POSH stimulates the ubiquitination and the clathrin-independent endocytosis of ROMK1 channels

Abstract

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its terminus. To study the effect of POSH on ROMK1 channels, measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-α, β, and γ subunits. Moreover, POSH still decreased potassium current in oocytes injected with a ROMK1 mutant (R1Δ373-378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHΔRING, which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHΔRING diminished its ubiquitination HEK293T cells. The notion that POSH may serve as an ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.