Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1.

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.

The antioxidant quercetin inhibits cellular proliferation via HIF-1-dependent induction of p21WAF.

Flavonoids are dietary antioxidants that may play a role as adjunct nutritional supplements in cancer or during inflammatory disorders. Hypoxia and the transcription factor hypoxia-inducible factor-1alpha also appear to play a key role in many human cancers. In this study, we investigated the role of quercetin in the hypoxia-dependent HIF-1alpha induction. It was shown that quercetin induced HIF-1alpha expression and HIF-1 activity under normoxia and hypoxia in human HepG2 hepatoma cells. By using actinomycin D and cycloheximide, we showed that quercetin acted post-transcriptionally by prolonging the HIF-1alpha protein half-life. Thereby quercetin interfered with the proline hydroxylation-dependent HIF-1alpha protein destabilization in the N-terminal HIF-1alpha transactivation domain. Experiments with quercetin analogues revealed that a flavonol structure and the presence of hydroxyl groups at position 3' and 4' are a prerequisite for the HIF-1alpha stabilizing effects. Further, quercetin inhibited cell proliferation and induced expression of the cell cycle inhibitor p21WAF and knocking-down HIF-1alpha disrupted these effects. These results provide evidence that quercetin inhibits the cell cycle and that induction of the HIF-system contributes to these effects of quercetin.