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Proteomic‐based identification of upstream intermediates of Na+i,K+i‐independent death signaling triggered by interaction of cardiotonic steroids with alpha‐subunit of Na,K‐ATPase
Author(s) -
Akimova Olga A,
Hamet Pavel,
Orlov Sergei N
Publication year - 2007
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.21.5.a534
Subject(s) - ouabain , signal transduction , g alpha subunit , chemistry , atpase , microbiology and biotechnology , protein subunit , western blot , receptor , immunoprecipitation , phosphatase , biology , phosphorylation , biochemistry , enzyme , sodium , organic chemistry , gene
During the last decade, it was shown that side‐by‐side with a major role in regulation of Na + i /K + i ratio, interaction of the Na,K‐ATPase with ouabain and other cardiotonic steroids (CTS) triggers diverse Na + i ,K + i ‐ independent signaling involved in cell proliferation and death. Thus, we demonstrated that chronic exposure to CTS results in massive death of renal epithelial, vascular endothelial and colon epithelial (Caco‐2) cells via their interaction with Na,K‐ATPase α‐subunit but independently of elevation of [Na + ] i /[K + ] i ratio. We designed this study to identify upstream intermediates of this novel CTS‐induce Na + i ,K + i ‐ independent signaling pathway using proteomic‐based approaches. We used cell lysates from control and ouabain‐treated Caco‐2 cells for co‐immunoprecipitation with anti‐α1 Na,K‐ATPase antibodies following by separation with 2D‐PAGE. From comparative analyse we detected more than 20 proteins sports whose interaction with Na,K‐ATPase was triggered by ouabain. 18 proteins were identified by mass spectrometry, including 8 signaling proteins from superfamilies of glucocorticoid receptors, Ser/Thr protein kinases and 2C phosphatases, Src and Rho GTPases. The role of these proteins in ouabain‐induced Na + i , K + i ‐ independent cell death signaling is currently under investigation. Supported by grants from the Canadian Institute of Health Research and the Kidney Foundation of Canada .