RESUMO
BACKGROUND: The pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode alpha-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression. RESULTS: Here we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim-1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion. CONCLUSION: Altogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Núcleo Celular/química , Chlorocebus aethiops , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Hematopoese/fisiologia , Humanos , Células Jurkat , Camundongos , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Ligação Proteica , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cytokines are the most important inducers of T helper (Th) cell differentiation. Interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) are responsible for human Th1-cell differentiation, while IL-4 is the critical cytokine promoting Th2-cell development. These two subsets of cells co-ordinate immunological responses to pathogens as well as autoimmune or allergic reactions. The pim family of proto-oncogenes encodes serine/threonine-specific kinases involved in cytokine-mediated signalling pathways in haematopoietic cells. Here we demonstrate that expression of pim-1 and pim-2 mRNAs is selectively up- or down-regulated in human cord-blood-derived CD4+ cells freshly induced to polarize towards Th1 or Th2 cells, respectively, whereas their expression is inhibited in both cell types by the immunosuppressive transforming growth factor beta (TGF-beta). Moreover, the Th1-specific cytokines IL-12 and IFN-alpha, but not the Th2-specific cytokine IL-4, transiently up-regulate pim-1 and pim-2 mRNA expression in human peripheral blood T cells and natural killer cells. In addition, the Pim-1 protein levels are strongly up-regulated by Th1-specific cytokines in all of these cell types. Taken together, our results suggest that pim genes and their protein products are involved in the early differentiation process of T helper cells.
Assuntos
Citocinas/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Células Th1/imunologia , Regulação para Cima/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Interferon-alfa/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-pim-1 , RNA Mensageiro/genética , Células Th2/imunologiaRESUMO
The activity of NFATc family transcription factors is tightly regulated in T cells via signaling pathways initiated by stimulation of the T cell receptor or its downstream effectors such as the Pim-1 serine/threonine kinase. Here, we demonstrate that NFATc-dependent transcription is inducible also in NGF-differentiated rat PC12 pheochromocytoma cells treated with phorbol esthers, calcium ionophores and/or forskolin and that the Pim-1 kinase can further potentiate the effects of these agents. PC12 cells share many characteristics with sympathetic neurons and can be induced to produce and release catecholamines, such as dopamine and noradrenaline, and inflammatory cytokines, such as interleukin 6. Interestingly, Pim-1 can synergize with forskolin-induced signaling pathways to stimulate also neuroendocrine functions of PC12 cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Sistemas Neurossecretores/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/fisiologia , Animais , Catecolaminas/metabolismo , Colforsina/farmacologia , Citocinas/metabolismo , Sinergismo Farmacológico , Ionóforos/farmacologia , Fatores de Transcrição NFATC , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Células PC12 , Ésteres de Forbol/farmacologia , Proteínas Proto-Oncogênicas c-pim-1 , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sistema Nervoso Simpático/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologiaRESUMO
Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/antagonistas & inibidores , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células COS , Proteínas de Transporte/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Interleucina-3/farmacologia , Cinética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-pim-1 , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína de Morte Celular Associada a bclRESUMO
Signal transducer and activator of transcription 5 (STAT5) plays a critical role in cytokine-induced survival of hematopoietic cells. One of the STAT5 target genes is pim-1, which encodes an oncogenic serine/threonine kinase. Here we demonstrate that Pim-1 inhibits STAT5-dependent transcription in cells responsive to interleukin-3, prolactin, or erythropoietin. Ectopic expression of Pim-1 in cytokine-dependent FDCP1 myeloid cells results in reduced tyrosine phosphorylation and DNA binding of STAT5, indicating that Pim-1 interferes already with the initial steps of STAT5 activation. However, the Pim-1 kinase does not directly phosphorylate or bind to STAT5. By contrast, Pim-1 interacts with suppressor of cytokine signaling 1 (SOCS1) and SOCS3 and potentiates their inhibitory effects on STAT5, most likely via phosphorylation-mediated stabilization of the SOCS proteins. Thus, both Pim and SOCS family proteins may be components of a negative feedback mechanism that allows STAT5 to attenuate its own activity.