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 DNAPKcs. 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.endosome | DDR | transmembrane domain R ing Finger protein 144A (RNF144A) and RNF144B belong to the RNF144 family and are very conserved in higher eukaryotes. Both proteins contain an RING1-in between rings (IBR) -RING2 [termed the ring between ring fingers (RBR)] domain in the N terminus and a potential single-transmembrane (TM) domain in the C terminus. RBR domain-containing proteins usually possess an E3 ubiquitin ligase activity and are involved in regulation of the cell cycle and apoptosis (1-3). These proteins function like RING (Really Interesting New Gene)/HECT (Homologous to the E6AP Carboxyl Terminus) hybrids (i.e., they use their RING1 to bind E2s) but then transfer ubiquitin onto a conserved cysteine residue in the RING2 domain through a thioester linkage similar to the E3 thioester-linked ubiquitin intermediates in HECT-type E3 ligases (4). It has been shown that DNA damage induces RNF144B expression to promote cell apoptosis through regulation of the stability of p21 (5), BAX (1), and p73 (6). RNF144B shares 71% homology of amino acids with RNF144A.

Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of UCH family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we are able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant (catalytic Cys to Ala, Nishio K, Kim SW, Kawai K, Mizushima T, Yamane T, Hamazaki J, Murata S, Tanaka K and Morimoto Y (2009) Biochem Biophys Res Commun 390, 855-860), which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.

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.

Background: Posttranslational modifications of E2F1 alter its transcriptional activity. Results: UCH37 binds E2F1 and deubiquitinates its UbK63-specific linkages to enhance the transcriptional activation of E2F1 target genes, particularly upon DNA damage. Conclusion: Ubiquitination and deubiquitination of UbK63-specific linkages provide an additional layer of regulation for E2F1 transcriptional activity. Significance: UCH37 is the first known deubiquitinating enzyme to directly regulate E2F1.