Question:

B-RAF, a serine/threonine protein kinase, is integrated in the signalling of insulin like growth factor IGF-1. Renal effects of IGF-1 include stimulation of proximal renal tubular phosphate transport, which is accomplished in large part by the Na⁺-coupled phosphate transporter NaPiIIa. The present study thus explored whether B-RAF influences the activity and protein abundance of the Na⁺-coupled phosphate transporter NaPiIIa.

Methods:

cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional co-injection of cRNA encoding wild-type B-RAF. Electrogenic phosphate transport was determined by dual electrode voltage clamp and NaPiIIa protein abundance in the cell membrane was quantified by chemiluminescence experiments.

Results:

In NaPiIIa-expressing Xenopus oocytes but not in oocytes injected with water, the addition of phosphate to extracellular bath generated a current (I_P), which was significantly increased following co-expression of wild-type B-RAF. According to kinetic analysis, co-expression of B-RAF enhanced the maximal current without significantly modifying the current required for half maximal current. According to chemiluminescence experiments, B-RAF enhanced the Na⁺-coupled phosphate transporter NaPiIIa protein abundance in the cell membrane. Exposure of the Xenopus oocytes to Brefeldin A (5 µM), an inhibitor of vesicle insertion, was followed by a decline of I_P, which was similar in Xenopus oocytes co-expressing NaPiIIa and B-RAF and in Xenopus oocytes expressing NaPiIIa alone.

Conclusions:

B-RAF is a powerful stimulator of the Na⁺-coupled phosphate transporter NaPiIIa.