Up-regulation of the betaine/GABA transporter BGT1 by JAK2 ManzarShojaiefard, Zohreh Hosseinzadeh, Shefalee K.Bhavsar, Florian Lang Physiology Department of Fasa University of Medical Science , Iran Department of Physiology, University of Tübingen , Gmelinstr . 5, D-72076 Tübingen , Germany

The Janus-activated kinase-2 JAK2 contributes to the signaling of several hormones and cytokines [1–3] including leptin [4], growth hormone [5,6], erythropoietin [2], thrombopoietin [2] and granulocyte colony-stimulating factor [2].

Excessive JAK2 activity may lead to the development of malignancy and JAK2 inhibitors may be effective in the treatment of myeloproliferative disorders[7–12]. The gain of function mutation V617F JAK2 has been detect in and presumably contributes to development of myeloproliferative disease [13–16] .

JAK2 is activated by oxidative stress and ischemia [17] and by Hypertonicity [18,19]. The sensitivity to osmotic cell shrinkage renders the kinase a candidate signaling molecule participating in the regulation of cell volume.

Mechanisms up-regulated during osmotic cell shrinkage include the activity of Na+ coupled osmolyte transporters, such as the betaine/GABA transporter BGT1 (SLC6A12) [20,21].

BGT1 belongs a superfamily of Na+ Cl – coupled transporters for neurotransmitters (e.g. dopamine, GABA, serotonin and norepinephrine), amino acids (e.g. glycine)[22] , creatine[22] , and the organic osmolytes betaine [23] and taurine [24] .BGT1 is expressed in liver[25–27] , kidney [27] , airways [28] , and brain [27,29].

Osmotic cell shrinkage up-regulates BGT1 expression[30–32] Osmotic cell shrinkage up-regulates BGT1 expression[30–32] .Beyond that, the carrier is regulated by ATP[33] ,Ca2+[34] , protein kinase C [35] , prostaglandin E2[36,37] and the cytoskeleton [38,39]

Cellular accumulation of osmolytes protects against apoptosis and thus participates in the regulation of cell survival[40] .

The present study explored whether the activity of BGT1 is influenced by regulated by JAK2. To this end, BGT1 was expressed in Xenopus oocytes with or without wild type JAK2, constitutively active V617F JAK2 or inactive K882E JAK2 and the electrogenic GABA transport determined utilizing dual electrode voltage clamp.

Materials and methods

Constructs were used encoding wild type BGT1[41] , and wild type human JAK2 (Imagenes, Berlin, Germany). Further, an inactive K882E JAK2 mutant [42] and the V617F JAK2 mutant [16] were generated by site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit; Stratagene, Heidelberg, Germany) according to the manufacturer’s instructions [43] . The following primers were used mutant [42] and the V617F JAK2 mutant [16] were

K882E JAK2: 5-GGGAGGTGGTCGCTGTA GAAAAGCTTCAGCATAGT-3; V617FJAK2: 5-AGCATTTGGTTTTAAATTATGGAGTATGT TTCTGTGGAGACGAGA-3; V617F JAK2: 5-TCTCGTCTCCACAGA AACATACTCCATAATTTAAAACCAAATGCT-3; K882E JAK2: 5-GGGAGGTGGTCGCTGTA GAAAAGCTTCAGCATAGT-3; K882E JAK2: 5- ACTATGCTGAAGCTTTT CTACAGCGACCACCTCCC-3 Underlined bases indicate mutation sites. The mutants were sequenced to verify the presence of the desired mutation. The mutants were used for generation of cRNA as described previously

Xenopus oocytes were prepared as previously described [45] . Where not indicated otherwise, 15 ng BGT1 cRNA on the first day and 10 ng of wild type JAK2 cRNA were injected on the second day or same day after preparation of the oocytes [46] . The oocytes were maintained at 17C in ND96 solution . where indicated, the JAK2inhibitor AG490 (40 uM), actinomycin D (10 uM) or brefeldin A (5 uM) were added to the respective solutions.

The voltage clamp experiments were performed at room temperature 4 days after injection [47] . Two-electrode voltage-clamp recordings were performed at a holding potential of -60mV. The data were filtered at 10 Hz and recorded with a Digidata A/D–D/A converter and Clampex V.9 software for data acquisition and analysis G -Amino-butyric acid (GABA) was added to the solutions at a concentration of 1 mM , unless otherwise stated . The flow rate of the superfusion was approx. 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s

Data are provided as means ± SEM, n represents the number oocytes investigated. All experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA or t-test, as appropriate. Results with p < 0.05 were considered statistically significant

Results

Fig. 1. Coexpression of JAK2 increases electrogenic GABA transport in BGT1-expressing Xenopus oocytes. (A) Representative original tracings showing GABA (1 mM)-induced current (BGT)in Xenopus oocytes injected with water (a), expressing wild type JAK2 alone (b), or expressing BGT1 without (c) or with (d) additional coexpression of wild type JAK2 (B) Arithmetic means ± SEM (n = 7) of GABA (1 mM)-induced current ( IBGT)in Xenopus oocytes injected with water (H2O, dotted bar), expressing JAK2 alone (JAK2, dark gray bar), or expressing BGT1 without (BGT1, white bar) or with (BGT1 + JAK2, dark gray bar) additional coexpression of wild type JAK2. ⁄⁄⁄ Indicates statistically significant(p < 0.001) difference from the absence of BGT1. ### Indicates statistically significant ( p < 0.001) difference from the absence of JAK2. (C) Arithmetic means ± SEM ( n =3)of GABA-induced current (IBGT) as a function of GABA concentration in Xenopus oocytes expressing BGT1 without (open circles, dashed line), or with (closed circles, solid line) additional coexpression of wild type JAK2.

Fig. 2. The effect of JAK2 is mimicked by V617F JAK2 but not by the inactive mutant K882E (A) Representative original tracings showing GABA (1 mM)-induced current ( IBGT )in Xenopus oocytes injected with water (a), or expressing JAK2 alone (b) ,BGT1 alone (c), BGT1 with JAK2(d),with constituvely active JAK2 (f). mutant K882E V617F JAK2 (e), or with the inactive (B) Arithmetic means ± SEM ( n = 15) of GABA (1 mM)-induced current ( IBGT)in Xenopus oocytes injected with water (H2O, dotted bar), expressing JAK2 alone (JAK2, dark gray bar), expressing BGT1 without (BGT1, white bar) or with wild type JAK2 (BGT + JAK2, dark gray bar), with constitutively active V617F JAK2 (BGT1 +V617F JAK2, black bar) or with the inactive mutant K882E JAK2 (BGT1 + K882E JAK2, light gray bar)/,indicates statistically significant ( p < 0.5, p < 0.001) difference to expression of BGT1 alone.

Fig. 3. The effect of V617f jak2 is reversed by the JAK2 inhibitor AG490. (A) Representative original tracings showing GABA (1 mM)-induced current ( IBGT) in Xenopus oocytes injected with BGT1 (a) or BGT1 + V617F JAK2 without (b) or with a 6 h (c) or 24 h (d) pretreatment with JAK2 inhibitor AG490 (40 UM). (B) Arithmetic means ± SEM (n = 15) of GABA (1 mM)-induced current ( IBGT )in Xenopus oocytes injected with water (H2O, dotted bar), or expressing BGT1 with constitutive active V617F JAK2 (black and gray bars) in the absence of inhibitor (black bar, 0H) or following pretreatment with the JAK2 inhibitor AG490 (40 UM, light gray bars) for the indicated time periods. #,## indicates statistically significant (p < 0.05, p < 0.01) difference from the absence of JAK2 inhibitor AG490. ***Indicates statistically significant ( p < 0.001) difference from BGT1 alone (absence of JAK2).

(A) Arithmetic means ± SEM ( n = 15) of GABA (1 mM)-induced current (I Fig. 4. Effects of brefeldin A and actinomycin D on BGT1 and BGT1 + V617F JAK2 expressing Xenopus oocytes. (A) Arithmetic means ± SEM ( n = 15) of GABA (1 mM)-induced current (I JAK2 ـ BGT) in Xenopus oocytes injected with cRNA encoding BGT1 with V617F and exposed to 5 uM brefeldin A for the indicated time periods in the absence (black bars)and presence light gray bars) of Brefeldin A (5). *,**Indicates statistically significant ( p < 0.05, p < 0.01) from the absence of JAK2; from the absence of brefeldin p < 0. 01, p < 0.001) ( different ## , ### indicates statistically significant A (B) Arithmetic means ± SEM ( n = 7) of GABA (1 mM)-induced current (IBGT )in Xenopus oocytes injected with cRNA encoding BGT1 without (white bars) and with JAK2 (black bars) in the presence and absence of significant different 10 uM actinomycin D 1–2 days prior to the measurement . *** Indicates statistically (p < 0.001) from the absence of V617F JAK2.

Discussion

The present study reveals a novel regulator of the betaine/GABA transporter BGT1 (SLC6A12). In BGT1 expressing Xenopus oocytes coexpression of the Janus Kinase 2 (JAK2) significantly increased the electrogenic transport of GABA. The effect of wild type JAK2 was mimicked by the gain of function mutant V617F JAK2 but not by the inactive K882EJAK2. Thus, kinase activity was required for this effect. Coexpression of the kinase did not significantly affect the substrate affinity but significantly enhanced the maximal transport rate.

The ability of JAK2 to modify transport has been demonstrated earlier [49,50]. More recent studies revealed the ability of JAK2 to up-regulate the Na +coupled glucose transporter SGLT1 (SLC5A1) [51] , the Na+ coupled neutral amino acid transporter B(0)AT (SLC6A19) [52] and the glutamate transporters EAAT1–4[48] .

The decline of BGT1 activity following inhibition of carrier insertion into the cell membrane by brefeldin A was similar in the presence and absence of the JAK2 inhibitor AG490, an observation suggesting that JAK2 did modify the clearance of carrier protein from the cell membrane. The observation may point to the ability of JAK2 to enhance carrier insertion into the cell membrane. In view of the impact of brefeldin A on the trafficking of a wide variety of molecules, the evidence is, however, rather circumstantial.

Exposure of JAK2 and BGT1 expressing oocytes to the JAK2 inhibitor AG490 does not appreciably affect BGT1 activity within the first 3 h, an observation pointing to indirect effects of JAK2 on carrier function. JAK2 may regulate carrier activity by influencing protein abundance rather than direct inactivation of the carrier. Alternatively, the inhibitor may gain access to the carrier only with delay. The effect of JAK2 on BGT1 is observed in the presence of actinomycin, which disrupts gene transcription. Thus, the observed effects of JAK2 are not explained by effects on gene transcription.

As JAK2 activity is activated by hypertonicity [18,19], the kinase presumably contributes to the up-regulation of BGT1 activity during osmotic cell shrinkage. Osmotic cell shrinkage is well known to up-regulate the activity of osmolyte transporters [53–55] . It must be kept in mind that the osmolyte carriers are up-regulated by stimulation of gene expression [53] , which apparently does not play a major role in the present observations. JAK2 has previously been shown to enhance the activity of cell volume regulatory Na+ H+ exchanger (NHE), which similarly servers to increase cell volume following osmotic cell shrinkage[57]

conclusion In conclusion, the present paper presents evidence, for the first time, that JAK2 up-regulates the GABA transporter BGT1 (SLC6A12). The observed JAK2 sensitive regulation of BGT1 could well contribute to cellular osmoregulation.

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