Warsi Jamshed, Elvira Bernat, Hosseinzadeh Zohreh, Shumilina Ekaterina, Lang Florian
Department of Physiology I, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
J Membr Biol. 2014 May;247(5):387-93. doi: 10.1007/s00232-014-9645-0. Epub 2014 Mar 11.
Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation (A572V)JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit ((K855A)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, (A568V)JAK3 or (K851A)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 (A568V)JAK3, but not by coexpression of (K851A)JAK3. Exposure of the oocytes expressing ClC-2 together with (A568V)JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of (A568V)JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and (A568V)JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (A568V)JAK3 and oocytes expressing ClC-2 alone, indicating that (A568V)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.
Janus激酶3（JAK3）促进淋巴细胞和肿瘤细胞的增殖并对抗其凋亡。在急性巨核细胞白血病中发现了功能获得性突变（A572V）JAK3。JAK3在催化亚基中被丙氨酸取代赖氨酸而失活（（K855A）JAK3）。细胞增殖和凋亡的调节涉及Cl(-)通道活性的改变。因此，本研究探讨了JAK3是否会改变小电导Cl(-)通道ClC-2的功能。为此，将ClC-2在非洲爪蟾卵母细胞中表达，分别共表达野生型JAK3、（A568V）JAK3或（K851A）JAK3，并用双电极电压钳测定Cl(-)通道活性。利用化学发光法测定细胞膜中通道蛋白的丰度。结果，ClC-2表达后细胞膜电导显著增加。共表达JAK3或（A568V）JAK3后电导显著降低，但共表达（K851A）JAK3则无此现象。将同时表达ClC-2和（A568V）JAK3的卵母细胞暴露于JAK3抑制剂WHI-P154（4-[(3'-溴-4'-羟基苯基)氨基]-6,7-二甲氧基喹唑啉，22 μM）可增加电导。共表达（A568V）JAK3可降低表达ClC-2的卵母细胞膜中ClC-2蛋白的丰度。在表达ClC-2和（A568V）JAK3的卵母细胞以及单独表达ClC-2的卵母细胞中，用布雷菲德菌素A（5 μM）抑制通道蛋白插入后，共表达ClC-2和（A568V）JAK3的卵母细胞中电导的下降情况相似，这表明（A568V）JAK3可能会减缓通道蛋白插入细胞膜的速度，而不是加速通道蛋白从细胞膜的回收。总之，JAK3下调ClC-2活性，从而对抗Cl(-)外流——这一效应可能影响细胞增殖和凋亡。
