RESUMO
γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Derivados de Benzeno/farmacologia , Monitoramento de Medicamentos , Folículo Piloso/efeitos dos fármacos , Propionatos/farmacologia , Inibidores de Proteases/farmacologia , Receptores Notch/antagonistas & inibidores , Sulfonas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Adolescente , Adulto , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Baltimore , Derivados de Benzeno/administração & dosagem , Derivados de Benzeno/sangue , Derivados de Benzeno/farmacocinética , Biomarcadores Farmacológicos/sangue , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Monitoramento de Medicamentos/métodos , Perfilação da Expressão Gênica/métodos , Folículo Piloso/metabolismo , Voluntários Saudáveis , Humanos , Macaca mulatta , Masculino , Modelos Animais , Terapia de Alvo Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Propionatos/administração & dosagem , Propionatos/sangue , Propionatos/farmacocinética , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/sangue , Inibidores de Proteases/farmacocinética , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Receptores Notch/metabolismo , Sulfonas/administração & dosagem , Sulfonas/sangue , Sulfonas/farmacocinética , Adulto JovemRESUMO
Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteínas Tirosina Quinases/metabolismo , Tirosina , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Ativação Enzimática , Humanos , Leucemia de Células T , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Fosforilação , Fosfotirosina , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Tirosina/metabolismo , Animais , Western Blotting , Catálise , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Células Jurkat , Fosforilação , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
Lck, a lymphocyte-specific tyrosine protein kinase, is bound to cellular membranes as the result of myristoylation and palmitoylation of its amino terminus. Its activity is inhibited by phosphorylation of tyrosine 394. The Tyr-505 --> Phe mutant of Lck (F505Lck) exhibits elevated biological activity and constitutive phosphorylation of Tyr-394 in vivo. Mutations at sites of fatty acylation that prevent F505Lck from associating with cellular membranes abolish the biological activity as a protein kinase in vivo and in vitro, and eliminate the phosphorylation of Tyr-394. Here, we show that exposure of cells expressing cytoplasmic or nuclear forms of F505Lck to H2O2, a general inhibitor of tyrosine protein phosphatases, restores the catalytic activity of these mutant proteins through stimulation of phosphorylation of Tyr-394. H2O2 treatment induced the phosphorylation of Tyr-394 therefore need not occur by autophosphorylation. Thus, there appear to be two mechanisms through which the phosphorylation of Lck at Tyr-394 can occur. One is restricted to the plasma membrane and does not require the presence of oxidants. The other is operational in the nucleus as well as the cytosol and is responsive to oxidants.
Assuntos
Reativadores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Tirosina/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Linhagem Celular , Membrana Celular/enzimologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Fosforilação , Ratos , Especificidade por SubstratoRESUMO
The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Imunofilinas/antagonistas & inibidores , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/metabolismo , Humanos , Células Jurkat , Cinética , Toxinas Marinhas , Modelos Biológicos , Fosforilação , Proteína Fosfatase 2 , Proteínas Recombinantes de Fusão/metabolismo , Serina-Treonina Quinases TOR , Proteínas de Ligação a Tacrolimo , TransfecçãoRESUMO
The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.
Assuntos
Proteínas Fúngicas/metabolismo , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Príons , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sirolimo/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas de Ciclo Celular , Ciclo do Ácido Cítrico/fisiologia , Meios de Cultura , Perfilação da Expressão Gênica , Glucose/metabolismo , Glutationa Peroxidase , Glicólise/fisiologia , Nitrogênio/metabolismo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.