Protein kinase C activation promotes the internalization of the human cationic amino acid transporter hCAT-1. A new regulatory mechanism for hCAT-1 activity.
The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gräf, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.
Rotmann
Alexander
A
Departments of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany.
Strand
Dennis
D
Martiné
Ursula
U
Closs
Ellen I
EI
D000818:Animals; D019921:Biotinylation; D027201:Cationic Amino Acid Transporter 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors; D015870:Gene Expression; D005909:Glioblastoma; D049452:Green Fluorescent Proteins / Q000235:genetics; D006706:Homeostasis; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis
D027201:Cationic Amino Acid Transporter 1; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D011993:Recombinant Fusion Proteins; C420825:SLC7A1 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate