Identification of β‐Catenin Interacting Proteins in the Nuclei of Native Rat Renal Inner Medullary Collecting Duct Cells

Abstract

The gene encoding the aquaporin‐2 water channel (AQP2) is regulated transcriptionally in response to vasopressin. Vasopressin increases phosphorylation of many proteins in collecting duct cells including AQP2 and β‐catenin (at Serine‐552), a multifunctional protein that can function as a transcriptional co‐regulator in the nucleus. β‐catenin phosphorylation is associated with translocation of β‐catenin from the cytoplasm to the nucleus of collecting duct cells where it may bind to transcription factors and other regulatory proteins. The purpose of this study was to identify β‐catenin interacting proteins in the nuclei of inner medullary collecting duct (IMCD) cells isolated from rat kidneys using chromatin immunoprecipitation coupled to mass spectrometry (ChIP‐MS). Samples were analyzed from rat IMCD suspensions treated either with the vasopressin analog dDAVP or its vehicle for 30 minutes in vitro. After crosslinking and nuclear isolation, chromatin immunoprecipitation was performed using a well characterized β‐catenin antibody (or with pre‐immune IgG). The co‐immunoprecipitated proteins were identified by protein mass spectrometry (n=4 for both dDAVP and control). We reproducibly identified 43 β‐catenin binding proteins present with β‐catenin antibody but not pre‐immune IgG, which included a number of known β‐catenin binding partners as well as many novel interacting proteins. There were no sequence‐specific transcription factors, but there were multiple proteins known to be involved in transcriptional regulation (Jup, Tdrd3, Lgr4, Cdh1, Cenpj, Taf1, and several histones). Binding of some proteins to β‐catenin changed in response to dDAVP (Minimum Bayes' Factor < 0.01) (Plec [down], Cdh1 [up], Krt4 [up], Lrba [up], Sptbn2 [up], and Tenm3 [up]). These findings provide key information necessary for modeling the transcriptional response to vasopressin. [JH supported by APS Undergraduate Summer Research Fellowship Program.]Support or Funding InformationThe work was supported by the APS Undergraduate Summer Research Fellowship and NHLBI Intramural Research Program (project ZIA‐HL001285 and ZIA‐HL006129, M.A.K.).