Lysine 63-linked Polyubiquitination of the Dopamine Transporter Requires WW3 and WW4 Domains of Nedd4-2 and UBE2D Ubiquitin-conjugating Enzymes

Abstract

RNA interference screen previously revealed that a HECTdomain 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.Post-translational modification of proteins by ubiquitination has recently emerged as an important mechanism governing membrane trafficking processes. Ubiquitin moieties attached to integral membrane proteins serve to target these proteins for rapid internalization at the cell surface and, after internalization, to lysosomes for degradation (1-3). In addition, ubiquitination of the components of internalization and endosomal sorting machineries regulate interactions and turnover of these proteins (1, 4 -5).Ubiquitination is accomplished through sequential actions of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase, the latter typically determining the substrate specificity of the reaction (6 -7). Proteins can be mono-and polyubiquitinated. Polyubiquitin chains can be formed by conjugation of ubiquitin to any of seven lysines in the ubiquitin molecule, although chains linked through Lys We have recently demonstrated that ubiquitination mediates protein kinase C (PKC) 2 -dependent endocytosis of the human dopamine transporter (DAT) (13-15). Upon PKC activation by phorbol esters, heterologously expressed DAT is internalized via a clathrin-dependent endocytic pathway and degraded in lysosomes (16,17). Ubiquitination of thre...

Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1

“…Immunoprecipitation-The cells grown in 35-mm dishes were placed on ice and washed with CMF-PBS, and the proteins were solubilized in Triton X-100/glycerol/HEPES lysis buffer supplemented with 100 mM NaCl, 10 mM N-ethylmaleimide and protease inhibitors for 20 min at 4°C (37). The lysate was then centrifuged at 100,000 ϫ g for 20 min to remove the insoluble material.…”

“…Single cell clones of HEK293 and PAE cells stably expressing CAT-1-HA-GFP were generated by selection in the presence of G418 (400 g/ml). HEK293 stably expressing CFP-HA-DAT were previously described (37). PAE stably expressing YFP-HA-DAT were previously described (38).…”

“…All siRNAs were purchased from Dharmacon. Nedd4-2, Nedd4-1, and clathrin heavy chain were depleted by siRNAs as previously described (37). siGENOME Non-Targeting duplex 3 (NT) was used as a control.…”

“…We have previously reported that Nedd4-2 is essential for DAT ubiquitination (37,38). Therefore, we tested whether NEDD4-2 is also the E3 ubiquitin ligase involved in the PKCdependent ubiquitination of CAT-1.…”

Ubc13 and COOH Terminus of Hsp70-interacting Protein (CHIP) Are Required for Growth Hormone Receptor Endocytosis

“…In many instances, Ubc13 acts as the E2 enzyme that, together with pseudo E2s, UEV1A, or MMS2, assembles Lys 63 -linked ubiquitin chains on the substrates (8,9). The dopamine transporter is provided with four-ubiquitin Lys 63 -linked chains, presumably via Nedd4-2 (10). UEV-1/Ubc13 is implicated in the regulation of an AMPA-type glutamate receptor in Caenorhabditis elegans (11).…”