RESUMO
Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Ubiquitinação/genética , Animais , Bovinos , Proteínas Culina/genética , Proteínas Culina/metabolismo , DNA Nucleotidilexotransferase/genética , Biblioteca Gênica , Células HeLa , Humanos , Fígado/citologia , Fígado/metabolismo , Plasmídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Timo/citologia , Timo/metabolismo , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Terminal deoxynucleotidyltransferase (TdT) interacting factor 2 (TdIF2) is an acidic protein that binds to TdT. TdIF2 binds to DNA and core histones and contains an acidic-amino acid-rich region in its C-terminus. It has therefore been suggested to function as a histone chaperone within the nucleus. TdIF2 localized within the nucleolus in HEK 293T cells, and its N-terminal (residues 1-234) and C-terminal (residues 606-756) regions were crucial for the nucleolar localization. A chromatin immunoprecipitation (ChIP) assay showed that TdIF2 associated with the promoter of human ribosomal RNA genes (hrDNAP), and an in vitro luciferase assay system showed that it promoted hrDNAP activity. Using the yeast two-hybrid system with TdIF2 as the bait, we isolated the cDNA encoding HIV Tat interactive protein 60 (Tip60), which has histone acetyltransferase (HAT) activity, as a TdIF2-binding protein. TdIF2 bound to Tip60 in vitro and in vivo, inhibited the Tip60 HAT activity in vitro and co-localized with Tip60 within the nucleolus. In addition, TdIF2 promotes upstream binding factor (UBF) acetylation in vivo. Thus, TdIF2 might promote hrDNAP activity by suppressing Tip60's HAT activity and promoting UBF acetylation.
Assuntos
Proteínas de Transporte/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , RNA Ribossômico/genética , Animais , Proteínas de Transporte/química , Citoplasma/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Regulação para Baixo , Células HEK293 , Histona Acetiltransferases/metabolismo , Humanos , Lisina Acetiltransferase 5 , Proteínas Nucleares/química , Ligação Proteica , Transporte Proteico , Proteínas de Ligação a RNA , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
We isolated human cDNA clone encoding Bood POZ containing gene type 2 (BPOZ-2) as a gene with a product that binds to TdT interacting factor 1 (TdIF1) using a yeast two-hybrid system. BPOZ-2 is an adaptor for E3 ligase CUL3 and participates in developmental processes. The binding between BPOZ-2 and TdIF1 was confirmed by GST pull-down and immunoprecipitation assays using specific antibodies against BPOZ-2 and TdIF1 in vitro and in vivo. Although when BPOZ-2 solely was expressed in COS7 cells, BPOZ-2 was observed mainly within the cytoplasm, co-transfection of pEGFP-BPOZ-2 and pDsRed-TdIF1 into COS7 cells resulted in co-localization of EGFP-BPOZ-2 and DsRed-TdIF1 within the nucleus. TdIF1 may recruit BPOZ-2 into the nucleus from the cytoplasm by directly binding to BPOZ-2. BPOZ-2 enhanced TdT ubiquitylation when TdIF1 was expressed together with BPOZ-2 in 293T cells, strongly suggesting that the recruitment of BPOZ-2 into the nucleus from the cytoplasm is significant for the TdT ubiquitylation within the nucleus.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , DNA Nucleotidilexotransferase/genética , Proteínas de Ligação a DNA , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
Bood POZ containing gene type 2 (BPOZ-2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2 bound to E3 ligase CUL3 in vitro and in vivo. BPOZ-2 itself was ubiquitinated through the CUL3-based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ-2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ-2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ-2-binding protein using a yeast two-hybrid system by screening a human thymus cDNA library. TdT, BPOZ-2, and CUL3 formed a ternary complex in vivo. TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co-expression of BPOZ-2 and CUL3 or BPOZ-2 in 293T cells, respectively.
