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The janus-activated kinase 2 JAK2 participates in the signalling of several hormones including interferon, a powerful regulator of lymphocyte function. Lymphocyte activity and survival depend on the activity of the voltage-gated K channel KCNA3 (Kv1.3). The present study thus explored whether JAK2 modifies the activity of voltage-gated K channel KCNA3. To this end, cRNA encoding KCNA3 was injected in Xenopus oocytes with or without additional injection of cRNA encoding wild-type human JAK2, human inactive JAK2 mutant, or human gain-of-function JAK2 mutant. KCNA3-dependent depolarization-induced current was determined utilizing dual-electrode voltage clamp, and protein KCNA3 abundance in the cell membrane was quantified by chemiluminescence. Moreover, the effect of interferon-γ on voltage-gated K current was determined by patch clamp in mainly KCNA3-expressing Jurkat T cells with or without prior treatment with JAK2 inhibitor AG490 (40 µM). As a result, KCNA3 channel activity and protein abundance were up-regulated by coexpression of JAK2 or JAK2 but not JAK2. The effect of JAK2 coexpression was reversed by AG490 treatment. In human Jurkat T lymphoma cells, voltage-gated K current was up-regulated by interferon-γ and down-regulated by AG490 (40 µM). In conclusion, JAK2 participates in the signalling, regulating the voltage-gated K channel KCNA3. [ABSTRACT FROM AUTHOR]

The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active SPAK, WNK-insensitive SPAK or catalytically inactive SPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice ( spak) and from gene-targeted mice carrying WNK-insensitive SPAK ( spak). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current ( I) was significantly decreased following coexpression of wild-type SPAK and SPAK, but not by coexpression of SPAK or SPAK. Kinetic analysis revealed that SPAK decreased maximal I without significantly modifying the glucose concentration required for halfmaximal I ( K). According to the chemiluminescence experiments, wild-type SPAK but not SPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak than in spak mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport. [ABSTRACT FROM AUTHOR]

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3 is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3. Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3 or inactive JAK3, and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 ( jak3) and from corresponding wild-type mice ( jak3). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current ( I), which was significantly increased following coexpression of JAK3. I in oocytes expressing EAAT3 was further increased by JAK3 but not by JAK3. I in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal I and significantly reduced the glutamate concentration required for half maximal I ( K). Intestinal electrogenic glutamate transport was significantly lower in jak3 than in jak3 mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms. [ABSTRACT FROM AUTHOR]

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit (JAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, JAK3 or JAK3, and the Cl channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or JAK3, but not by coexpression of JAK3. Exposure of the oocytes expressing ClC-2 together with JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with JAK3 and oocytes expressing ClC-2 alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl exit-an effect possibly influencing cell proliferation and apoptosis. [ABSTRACT FROM AUTHOR]

The tyrosine kinase Janus kinase 3 (JAK3) contributes to signaling regulating the proliferation and apoptosis of lymphocytes and tumor cells. Replacement of lysine by alanine in the catalytic subunit yields the inactive K851AJAK3 mutant that underlies severe combined immune deficiency. The gain-of-function mutation A572VJAK3 is found in acute megakaryoplastic leukemia and T cell lymphoma. The excessive nutrient demand of tumor cells requires upregulation of transporters in the cell membrane including peptide transporters PEPT1 and PEPT2. The carriers further accomplish intestinal peptide transport. Little is known about signaling regulating peptide transport. The present study explored whether PEPT1 and PEPT2 are upregulated by JAK3. PEPT1 or PEPT2 was expressed in Xenopus oocytes with or without additional expression of JAK3, and electrogenic peptide (glycine–glycine) transport was determined by dual-electrode voltage clamp. PEPT2-HA membrane protein abundance was analyzed by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide-induced current in Ussing chamber experiments. In PEPT1- and PEPT2-expressing oocytes, but not in water-injected oocytes, the dipeptide gly–gly generated an inward current, which was significantly increased following coexpression of JAK3. The effect of JAK3 on PEPT1 was mimicked by A568VJAK3 but not by K851AJAK3. JAK3 increased maximal peptide-induced current in PEPT1-expressing oocytes but rather decreased apparent affinity of the carrier. Coexpression of JAK3 enhanced the PEPT2-HA protein abundance in the cell membrane. In JAK3- and PEPT1-expressing oocytes, peptide-induced current was blunted by the JAK3 inhibitor WHI-P154, 4-[(3′-bromo-4′-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (22 μM). In intestinal segments gly–gly generated a current which was significantly smaller in JAK3-deficient mice ( jak3−/−) than in wild-type mice ( jak3+/+). In conclusion, JAK3 is a powerful regulator of peptide transporters PEPT1 and PEPT2. [ABSTRACT FROM AUTHOR]