title: Regulation of DENND3, the exchange factor for the small GTPase Rab12 through an intramolecular interaction.
abstract: The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking. Proteins bearing adifferentiallyexpressed innormal andneoplastic cells (DENN) domain have emerged as the largest family of Rab-activating guanine nucleotide exchange factors (GEFs). Rab12 functions in the initiation of starvation-induced autophagy, and our previous work revealed that its activator, DENN domain-containing protein 3 (DENND3), is phosphorylated and activated upon starvation. However, how the GEF activity of DENND3 toward Rab12 is regulated at the molecular level is still not understood. Here, we combine size-exclusion chromatography, Förster resonance energy transfer, pulldown, andin vitroGEF assays to demonstrate that regulation of GEF activity is achieved through an intramolecular interaction that is controlled by a key residue in DENND3, tyrosine 940. Our study sheds light on the regulation of Rab12 activation and lays the groundwork for characterizing the regulation of other DENN domain-containing proteins.
authors.affiliation: From the Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada jie.xu3@mail.mcgill.ca.
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.affiliation: From the Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada peter.mcpherson@mcgill.ca.
authors.forename: Peter S
authors.initials: PS
authors.lastname: McPherson
mesh_terms: D000818:Animals; D001343:Autophagy; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D014443:Tyrosine / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C000605578:DENND3 protein, human; C000593695:Dennd3 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D014443:Tyrosine; D006160:Guanosine Triphosphate; C000595432:Rab12 protein, human; D020691:rab GTP-Binding Proteins
keywords: DENN domain; DENND3; Rab12; autophagy; guanine nucleotide exchange factor (GEF); intramolecular interaction; membrane trafficking; oligomerization; phosphorylation; phosphotyrosine