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

• Published in: The Journal of biological chemistry Vol. 286; no. 10; pp. 8697 - 8706
• Main Authors: Vina-Vilaseca, Arnau, Bender-Sigel, Julia, Sorkina, Tatiana, Closs, Ellen Ildicho, Sorkin, Alexander
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.03.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Transport; Animals; Cationic Amino Acid Transporter 1 - genetics; Cationic Amino Acid Transporter 1 - metabolism; Clathrin - genetics; Clathrin - metabolism; Clathrin-Coated Vesicles - genetics; Clathrin-Coated Vesicles - metabolism; E3 Ubiquitin Ligase; Endocytosis; Endocytosis - physiology; Endosomal Sorting Complexes Required for Transport - genetics; Endosomal Sorting Complexes Required for Transport - metabolism; HEK293 Cells; Humans; Membrane Biology; Nedd4 Ubiquitin Protein Ligases; Protein Kinase C (PKC); Protein Kinase C - genetics; Protein Kinase C - metabolism; Protein Structure, Secondary; Swine; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; Ubiquitination - physiology; E3 Ubiquitin Ligase; Endocytosis; Ubiquitination; Amino Acid Transport; Protein Kinase C (PKC)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.186858

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.