RESUMO
Vascular endothelial growth factor (VEGF) has a central role in normal as well as in tumor angiogenesis. As such, VEGF is subjected to multi-level regulation at the transcriptional, post-transcriptional, translational, and post-translational levels to ensure proper expression during embryogenesis and adulthood. Its mRNA contains an exceptionally long (1038 bp) 5' untranslated region (5'UTR), which has a role in transcriptional as well as translational regulation of VEGF expression. In this communication, we provide new evidence showing that an open reading frame (ORF) present in the 5'UTR encodes for new putative isoforms of VEGF due to alternative translational initiation from CUG codons. Like VEGF, the translation of the new isoforms is not sensitive to stress signals such as anoxia. Most likely, these isoforms either possess new capabilities, which are different from the activity of the classical VEGF isoforms, or affect the efficiency and capacity of translational initiation from the canonical AUG codon.
Assuntos
Códon de Iniciação , Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Animais , Anticorpos/imunologia , Células COS , Fatores de Crescimento Endotelial/imunologia , Humanos , Linfocinas/imunologia , Peso Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Transfecção , Células U937 , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
IFN regulatory factors (IRFs) constitute a family of transcription factors that are involved in IFN signaling and the development and differentiation of the immune system. Targeted gene disruption studies in mice assigned their primary role to the immune system. Two lymphoid-specific IRF members, IFN consensus sequence binding protein (ICSBP) and IRF-4, bind target DNA with greater efficiency following interaction with two transcription factors, PU.1 and E47, leading to transcriptional synergy. PU.1 and E47 are essential for proper differentiation and maturation of lymphoid cells. In addition, ICSBP interacts with two IRF members, IRF-1 and IRF-2, which also have central roles in the regulation of cell-mediated immunity. Previously, we identified a region in ICSBP, termed the IRF association domain (IAD), that is conserved in all IRFs (excluding IRF-1 and IRF-2) and is essential for its interactions with other IRF proteins. Here we show that the IAD is an independent module used by ICSBP and IRF-4 for protein-protein interactions. In addition, an IAD of IRF-2 (IAD2), necessary for interaction with ICSBP, was identified and found to be conserved in IRF-1. The IAD2 shares similar characteristics with the PEST domain that is essential for the interaction of PU.1 with IRF-4. We also show that the ICSBP DNA binding domain is indispensable for the formation of DNA binding heterocomplexes and transcriptional activity. Therefore, our results shed light on the molecular mechanisms that affect IRF activities in the immune system via discrete functional domains.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Interferons/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Animais , Sequência Consenso/imunologia , DNA/fisiologia , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/fisiologia , Fator Regulador 2 de Interferon , Fatores Reguladores de Interferon , Interferons/fisiologia , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Fatores de Transcrição TCF , Transativadores/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição , Transcrição Gênica/imunologiaRESUMO
Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.