title: Regulation of RNF144A E3 Ubiquitin Ligase Activity by Self-association through Its Transmembrane Domain.
abstract: RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G(252)XXXG(256) motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.
authors.affiliation: From the Section of Hematology/Oncology, Department of Medicine.
authors.forename: Shiuh-Rong
authors.initials: SR
authors.lastname: Ho
authors.affiliation: Interdepartmental Program in Translational Biology and Molecular Medicine.
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: From the Section of Hematology/Oncology, Department of Medicine, Interdepartmental Program in Translational Biology and Molecular Medicine, Department of Molecular and Cellular Biology, and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030 weeichil@bcm.edu.
authors.forename: Weei-Chin
authors.initials: WC
authors.lastname: Lin
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C571196:RNF144A protein, human; D044767:Ubiquitin-Protein Ligases
keywords: E3 ubiquitin ligase; GXXXG motif; RBR; RNF144 subfamily; cell biology; membrane protein; self-association; transmembrane domain; ubiquitylation (ubiquitination)