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.

Background: RNF144A is a membrane-associated E3 ubiquitin ligase. Results: The GXXXG motif within the TM domain of RNF144A mediates its self-association and activation of E3 ligase activity. Conclusion: The TM domain regulates RNF144A through two independent steps: membrane localization and GXXXG motifmediated self-association. Significance: The GXXXG motif is conserved among all RBR-TM proteins, suggesting a similar regulation for other RBR-TM proteins.

Haploinsufficiency of RAI1 is responsible for Smith-Magenis Syndrome (SMS), a childhood neurodevelopmental disorder associated with hyperphagia, obesity, and autistic features. We previously showed that constitutive inactivation of one or both copies of Rai1 in the germline or developing brain induces SMS-like neurobehavioral deficits and obesity in mice. By contrast, the postnatal function of Rai1 is unclear. Here, we globally deleted one or both copies of Rai1 during two postnatal developmental windows by generating an inducible Rai1 knockout mouse model. We found that delayed Rai1 deletion at 3 or 8 weeks of age had no effect on neurobehavioral functions but resulted in adult-onset obesity and decreased expression of brain-derived neurotrophic factor (Bdnf) in the hypothalamus. Remarkably, genetic overexpression of human Bdnf in Rai1 heterozygous mice reversed SMS-like obesity, hyperphagia, metabolic syndrome-like features, and hyposociability. Increasing Bdnf signaling in the paraventricular nucleus of the hypothalamus (PVH) or the ventromedial nucleus of the hypothalamus (VMH) was sufficient to mediate the anti-obesity effect. Our work identifies the function of Rai1 in different temporal windows after birth and provides in vivo evidence that increasing Bdnf signaling is therapeutically effective in a preclinical mouse model of SMS.

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