Serum- and glucocorticoid-inducible kinase SGK2 regulates human organic anion transporters 4 via ubiquitin ligase Nedd4-2

Abstract

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the kidney and placenta. In the current study, we examined the regulation of hOAT4 by serum- and glucocorticoid-inducible kinase 2 (sgk2) in the kidney COS-7 cells. We showed that sgk2 stimulated hOAT4 transport activity. Such stimulation mainly resulted from an increased cell surface expression of the transporter, kinetically revealed as an increased maximal transport velocity Vmax without significant change in substrate-binding affinity Km. We further showed that regulation of hOAT4 activity by sgk2 was mediated by ubiquitin ligase Nedd4-2. Overexpression of Nedd4-2 enhanced hOAT4 ubiquitination, and inhibited hOAT4 transport activity, whereas overexpression of ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on hOAT4. Our co-immunoprecipitation experiment revealed that sgk2 weakened the association between hOAT4 and Nedd4-2. In conclusion, our study demonstrated for the first time that sgk2 stimulated hOAT4 transport activity by abrogating the inhibition effect of Nedd4-2 on the transporter.

Posttranslational Regulation of Organic Anion Transporters by Ubiquitination: Known and Novel

“…We showed that in contrast to the inhibitory effect of PKC, sgk2 stimulated OAT1 and OAT4 transport activity. Such stimulation mainly resulted from an increased cell surface expression of the transporter, kinetically revealed as an increased maximal transport velocity Vmax without significant change in substrate‐binding affinity Km . We further showed that the regulation of OAT4 activity by sgk2 was mediated by ubiquitin ligase Nedd4‐2.…”

“…In addition to PKC, we recently demonstrated that the serum‐ and glucocorticoid‐inducible kinase 2 (sgk2) also regulates OAT activity via Nedd4‐2 . The sgk family of protein kinases has three isoforms: sgk1, sgk2, and sgk3 that are involved in controlling diverse cellular processes including sodium Na + homeostasis, osmoregulation, cell survival, and cell proliferation .…”

“…Our co‐immunoprecipitation experiment revealed that sgk2 weakened the association between OAT4 and Nedd4‐2. Therefore, sgk2 stimulates OAT4 transport activity by abrogating the inhibition effect of Nedd4‐2 on the transporter …”

SGK1/Nedd4–2 signaling pathway regulates the activity of human organic anion transporters 3

“…The detailed procedure has been reported previously (Wang et al, ; Xu et al, ). The cells were lysed with lysis buffer and then precleared with protein G‐agarose beads (Pierce, Rockford, IL) at 4°C for 2 h to reduce nonspecific binding.…”

“…In contrast, sgk2 is insensitive to such regulation. Our laboratory recently demonstrated that sgk2 plays a significant role in the regulation of OATs (Wang, Xu, Toh, Pao, & You, ; Xu et al, ). However, the role of sgk1 in OAT function has not been explored.…”