Up-Regulation of Amino Acid Transporter SLC6A19 Activity and Surface Protein Abundance by PKB/Akt and PIKfyve

Abstract

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive ^T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant ^S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (I_le), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of ^T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of ^S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of I_le following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells.

Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2

“…The cRNA was generated, and the Xenopus oocytes were prepared for voltage-clamp analysis, as previously described (13). Oocytes were injected with wild-type JAK2 cRNA, V617F JAK2, or K882E JAK2 cRNA (10 ng) on the first day after preparation of the Xenopus oocytes.…”

SPAK and OSR1 Dependent Down-Regulation of Murine Renal Outer Medullary K⁺ Channel ROMK1

“…The oocytes were maintained at 17°C in ND96-A solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1 2 , 1.8 CaC1 2 , 5 HEPES, tretracycline (Sigma, 0.11 mM), ciprofloxacin (Sigma, 4 μM), gentamycin (Refobacin, 0.2 mM) and theophylin (Euphylong, 0.5 mM) as well as sodium pyruvate (Sigma, 5 mM) were added to the ND96, pH was adjusted to 7.5 by addition of NaOH. The voltage clamp experiments were performed at room temperature 4 days after the first injection [36]. ROMK1 channel currents were elicited every 20 s with 3 s pulses from -160 mV to +60 mV applied from a holding potential of -80 mV.…”

Amino Acid Transporters in Drug Discovery

“…Moreover, the function of B0AT1 is up-regulated by PKB/Akt also in tumor cells (Bogatikov et al, 2012). A specific allosteric regulation by K + has been described: K + indeed, shows a biphasic modulation at the intracellular site with stimulation up to 50 mM internal (intracellular) K + and inhibition at higher K + concentrations (Oppedisano and Indiveri, 2008).…”