A plasma membrane amino acid transporter B0,+ (ATB0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB0,+ trafficking. Rat ATB0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export “RI” motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.
Cancer cells need a constant supply of nutrients. SLC6A14, an amino acid transporter B0,+ (ATB0,+) that is upregulated in many cancers, transports all but acidic amino acids. In its exit from the endoplasmic reticulum (ER), it is recognized by the SEC24C subunit of coatomer II (COPII) for further vesicular trafficking to the plasma membrane. SEC24C has previously been shown to be phosphorylated by protein kinase B/AKT, which is hyper-activated in cancer; therefore, we analyzed the influence of AKT on SLC6A14 trafficking to the cell surface. Studies on overexpressed and endogenous transporters in the breast cancer cell line MCF-7 showed that AKT inhibition with MK-2206 correlated with a transient increase of the transporter in the plasma membrane, not resulting from the inhibition of ER-associated protein degradation. Two-dimensional electrophoresis demonstrated the decreased phosphorylation of SLC6A14 and SEC24C upon AKT inhibition. A proximity ligation assay confirmed this conclusion: AKT inhibition is correlated with decreased SLC6A14 phosphothreonine and SEC24C phosphoserine. Augmented levels of SLC6A14 in plasma membrane led to increased leucine transport. These results show that the inactivation of AKT can rescue amino acid delivery through SLC6A14 trafficking to the cell surface, supporting cancer cell survival. The regulation of the ER export of the amino acid transporter seems to be a novel function of AKT.
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