The role of ubiquitination of the type IIa sodium phosphate cotransporter (Npt2a) in protein trafficking

Abstract

In the apical membrane of OK cells, Npt2a is associated with the PTH receptor, PKA, AKAP 79/150, NHERF, ezrin, and protein phosphatase 2a. PTH causes phosphorylation of several apical membrane proteins and ubiquitination of Npt2a We hypothesized that PTH‐stimulated phosphorylation of associated proteins was required for dissociation of Npt2a from the protein complex and that ubiquitination was required for Npt2a trafficking to the endosome. To address this hypothesis, we treated OK cells with vehicle or PTH, compared Npt2a‐associated proteins by immunoprecipitation (IP), protein phosphorylation, and Npt2a trafficking. PTH resulted in a rapid (within 15 min) expression of Npt2a in late endosomes (rab7, mannose6phosphate receptor positive), but not early endosomes (rab5 positive). Removal of PTH resulted in insertion of new proteins, not recycled Npt2a, into the apical membrane. Gel filtration of vehicle and PTH‐treated OK cell lysates demonstrated a shift in the fractions expressing NHERF and Npt2a. IP studies showed decreased association of Npt2a with NHERF, ezrin, RII, and the PTH receptor and decreased association of the PTH receptor with NHERF. In contrast, Npt2a continued to co‐associate with AKAP. PTH stimulated ubiquitination of Npt2a and association with VCP, a chaperone for ubiquitinated proteins. Under basal conditions, Npt2a degradation was blocked by inhibitors of either lysosomal or proteasomal degradation, while PTH‐stimulated Npt2a degradation was resistant to proteasomal inhibitors. We conclude that PTH stimulates endocytosis of Npt2a by ubiquitination of Npt2a, resulting in release of Npt2a from its anchoring complex in the apical membrane and targeting to the late endosomal fraction for degradation.