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POSH (Plenty of SH3) is an E3 ligase of ROMK (Kir1.1) channels in cortical collecting duct (CCD)
Author(s) -
Pan Chunyang,
Lin Daohong,
Yue Peng,
Wang Wenhui
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.998.40
Subject(s) - ubiquitin ligase , chemistry , microbiology and biotechnology , ubiquitin , transfection , potassium channel , immunoprecipitation , immunostaining , biology , biophysics , biochemistry , gene , immunology , immunohistochemistry
The aim of the present study is to explore the role of POSH in the regulating ROMK channels. Immunostaining shows that POSH is expressed in the CCD. Moreover, GST pull‐down, immunoprecipitation and in vitro protein chemistry studies have demonstrated that POSH is associated with ROMK channels in its N‐terminus. Co‐expression of POSH decreased K current in a dose‐dependent manner in oocytes injected with ROMK1 channels. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit Na channels in oocytes injected with ENaC‐alpha, beta and gamma subunits. Surface labeling with biotin shows that POSH decreased the membrane expression of ROMK channels in 293T cell transfected with both. The ubiquitinlation assay has further demonstrated that POSH enhanced the ubiquitinlation of ROMK channels in 293T cells transfected with ROMK1 and POSH. The notion that POSH may act as an E3 ubiquitinligase of ROMK is also indicated by in vitro ubiquitin assay in which adding POSH increased the ubiquitination of ROMK1. Finally, ubiquitinlation of ROMK1 is absent in cells transfected with truncated POSH in which RING domain was deleted. We conclude that POSH is an E3 ubiquitin ligase for ROMK channels.