RESUMO
Photoreceptor cell death is the definitive cause of vision loss in retinal detachment (RD). Mammalian STE20-like kinase (MST) is a master regulator of both cell death and proliferation and a critical factor in development and tumorigenesis. However, to date the role of MST in neurodegeneration has not been fully explored. Utilizing MST1(-/-) and MST2(-/-) mice we identified MST2, but not MST1, as a regulator of photoreceptor cell death in a mouse model of RD. MST2(-/-) mice demonstrated significantly decreased photoreceptor cell death and outer nuclear layer (ONL) thinning after RD. Additionally, caspase-3 activation was attenuated in MST2(-/-) mice compared to control mice after RD. The transcription of p53 upregulated modulator of apoptosis (PUMA) and Fas was also reduced in MST2(-/-) mice post-RD. Retinas of MST2(-/-) mice displayed suppressed nuclear relocalization of phosphorylated YAP after RD. Consistent with the reduction of photoreceptor cell death, MST2(-/-) mice showed decreased levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 and interleukin 6 as well as attenuated inflammatory CD11b cell infiltration during the early phase of RD. These results identify MST2, not MST1, as a critical regulator of caspase-mediated photoreceptor cell death in the detached retina and indicate its potential as a future neuroprotection target.
Assuntos
Apoptose , Caspase 3/metabolismo , Células Fotorreceptoras de Vertebrados/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Descolamento Retiniano/enzimologia , Animais , Caspase 3/genética , Camundongos , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/patologia , Proteínas Serina-Treonina Quinases/genética , Descolamento Retiniano/genética , Descolamento Retiniano/patologia , Serina-Treonina Quinase 3 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans.
Assuntos
Neoplasias Hepáticas/patologia , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas/enzimologia , Camundongos , Tamanho do Órgão , Fosfoproteínas/fisiologia , Serina-Treonina Quinase 3 , Proteínas de Sinalização YAPRESUMO
Target of Rapamycin (TOR), a giant protein kinase expressed by all eucaryotic cells, controls cell size in response to nutrient signals. In metazoans, cell and organismal growth is controlled by nutrients and the insulin/insulin-like growth factor (IGF) system, and the understanding of how these inputs coordinately regulate TOR signaling has advanced greatly in the past 5 years. In single-cell eucaryotes and Caenorhabditis elegans, TOR is a dominant regulator of overall mRNA translation, whereas in higher metazoans, TOR controls the expression of a smaller fraction of mRNAs that is especially important to cell growth. TOR signals through two physically distinct multiprotein complexes, and the control of cell growth is mediated primarily by TOR complex 1 (TORC1), which contains the polypeptides raptor and LST8. Raptor is the substrate binding element of TORC1, and the ability of raptor to properly present substrates, such as the translational regulators 4E-BP and p70 S6 kinase, to the TOR catalytic domain is essential for their TOR-catalysed phosphorylation, and is inhibited by the Rapamycin/FKBP-12 complex. The dominant proximal regulator of TORC1 signaling and kinase activity is the ras-like small GTPase Rheb. Rheb binds directly to the mTOR catalytic domain, and Rheb-GTP enables TORC1 to attain an active configuration. Insulin/IGF enhances Rheb GTP charging through the ability of activated Akt to inhibit the Rheb-GTPase-activating function of the tuberous sclerosis heterodimer (TSC1/TSC2). Conversely, energy depletion reduces Rheb-GTP charging through the ability of the adenosine monophosphate-activated protein kinase to phosphorylate TSC2 and stimulate its Rheb-GTPase activating function, as well as by HIFalpha-mediated transcriptional responses that act upstream of the TSC1/2 complex. Amino-acid depletion inhibits TORC1 acting predominantly downstream of the TSC complex, by interfering with the ability of Rheb to bind to mTOR. The components of the insulin/IGF pathway to TORC1 are now well established, whereas the elements mediating the more ancient and functionally dominant input of amino acids remain largely unknown.
Assuntos
Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Quinases/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TORRESUMO
The p70 S6 kinase (p70 S6K) was the first signaling element in mammalian cells shown to be inhibited by rapamycin. The activity of the p70 S6K in mammalian cell is upregulated by extracellular amino acids (especially leucine) and by signals from receptor tyrosine kinases (RTKs), primarily through activation of the type 1A PI-3 kinase. The amino acid-/rapamycin-sensitive input and the PI-3 kinase input are co-dominant but largely independent, in that deletion of the amino-terminal and carboxy-terminal noncatalytic sequences flanking the p70 S6K catalytic domain renders the kinase insensitive to inhibition by both rapamycin and by withdrawal of amino acids, whereas this p70 S6K mutant remains responsive to activation by RTKs and to inhibition by wortmannin. At a molecular level, this dual control of p70 S6K activity is attributable to phosphorylation of the two p70 S6K sites: The Ptd Ins 3,4,5P3-dependent kinasel (PDK1) phosphorylates p70 S6K at a Thr on the activation loop, whereas mTOR phosphorylates a Thr located in a hydrophobic motif carboxyterminal to the catalytic domain. Together these two phosphorylations engender a strong, positively cooperative activation of p70 S6K, so that each is indispensable for physiologic regulation. Like RTKs, the p70 S6K appears early in metazoan evolution and comes to represent an important site at which the more ancient, nutrient-responsive TOR pathway converges with the RTK/PI-3 kinase pathway in the control of cell growth. Dual regulation of p70 S6K is seen in Drosophila; however, this convergence is not yet evident in Caenorhabditis elegans, wherein nutrient activation of the insulin receptor (InsR) pathway negatively regulates dauer development and longevity, whereas the TOR pathway regulates overall mRNA translation through effectors distinct from p70 S6K, as in yeast. The C. elegans TOR and InsR pathways show none of the cross- or convergent regulation seen in mammalian cells. The nature of the elements that couple nutrient sufficiency to TOR activity remain to be discovered, and the mechanisms by which RTKs influence TOR activity in mammalian cells require further study. One pathway for RTK control involves the tuberous sclerosis complex, which is absent in C. elegans, but of major importance in Drosophila and higher metazoans.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteínas Quinases/fisiologia , Animais , Cricetinae , Receptor de Insulina/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Sirolimo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5' termini (5'TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. We now show that the translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and that this nutritional control depends strictly on the integrity of the 5'TOP motif. However, neither phosphorylation of ribosomal protein (rp) S6 nor activation of S6K1 per se is sufficient to relieve the translational repression of TOP mRNAs in amino acid-starved cells. Likewise, inhibition of S6K1 activity and rpS6 phosphorylation by overexpression of dominant-negative S6K1 mutants failed to suppress the translational activation of TOP mRNAs in amino acid-refed cells. Furthermore, TOP mRNAs were translationally regulated by amino acid sufficiency in embryonic stem cells lacking both alleles of the S6K1 gene. Inhibition of mTOR by rapamycin led to fast and complete repression of S6K1, as judged by rpS6 phosphorylation, but to only partial and delayed repression of translational activation of TOP mRNAs. In contrast, interference in the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway by chemical or genetic manipulations blocked rapidly and completely the translational activation of TOP mRNAs. It appears, therefore, that translational regulation of TOP mRNAs, at least by amino acids, (i) is fully dependent on PI3-kinase, (ii) is partially sensitive to rapamycin, and (iii) requires neither S6K1 activity nor rpS6 phosphorylation.
Assuntos
Aminoácidos/química , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas , Pirimidinas/química , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Alelos , Motivos de Aminoácidos , Antifúngicos/farmacologia , Sequência de Bases , Western Blotting , Linhagem Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Humanos , Toxinas Marinhas , Modelos Biológicos , Dados de Sequência Molecular , Oxazóis/farmacologia , Fosforilação , Plasmídeos/metabolismo , Polirribossomos/metabolismo , Testes de Precipitina , RNA/metabolismo , Proteína S6 Ribossômica , Fatores de Tempo , TransfecçãoAssuntos
Biossíntese de Proteínas , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Proteínas Fúngicas/metabolismo , Expressão Gênica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas/química , Proteínas Quinases S6 Ribossômicas/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade por Substrato , Serina-Treonina Quinases TORRESUMO
BACKGROUND: The p70 S6 kinase, like several other AGC family kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 kinase (alpha 1). Phosphorylation of the former site is catalyzed by PDK1, whereas the kinase responsible for the phosphorylation of the latter site is not known. RESULTS: The major protein kinase that is active on the p70 S6 kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 kinase at Thr412 and other sites and activates the p70 S6 kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 kinase inhibitors. CONCLUSION: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 kinase.
Assuntos
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Insulina/farmacologia , Fígado/enzimologia , Dados de Sequência Molecular , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas/química , Homologia de Sequência de Aminoácidos , Treonina/metabolismoRESUMO
ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:
Assuntos
Trifosfato de Adenosina/farmacologia , Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Indóis/farmacologia , Cinética , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Maleimidas/farmacologia , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Norepinefrina/farmacologia , Ratos , Serotonina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation. In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Ser/Thr phosphorylation reflected the activation of protein (Ser/Thr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (Ser/Thr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effectors. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (Ser/Thr) kinases to execute their cellular programs. The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance of the Ras-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Proteínas 14-3-3 , Animais , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Ligação Proteica , Proto-Oncogene Mas , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The molecular details of mammalian stress-activated signal transduction pathways have only begun to be dissected. This, despite the fact that the impact of these pathways on the pathology of chronic inflammation, heart disease, stroke, the debilitating effects of diabetes mellitus, and the side effects of cancer therapy, not to mention embryonic development, innate and acquired immunity, is profound. Cardiovascular disease and diabetes alone represent the most significant health care problems in the developed world. Thus it is not surprising that understanding these pathways has attracted wide interest, and in the past 10 years, dramatic progress has been made. Accordingly, it is now becoming possible to envisage the transition of these findings to the development of novel treatment strategies. This review focuses on the biochemical components and regulation of mammalian stress-regulated mitogen-activated protein kinase (MAPK) pathways. The nuclear factor-kappa B pathway, a second stress signaling paradigm, has been the subject of several excellent recent reviews (258, 260).
Assuntos
Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Fisiológico/fisiopatologia , Animais , Citocinas/fisiologia , Humanos , Mamíferos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxazóis/farmacologia , Transdução de Sinais , Tirosina 3-Mono-Oxigenase/metabolismo , Vimentina/metabolismo , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Células COS , Inibidores Enzimáticos/metabolismo , Toxinas Marinhas , Dados de Sequência Molecular , Oxazóis/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Ligação Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
Assuntos
Astrócitos/metabolismo , Ativadores de Enzimas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-raf/biossíntese , Agonistas do Receptor Purinérgico P2 , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tionucleotídeos/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation.
Assuntos
Fator Neurotrófico Ciliar/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fosfosserina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteínas Quinases , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Transativadores/metabolismo , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Fragmentos de Peptídeos/farmacologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Fator de Transcrição STAT3 , Serina-Treonina Quinases TORRESUMO
Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.
Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Treonina/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Fator de Crescimento Insulin-Like I/farmacologia , Isoenzimas/fisiologia , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , OvinosRESUMO
p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Immunoblotting , Mitógenos/farmacologia , Mutação , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Serina-Treonina Quinases TORRESUMO
Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells.
Assuntos
Fosfatidilinositóis/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Amino acid deprivation of Chinese hamster ovary cells overexpressing human insulin receptors results in deactivation of p70 S6 kinase (p70) and dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which become unresponsive to insulin; readdition of amino acids restores these responses in a rapamycin-sensitive manner, suggesting that amino acids and mammalian target of rapamycin signal through common effectors. Contrarily, withdrawal of medium amino acids from the hepatoma cell line H4IIE does not abolish the ability of insulin to stimulate p70 and 4E-BP1. The addition of 3-methyladenine (3MA) to H4IIE cells deprived of amino acids inhibited the increment in protein degradation caused by amino acid withdrawal nearly completely at 10 mM and also strongly inhibited the ability of insulin to stimulate p70 and 4E-BP1 at 10 mM. Treatment of H4IIE cells with 3MA did not alter the ability of insulin to activate tyrosine phosphorylation, phosphoinositide 3-kinase, or mitogen-activated protein kinase. In conclusion, the ability of H4IIE cells to maintain the insulin responsiveness of the mammalian target of rapamycin-dependent signaling pathways impinging on p70 and 4E-BP1 without exogenous amino acids reflects the generation of amino acids endogenously through a 3MA-sensitive process, presumably autophagy, a major mechanism of facultative protein degradation in liver.
Assuntos
Aminoácidos/metabolismo , Autofagia , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais , Sirolimo/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Humanos , Insulina/farmacologia , Fosforilação , Ratos , Receptor de Insulina/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Fatores de Transcrição E2F , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Isoenzimas , Dados de Sequência Molecular , Mutação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/efeitos dos fármacos , Proteína 1 de Ligação ao Retinoblastoma , Sirolimo/farmacologia , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.
Assuntos
Proteína Quinase C/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Animais , Células COS , Dimerização , Ativação Enzimática , Mutação , Fosfopeptídeos/farmacologia , Fosforilação , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes/metabolismo , Serina/metabolismo , TransfecçãoRESUMO
The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p70Delta2-46/DeltaCT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase.