Abstract
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.
Highlights
Growing and proliferating cancer cells rely on the delivery of nutrients for anabolic processes and energy delivery [1]
The activation of AKT kinase starts with its phosphorylation on Thr308 via phosphoinositide-dependent kinase 1 (PDK-1) [33], followed by phosphorylation on Ser473 with a major contribution from the Rictor–mammalian target of rapamycin complex [34,35]
A lack of active AKT correlated with a two-fold increase in both SLC6A14 species after 30 min of treatment with MK-2206, the amount of the transporter almost returned to the control level after 1 h of treatment, and the level of both bands increased by 40% after 24 h (Figure 1B, right panel, Figure 1C right panel)
Summary
Growing and proliferating cancer cells rely on the delivery of nutrients for anabolic processes and energy delivery [1]. Beside the GLUT1–SLC2A1 transporting glucose, the main energetic substrate for aerobic glycolysis, there are several amino acid transporters upregulated in cancer, supporting protein synthesis, and the biosynthesis of nucleotides and energy metabolism (for review, see [2]). The budding of vesicles, as well as the formation of large membrane carriers for the transport of large-size cargo (procollagens), involves several proteins, and is started by Sec, which converts Sar into its GTP-bound form, and this potentiates membrane curvature. This recruits Sec23/Sec heterodimers, and vesicle formation is completed after the binding of Sec13/Sec heterodimers that form heterotetramers as the outer lattice of the coat. The experiments were performed with cell lines characterized by a high level of SLC6A14, i.e., the endogenous transporter in breast cancer MCF-7 cells, and the overexpressed transporter in HEK293 cells
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