but to date there has been little information on their function in disease. The generation of Socs3(-/Delta vav) mice, which lack SOCS-3 in the hematopoietic and endothelial cell compartment, allowed us to explore the role of endogenous SOCS-3 during acute inflammatory arthritis. Joint inflammation in Socs3(-/Delta vav) mice was particularly severe and was characterized by increased numbers of neutrophils in the inflamed synovium, bone marrow, peripheral blood, and spleen. These features were most likely due to increased production of and enhanced responsiveness to G-CSF and IL-6 during arthritis in these mice. Local osteoclast generation and bone destruction were also dramatically increased in the absence of SOCS-3, as was macrophage activation. Finally, SOCS-3 was found to negatively regulate CD4+ T lymphocyte activation, including production of the pleiotropic cytokine IL-17. The absence of SOCS-3 therefore had dramatic effects in this disease model, with a broader impact on cellular responses than SOCS-1 deficiency. These findings provide direct in vivo evidence that endogenous SOCS-3 is a critical negative regulator of multiple cell types orchestrating inflammatory joint disease.
Assuntos
Artrite Reumatoide/imunologia , Imunidade/fisiologia , Interleucina-1/imunologia , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Animais , Artrite Reumatoide/patologia , Bovinos , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Imunidade/imunologia , Interleucina-6/sangue , Articulações/citologia , Articulações/imunologia , Articulações/patologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/imunologia , Osteoclastos/citologia , Osteoclastos/metabolismo , Soroalbumina Bovina/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T/citologia , Linfócitos T/imunologiaAssuntos
Proteínas Serina-Treonina Quinases , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Ativação Enzimática , Humanos , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de TumorRESUMO
GSK3 (glycogen synthase kinase-3) regulation is proposed to play a key role in the hormonal control of many cellular processes. Inhibition of GSK3 in animal models of diabetes leads to normalization of blood glucose levels, while high GSK3 activity has been reported in Type II diabetes. Insulin inhibits GSK3 by promoting phosphorylation of a serine residue (Ser-21 in GSK3alpha, Ser-9 in GSK3beta), thereby relieving GSK3 inhibition of glycogen synthesis in muscle. GSK3 inhibition in liver reduces expression of the gluconeogenic genes PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase), as well as IGFBP1 (insulin-like growth factor binding protein-1). Overexpression of GSK3 in cells antagonizes insulin regulation of these genes. In the present study we demonstrate that regulation of these three genes by feeding is normal in mice that express insulin-insensitive GSK3. Therefore inactivation of GSK3 is not a prerequisite for insulin repression of these genes, despite the previous finding that GSK3 activity is absolutely required for maintaining their expression. Interestingly, insulin injection of wild-type mice, which activates PKB (protein kinase B) and inhibits GSK3 to a greater degree than feeding (50% versus 25%), does not repress these genes. We suggest for the first time that although pharmacological inhibition of GSK3 reduces hepatic glucose production even in insulin-resistant states, feeding can repress the gluconeogenic genes without inhibiting GSK3.
Assuntos
Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Insulina/farmacologia , Fígado/metabolismo , Transcrição Gênica , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Ingestão de Alimentos/fisiologia , Alimentos , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese , Glucose/biossíntese , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Insulina/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fígado/efeitos dos fármacos , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Elementos de Resposta , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Reduced insulin-mediated glucose transport in skeletal muscle is a hallmark of the pathophysiology of T2DM (Type II diabetes mellitus). Impaired intracellular insulin signalling is implicated as a key underlying mechanism. Attention has focused on early signalling events such as defective tyrosine phosphorylation of IRS1 (insulin receptor substrate-1), a major target for the insulin receptor tyrosine kinase. This is required for normal induction of signalling pathways key to many of the metabolic actions of insulin. Conversely, increased serine/threonine phosphorylation of IRS1 following prolonged insulin exposure (or in obesity) reduces signalling capacity, partly by stimulating IRS1 degradation. We now show that IRS1 levels in human muscle are actually increased 3-fold following 1 h of hyperinsulinaemic euglycaemia. Similarly, transient induction of IRS1 (3-fold) in the liver or muscle of rodents occurs following feeding or insulin injection respectively. The induction by insulin is also observed in cell culture systems, although to a lesser degree, and is not due to reduced proteasomal targeting, increased protein synthesis or gene transcription. Elucidation of the mechanism by which insulin promotes IRS1 stability will permit characterization of the importance of this novel signalling event in insulin regulation of liver and muscle function. Impairment of this process would reduce IRS1 signalling capacity, thereby contributing to the development of hyperinsulinaemia/insulin resistance prior to the appearance of T2DM.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Fosfoproteínas/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Humanos , Proteínas Substratos do Receptor de Insulina , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Proteínas Quinases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Regulação para CimaRESUMO
In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.
Assuntos
Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase/metabolismo , Miocárdio/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Transporte Biológico , Transportador de Glucose Tipo 4 , Glicogênio/análise , Quinase 3 da Glicogênio Sintase/genética , Insulina/farmacologia , Insulina/fisiologia , Camundongos , Camundongos Knockout , Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimologia , Miocárdio/química , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
The mechanism by which Suppressor of Cytokine Signaling-3 (SOCS3) negatively regulates cytokine signaling has been widely investigated using over-expression studies in cell lines and is thought to involve interactions with both the gp130 receptor and JAK1. Here, we compare the endogenous JAK/STAT signaling pathway downstream of Leukemia Inhibitory Factor (LIF) signaling in wild type (WT) Embryonic Stem (ES) cells and in ES cells lacking either the entire Socs3 gene or bearing a truncated form of SOCS3 (SOCS3DeltaSB) lacking the C-terminal SOCS box motif (SOCS3(DeltaSB/DeltaSB)). In SOCS3(DeltaSB/DeltaSB) cells phosphorylated JAK1 accumulated at much higher levels than in WT cells or even cells lacking SOCS3 (SOCS3(-/-)). In contrast enhanced activation of STAT3 and SHP2 was seen in SOCS3(-/-) cells. Size exclusion chromatography of cell extracts showed that in unstimulated cells, JAK1 was exclusively associated with receptors but following cytokine stimulation hyperphosphorylated JAK1 (pJAK1) appeared to dissociate from the receptor complex in a manner independent of SOCS3. In WT and SOCS3(DeltaSB/DeltaSB) cells SOCS3 was associated with pJAK1. The data suggest that dissociation of activated JAK1 from the receptor results in separate targeting of JAK1 for proteasomal degradation through a mechanism dependent on the SOCS3 SOCS box thus preventing further activation of STAT3.
Assuntos
Células-Tronco Embrionárias/metabolismo , Janus Quinase 1/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Motivos de Aminoácidos/genética , Animais , Diferenciação Celular/genética , Citocinas/metabolismo , Citocinas/farmacologia , Células-Tronco Embrionárias/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/química , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli ("crosstalk"). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor alpha (TNFalpha), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFalpha. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFalpha.
Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5(-/-) cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Semaforina-3A/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Dados de Sequência Molecular , Proteínas Musculares , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Ratos , Semaforina-3A/metabolismoRESUMO
Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution.
Assuntos
Apoptose , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Supressoras da Sinalização de Citocina/deficiência , Animais , Ativação Enzimática , Células Epiteliais/citologia , Feminino , Deleção de Genes , Marcação de Genes , Integrases/metabolismo , Lactação/fisiologia , Fator Inibidor de Leucemia/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
SOCS3 is essential for regulating the extent, duration, and specificity of cellular responses to cytokines such as G-CSF and IL-6. Here we describe the solution structure of SOCS3, the first structure determined for any SOCS protein, in complex with a phosphotyrosine-containing peptide from the IL-6 receptor signaling subunit gp130. The structure of the complex shows that seven peptide residues form a predominantly hydrophobic binding motif. Regions outside the SOCS3 SH2 domain are important for ligand binding, in particular, a single 15 residue alpha helix immediately N-terminal to the SH2 domain makes direct contacts with the phosphotyrosine binding loop and, in part, determines its geometry. The SH2 domain itself is remarkable in that it contains a 35 residue unstructured PEST motif insertion that is not required for STAT inhibition. The PEST motif increases SOCS3 turnover and affects its degradation pathway, implying that it has an important regulatory role inside the cell.
Assuntos
Mutagênese Insercional , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genética , Domínios de Homologia de src/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Genes Reporter , Meia-Vida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luciferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Análise Espectral Raman , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , TransfecçãoRESUMO
The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, we generated homozygous knockin mice in which the protein kinase B phosphorylation sites on GSK3alpha (Ser21) and GSK3beta (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice we show that inactivation of GSK3beta rather than GSK3alpha is the major route by which insulin activates muscle glycogen synthase. In contrast, we demonstrate that the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/Ser9. GSK3 also becomes inhibited in the Wnt-signalling pathway, by a poorly defined mechanism. In GSK3alpha/GSK3beta homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of beta-catenin and stimulation of Wnt-dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has previously been implicated.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Glicogênio/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3ARESUMO
We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.
Assuntos
Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Linhagem Celular , Perda do Embrião , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Ativação Enzimática , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Fenótipo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Methionine synthase and methylmalonyl-CoA mutase (mutase) are the only two known vitamin B(12) (B(12)) dependent enzymes in humans. A lower level of B(12) has been shown to be an independent maternal risk factor for neural tube defects (NTDs) prompting an investigation of common genetic variants within B(12) dependent enzymes. To investigate the role of methylmalonyl-CoA mutase variants we studied 279 complete NTD triads (NTD affected case and both parents) and 256 controls. Based on case-control and family based (transmission disequilibrium test) analyses we did not find an association between the mutase single nucleotide polymorphisms (SNPs) K212K (636A-->G), H532R (1595A-->G) and V671I (2011G-->A) and NTDs. However, there was a significant difference in the frequencies of these polymorphisms between a group of African Americans and American Caucasians (K212K, P=0.002; H532R, P=0.001; V671I, P=0.006). In conclusion, common variants in the mutase gene do not appear to be risk factors for NTDs but their allele frequencies are significantly different between ethnic groups.