RNF144A, an E3 ubiquitin ligase for DNA-PKcs, promotes apoptosis during DNA damage

Abstract

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.

Regulation of RNF144A E3 Ubiquitin Ligase Activity by Self-association through Its Transmembrane Domain

“…Deletion of the C-terminal TM domain of RNF144A significantly reduces its autoubiquitination. However, the mechanism was not known (18). In this study, we show that the TM domain is required for RNF144A self-association-dependent activation of ubiquitin ligase activity at least in part through a classic GXXXG motif.…”

“…Plasmid Construction and Mutagenesis-GST-tagged and FLAG-tagged RNF144A and RNF144B have been described previously (18). In brief, the mammalian expression pCMVTag2B vector was used for cloning of the FLAG-RNF144A and FLAG-RNF144A truncated mutants.…”

“…Protein purification was performed as described previously (18). Briefly, Escherichia coli strain DH5␣ transformed with either the pGEX-6P1 vector, pGEX-6P1-RNF144A-WT (a.a. 1-292), the pGEX-6P1-RNF144A-RING1 mutant (C20A/C23A), pGEX-6P1-RNF144A-⌬TM (⌬a.a.…”

“…In Vivo Ubiquitination Assay-The in vivo ubiquitination assay was performed as described previously (18). Briefly, HEK293T cells were cotransfected with HA-tagged ubiquitin and FLAG-tagged RNF144A (WT or mutants) for 24 h as indicated.…”

“…In addition to a regular RBR domain, RNF144 subfamily members contain a single transmembrane (TM) domain that is close to their C termini (17)(18)(19). Both RNF144A and RNF144B are induced by p53 and promote cell apoptosis under various DNA damages (17,18,20,21).…”