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

Content Provider	Journal of Biological Chemistry (JBC)
Author	Vina-Vilaseca, Arnau Bender-Sigel, Julia Sorkina, Tatiana Closs, Ellen Ildicho Sorkin, Alexander
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.
Related Links	http://www.jbc.org/content/286/10/8697.abstract
Ending Page	8706
Starting Page	8697
Page Count	10
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	10
Volume Number	286
DOI	10.1074/jbc.M110.186858
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2011-03-11
Access Restriction	Open
Subject Keyword	Amino Acid Transport E3 Ubiquitin Ligase Endocytosis Protein Kinase C (PKC) Ubiquitination Cell Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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