title: Upregulation of the Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb by B-RAF.
abstract: 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.
authors.affiliation: Department of Physiology I, University of Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Pakladok
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hosseinzadeh
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Lebedeva
authors.forename: Ioana
authors.initials: I
authors.lastname: Alesutan
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D017136:Ion Transport; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D010710:Phosphates; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000378:metabolism*; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D014981:Xenopus
chemical_list: D007211:Indoles; C528407:PLX 4720; D010710:Phosphates; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D013449:Sulfonamides; D048493:Proto-Oncogene Proteins B-raf