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 of 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 N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or 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 in 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-alpha, beta, and gamma subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Delta373-378), 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 POSHDeltaRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHDeltaRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 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 by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.

FIGURE 1.

Confocal images demonstrating the immunostaining of POSH and AQP2 in the cortex of the rat kidney.

FIGURE 2.

ROMK channels are associated with POSH. A, a Western blot demonstrating that ROMK channels are immunoprecipitated with POSH in the rat kidney. B, a GST pulldown experiment shows that GST-POSH fusion protein is able to pull down ROMK1 (left panel) and that GST-ROMK protein is also able to associate with POSH (right panel). GST-POSH or GST-ROMK1 was bound to glutathione-Sepharose beads and was incubated with either 10 μl of purified ROMK1 protein or POSH protein labeled with [³⁵S]methionine in NETN buffer. After washing, the bound proteins were resolved by 10% SDS-PAGE followed by autoradiography. C, GST pulldown experiments showing the association between GST-POSH and ROMK1. D, a Western blot showing that POSH is specifically immunoprecipitated with FLAG-tagged ROMK1 in 293T cells transfected with FLAG-R1+POSH but not in cells transfected with POSH alone. Two bands on the left side of the gel are molecular size markers. IP, immunoprecipitation; IB, immunoblot.

FIGURE 3.

POSH associates with the N terminus of ROMK1. The top panel is a Western blot showing that POSH is immunoprecipitated with FLAG-tagged ROMK1 or FLAG-tagged ROMK1-N terminus (FLAGR1-N) but not with FLAG-tagged ROMK1 C terminus (FLAGR1-C). The middle panel shows the expression of POSH in 293T cells. The bottom three panels demonstrate the expression of FLAG-tagged ROMK1(R1), FLAGR1-C, and FLAGR1-N, respectively. IP, immunoprecipitation; IB, immunoblot.

FIGURE 4.

POSH inhibits ROMK1 channels. A, effect of POSH on potassium currents measured with the perforated whole cell recording in HEK293 cells transfected with GFP-ROMK1 and POSH at different concentrations. B, a bar graph summarizes the results of the effect of POSH on ROMK1 channels (n = 4). C, a Western blot showing the expression of ROMK1 and POSH in HEK293 transfected with GFP-ROMK1 and POSH at different concentrations. The asterisk indicates significant different (p < 0.05) in comparing with the control (ROMK1 alone).

FIGURE 5.

Effect of POSH on ROMK1 channel activity and expression. A, effect of POSH on potassium current in oocytes injected with ROMK1 and POSH at different concentrations. The asterisks indicate a significant difference between control (without POSH) and POSH-injected oocytes. The experimental numbers are at least 11 for each condition. B, a confocal image showing the effect of POSH on the surface expression of GFP-ROMK1 in oocytes. C, POSH decreases the surface expression of ROMK1 channels in HEK293T cells transfected with POSH+GFP-ROMK1 or GFP-ROMK1 alone. Sulfo-NHS-SS biotin was used to label the surface ROMK1, and 50 μg of protein from cell lysates was used for the experiments. D, biotin labeling experiments showing the effect of increased POSH expression on the ROMK1 expression in plasma membranes of HEK293T cells. A bar graph summarizing the results from four experiments is shown on the bottom. IP, immunoprecipitation; IB, immunoblot.

FIGURE 6.

POSH stimulates dynamin (Dyn)-dependent endocytosis of ROMK1. A, a whole cell potassium currents measured with the perforated whole cell patch recording in HEK293T cells transfected with GFP-ROMK1(R1), R1+POSH, R1+wtDyn, R1+wtDyn+POSH, R1+DynK44A, and R1+DynK44A+POSH (n = 4). The voltage protocol is the same as shown in Fig. 4 (from −100 to 60 mV). B, the potassium currents at −100 mV measured in different experiments are summarized in a bar graph. An asterisk indicates significant different (p < 0.05) in comparison with the control (ROMK1 alone). C, a Western blot shows the expression of corresponding proteins in all groups.

FIGURE 7.

POSH stimulates the endocytosis of ROMK1. A, effect of POSH on potassium currents in oocytes injected with ROMK1 (R1)+POSH, R1+dominant negative dynamin (DynK44A) +POSH, and POSH+R1Δ373–378. B, effect of POSH on amiloride-sensitive sodium current in oocytes injected with ENaC (2 ng of α, 1 ng of β, and 1 ng of γ subunit).

FIGURE 8.

The RING domain is required for the effect of POSH. A, diagram shows a schematic structure of POSH. R, RING domain; SH, SH3 domain. The number above or below the corresponding domain indicates the deletion of amino acids in the constructs tested. B, effect of POSH and the truncated POSH on potassium current in oocytes injected with ROMK1 (R1), R1+POSH, and R1+truncated POSH. An asterisk indicates a significant difference in comparison with the control value (no POSH). **, p < 0.01; *, p < 0.05. The number in parentheses under each bar is the experimental number for each group. C, a Western blot demonstrating the association of ROMK1 and POSH or truncated POSH. IP, immunoprecipitation; IB, immunoblot.

FIGURE 9.

POSH stimulates ubiquitination of ROMK. A, in vitro ubiquitination assay shows the effect of POSH on ROMK1 ubiquitination. ROMK proteins (generated with TnT quick coupled transcription/translation system) were incubated at 37 °C for 30 min with exogenous ubiquitin, ATP, E1 and E2 ubiquitin ligase (UbcH5a) in the presence or absence of His-tagged POSH. After the reaction ROMK proteins were resolved by electrophoresis and Western blotting with either ROMK or ubiquitin antibodies. B, a Western blot shows the effect of POSH or POSHΔRING on ROMK1 ubiquitination. 293T cells were transfected with ROMK1 (R1), R1+POSH, or R1+ POSHΔRING. MG132, an inhibitor of proteosome was added during experiments. IP, immunoprecipitation; IB, immunoblot.

FIGURE 10.

A scheme illustrating the proposed mechanism by which POSH induces the ubiquitination and hence the inhibition of ROMK channels.