Upregulation of Peptide Transporters PEPT1 and PEPT2 by Janus Kinase JAK2

Abstract

Background/Aims: Janus-activated kinase-2 JAK2 participates in the signaling of several hormones including growth hormone, fosters tumor growth and modifies the activity of several Na⁺ coupled nutrient transporters. Peptide uptake into intestinal and tumor cells is accomplished by electrogenic peptide transporters PEPT1 and PEPT2. The present study thus explored whether JAK2 contributes to the regulation of PEPT1 and PEPT2 activity. Methods: cRNA encoding either PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2. The current created by the dipeptide glycine-glycine (I_gly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. Results: No appreciable I_gly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes I_gly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, I_gly-gly without significantly modifying the concentration required for halfmaximal I_gly-gly (K_M). Following disruption of carrier insertion with brefeldin A (5 µM) I_gly-gly declined similarly fast in Xenopus oocytes expressing PEPT1 with JAK2 and in Xenopus oocytes expressing PEPT1 alone. In oocytes expressing both, PEPT1 and ^V617FJAK2, I_gly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased I_gly-gly in mouse intestine. Conclusion: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.

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

“…The inactive K882E JAK2 mutant (30) and the gain of function V617F JAK2 mutant (42,52) were generated by site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit; Stratagene) as described previously (58).…”

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

“…Constructs encoding wild-type ROMK1 or ROMK1 containing an extracellular hemagglutinin epitope (ROMK1-HA) [29], wild-type SPAK, constitutively active T233E SPAK, catalytically inactive D212A SPAK [6,30], wild-type OSR1, constitutively active T185E OSR1, and catalytically inactive D164A OSR1 [31], were used for generation of cRNA as described previously [32]. The constructs were a kind gift from Dario Alessi (University of Dundee).…”

Up-Regulation of Kir2.1 (KCNJ2) by the Serum & Glucocorticoid Inducible SGK3

“…In two-electrode voltage-clamp experiments K ir 2.1 currents were elicited every 20 s with 1 s pulses from -150 mV to +30 mV applied from a holding potential of -60 mV. The oocytes were maintained at 17°C in a ND96 solution with antibiotics (ND96-A) containing 88.5 mM NaCl (Sigma-Aldrich Chemie, Steinheim, Germany), 2 mM KCl (Carl Roth, Karlsruhe, Germany), 1.8 mM CaCl 2 (Sigma-Aldrich Chemie, Steinheim, Germany), 1 mM MgCl 2 (Sigma-Aldrich Chemie, Steinheim, Germany) ) , 5 mM HEPES (Carl Roth, Karlsruhe, Germany), 0.11 mM tetracycline (Sigma-Aldrich Chemie, Steinheim, Germany), 4 μM ciprofloxacin (Fresenius Kabi Austria, Austria), 0.22 mM gentamycin (Refobacin, Merck Serono, Darmstadt, Germany), 0.5 mM theophylline (Euphylong, Nycomed, Konstanz, Germany) as well as 5 mM sodium pyruvate (Sigma-Aldrich Chemie, Steinheim, Germany) [48]. The pH was adjusted to 7.4 by addition of NaOH (AppliChem, Darmstadt, Germany).…”