ABSTRACT
E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead, UCH37, but not a catalytically dead mutant, decreases the Lys-63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces the gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1, and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase 3, caspase 7, PARP1, and Apaf-1 and activates their expression after DNA damage. Moreover, the expression of E2F1 proliferative and pro-apoptotic genes is correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its Lys-63-linked ubiquitination by UCH37.
Subject(s)
E2F1 Transcription Factor/metabolism , Gene Expression Regulation, Enzymologic/physiology , Promoter Regions, Genetic/physiology , Transcription, Genetic/physiology , Ubiquitin Thiolesterase/biosynthesis , Ubiquitination/physiology , E2F1 Transcription Factor/genetics , HEK293 Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , S Phase/physiology , Ubiquitin Thiolesterase/geneticsABSTRACT
Several ring between ring fingers (RBR) -domain proteins, such as Parkin and Parc, have been shown to be E3 ligases involved in important biological processes. Here, we identify a poorly characterized RBR protein, Ring Finger protein 144A (RNF144A), as the first, to our knowledge, mammalian E3 ubiquitin ligase for DNA-PKcs. We show that DNA damage induces RNF144A expression in a p53-dependent manner. RNF144A is mainly localized in the cytoplasmic vesicles and plasma membrane and interacts with cytoplasmic DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs plays a critical role in the nonhomologous end-joining DNA repair pathway and provides prosurvival signaling during DNA damage. We show that RNF144A induces ubiquitination of DNA-PKcs in vitro and in vivo and promotes its degradation. Depletion of RNF144A leads to an increased level of DNA-PKcs and resistance to DNA damaging agents, which is reversed by a DNA-PK inhibitor. Taken together, our data suggest that RNF144A may be involved in p53-mediated apoptosis through down-regulation of DNA-PKcs when cells suffer from persistent or severe DNA damage insults.
Subject(s)
Apoptosis/physiology , Carrier Proteins/physiology , Catalytic Domain/physiology , DNA Damage/physiology , DNA-Activated Protein Kinase/physiology , Gene Expression Regulation, Enzymologic/physiology , Ubiquitin-Protein Ligases/physiology , Blotting, Western , Carrier Proteins/metabolism , Colony-Forming Units Assay , DNA Primers/genetics , DNA-Activated Protein Kinase/metabolism , Fluorescent Antibody Technique , HEK293 Cells , Humans , Immunoprecipitation , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin-Protein Ligases/metabolismABSTRACT
E2F1 is a eukaryotic transcription factor that is known to regulate various cellular pathways such as cell cycle progression, DNA replication, DNA damage responses and induction of apoptosis. Given its versatile roles, a precise and tight regulation of E2F1 is very critical to maintain genomic stability. E2F1 is regulated both at transcriptional and posttranslational levels during cell cycle and upon DNA damage. After S phase, E2F1 is targeted for degradation and is kept at low levels or in an inactive state until the next G 1/S phase transition. Our studies show that APC/C ubiquitin ligase in conjunction with its co-activator Cdh1 (APC/C (Cdh1) ) can downregulate E2F1. We also identify an APC/C subunit APC5 that binds to E2F1 and is essential for E2F1 ubiquitination. We confirm an interaction between E2F1 and Cdh1 as well as an interaction between E2F1 and APC5 both in vivo and in vitro. In vitro GST pull-down assays have mapped the C-terminal 79 a.a. of E2F1 as Cdh1 interacting residues. Ectopically expressed Cdh1 downregulates the expression of E2F1-4. Our studies have also shown for the first time that E2F1 can be modified by K11-linkage specific ubiquitin chain formation (Ub-K11). The formation of Ub-K11 chains on E2F1 is increased in the presence of Cdh1 and accumulated in the presence of proteasome inhibitor, suggesting that APC/C (Cdh1) targets E2F1 for degradation by forming Ub-K11 chains. We also show that the effect of Cdh1 on E2F1 degradation is blocked upon DNA damage. Interestingly, Ub-K11-linked E2F1 accumulates after treatment of DNA damaging agents. The data suggest that DNA damage signaling processes do not inhibit APC/C (Cdh1) to ubiquitinate E2F1. Instead, they block the proteasomal degradation of Ub-K11-linked E2F1, and therefore lead to its accumulation.
