Lysine 63-linked polyubiquitination of the dopamine transporter requires WW3 and WW4 domains of Nedd4-2 and UBE2D ubiquitin-conjugating enzymes.
RNA interference screen previously revealed that a HECT-domain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys(63)-linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys(63)-linked chain that can be conjugated to various lysine residues of DAT.
Vina-Vilaseca
Arnau
A
Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, Colorado 80010, USA.
Sorkin
Alexander
A
D000818:Animals; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D050483:Dopamine Plasma Membrane Transport Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D007527:Isoenzymes; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C; D008239:Lysine