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 downregulation 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. Cationic amino acid transporter (CAT)-12 is a member of the SLC7 gene family of membrane transport proteins. The SLC7 family is divided into two groups, glycosylated (CAT) and nonglycosylated transporters (reviewed in Ref. 1). The latter associate with a glycosylated protein from the SLC3 family to form heteromeric amino acid transporters. There are four proven members in the CAT subfamily: CAT-1, CAT-2A, CAT-2B, and CAT-3 (reviewed in Ref. 2). All CATs have a predicted topology of 14 transmembrane domains with cytoplasmic amino and carboxyl termini. The largest, third extracellular loop is glycosylated in all CATs. CAT-2 proteins are also glycosylated in the second loop. CAT-1 is expressed ubiquitously in all adult tissues except the liver (reviewed in Ref.3). The transport properties of CAT-1 resemble those of system y ϩ , defined by the selectivity for cationic amino acids, K m of 0.1-0.2 mM, Na ϩ and pH independence, and strong trans-stimulation. CAT-1 is ubiquitously expressed and the main portal of entry for cationic amino acids into mammalian cells. Homozygous knockout of CAT-1 in mice is postnatally lethal (4). CAT-1 activity has been reported to be regulated through activation of protein kinase C (PKC) (5, 6). Even though there are some discrepancies, most studies conclude that activation of PKC leads to inhibition of L-Arg uptake due to the decreased activity of CAT-1. Studies of CAT-1 phosphorylation and mutations of the PKC phosphorylation consensus sites in CAT-1 suggested that CAT-1 inhibition by PKC is not due to CAT-1 phosp...

Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

“…The importance of ubiquitination in the endocytosis of various membrane proteins has been appreciated in recent years (5,17,18,25). Both monoubiquitination and polyubiquitination have been shown to serve as efficient internalization signals (15,17,18,25).…”

Phosphorylation and ubiquitylation are opposing players in regulating endocytosis of the water channel Aquaporin-2

“…For membrane proteins, induction of phosphorylation, including polyphosphorylation, is a mechanism for regulating the activity and intracellular trafficking of channels and transporters (Moeller et al, 2010;Rosenbaek et al, 2012;Tong et al, 2009;Yang et al, 2006). Ubiquitylation of membrane proteins through specific linkage is a common way of mediating internalization and promotion of protein degradation by the lysosomal pathway (Kazazic et al, 2009;Piper and Luzio, 2007;Vina-Vilaseca et al, 2011). 'Cross-talking' of phosphorylation and ubiquitylation has been proposed (Hunter, 2007), and phosphorylation often occurs as a priming event for ubiquitylation (Hunter, 2007;Murakami et al, 2009;Orford et al, 1997;Sehat et al, 2007).…”

Phosphorylation of RS1 (RSC1A1) Steers Inhibition of Different Exocytotic Pathways for Glucose Transporter SGLT1 and Nucleoside Transporter CNT1, and an RS1-Derived Peptide Inhibits Glucose Absorption

“…Post-translational short-term regulations of plasma membrane transporters adapt transporter activities according to physiologic demands (Zhang et al, 2008;Kock et al, 2010;Vina-Vilaseca et al, 2011;Gorboulev et al, 2012;Leto and Saltiel, 2012;Mühlfeld et al, 2012;Zhang et al, 2013). Physiologically important examples are insulin-induced recruitment of glucose transporter 4 (GLUT4) to the plasma membrane of adipocytes (Leto and Saltiel, 2012), protein kinase C-dependent endocytosis of organic anion transporter 1 (OAT1) (Zhang et al, 2008(Zhang et al, , 2013, and glucose-induced incorporation of the Na 1 -D-glucose cotransporter SGLT1 into brush-border membranes (BBMs) of small intestinal enterocytes (Gorboulev et al, 2012).…”