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

Arnau Vina-Vilaseca,Julia Bender-Sigel,Tatiana Sorkina,Ellen Ildicho Closs,Alexander Sorkin

doi:10.1074/jbc.m110.186858

Abstract

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.

Highlights

Grant DA014204 from the NIDA. □S The on-line version of this article contains supplemental Figs
HEK293 cells have endogenous cationic amino acid transporters but the uptake of L-[3H]Arg was significantly increased in cells stably expressing Cationic amino acid transporter 1 (CAT-1)-hemagglutinin antigen (HA)-green fluorescent protein (GFP) (Fig. 1B), indicating that Cationic amino acid transporter (CAT)-1HA-GFP mediates substrate transport
Because cationic amino acid transporters are expressed in all mammalian cells, transport parameters of HA-tagged and wild-type CAT-1 were compared in Xenopus oocytes

Summary

Endocytosis of Cationic Amino Acid Transporter

Three enzymes (E1, E2, and E3) [20]. The E3 ligase is the last enzyme responsible for the transfer of ubiquitin to the substrate. Two highly homologous E3 enzymes, NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) and to a lesser extent NEDD4-1, have been implicated in ubiquitination of many mammalian transporters and channels [21,22,23,24,25,26]. These enzymes contain the catalytic HECT (homologous E6-AP carboxyl-terminal) domain. CAT-1 was found to be ubiquitinated in a PKC-dependent manner, and this ubiquitination required NEDD4 family E3 ligases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION