title: Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1.
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.
authors.affiliation: From the Department of Pharmacology, Anschutz Medical Center, University of Colorado Denver, Aurora, Colorado 80010.
authors.forename: Arnau
authors.initials: A
authors.lastname: Vina-Vilaseca
authors.affiliation: the Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55101, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bender-Sigel
authors.affiliation: the Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Sorkina
authors.affiliation: the Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz 55101, Germany.
authors.forename: Ellen Ildicho
authors.initials: EI
authors.lastname: Closs
authors.affiliation: From the Department of Pharmacology, Anschutz Medical Center, University of Colorado Denver, Aurora, Colorado 80010,; the Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and. Electronic address: sorkin@pitt.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D000818:Animals; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013552:Swine; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology
chemical_list: D027201:Cationic Amino Acid Transporter 1; D002966:Clathrin; D056827:Endosomal Sorting Complexes Required for Transport; C420825:SLC7A1 protein, human; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C