Department of Physiology I, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
J Membr Biol. 2014 Feb;247(2):137-45. doi: 10.1007/s00232-013-9616-x. Epub 2013 Nov 21.
B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal IP. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased IP in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal IP. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.
B-RAF 是一种丝氨酸/苏氨酸蛋白激酶，有助于胰岛素样生长因子 IGF1 的信号转导。IGF1 的作用包括刺激近端肾小管磷酸盐转运，这在很大程度上是由 Na⁺-偶联磷酸盐共转运体 NaPi-IIa 完成的。相关的 Na⁺-偶联磷酸盐共转运体 NaPi-IIb 在肠道和肿瘤细胞中完成磷酸盐转运。本研究探讨了 B-RAF 是否影响 II 型 Na⁺-偶联磷酸盐共转运体 NaPi-IIa 和 NaPi-IIb 的蛋白丰度和/或活性。将编码野生型 NaPi-IIa 和野生型 NaPi-IIb 的 cRNA 注射到非洲爪蟾卵母细胞中，或在注射编码野生型 B-RAF 的 cRNA 之外进行注射，并通过双电极电压钳测定电致磷酸盐转运。通过共聚焦显微镜观察非洲爪蟾卵母细胞膜上 NaPi-IIa 蛋白的丰度，并通过化学发光进行定量。此外，在 HEK293 细胞中，使用细胞表面蛋白的生物素化和 Western 印迹法定量 B-RAF 抑制剂 PLX-4720 对 NaPi-IIa 细胞表面蛋白丰度的影响。在表达 NaPi-IIa 的非洲爪蟾卵母细胞中，但在注射水的卵母细胞中，向细胞外浴中添加磷酸盐会产生电流 (I P)，而共表达 B-RAF 会显著增加该电流。根据动力学分析，共表达 B-RAF 增强了最大 I P。共表达 B-RAF 进一步增强了非洲爪蟾卵母细胞膜上 NaPi-IIa 蛋白的丰度。用 PLX-4720 处理 HEK293 细胞 24 小时会显著降低 NaPi-IIa 细胞膜蛋白丰度。B-RAF 的共表达进一步显著增加了表达 NaPi-IIb 的非洲爪蟾卵母细胞中的 I P。同样，B-RAF 共表达增强了最大 I P。总之，B-RAF 分别是肾脏和肠道 II 型 Na⁺-偶联磷酸盐共转运体 NaPi-IIa 和 NaPi-IIb 的强大刺激物。
