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Mass spectrometric analysis of novel phosphorylation sites in the TRPC4β channel
Author(s) -
Lee Ji Eun,
Song MinYoung,
Shin Seok Kyo,
Bae Seong Han,
Park KangSik
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6305
Subject(s) - phosphorylation , trpc , transient receptor potential channel , chemistry , hek 293 cells , tyrosine phosphorylation , protein phosphorylation , microbiology and biotechnology , receptor , biochemistry , biology , protein kinase a
RATIONALE The transient receptor potential canonical (TRPC) channel 4β is a non‐selective cation channel that is regulated by intracellular Ca 2+ and G protein‐coupled receptors. Tyrosine phosphorylation of TRPC4β is important in mediating the activity and membrane expression of this channel protein. However, studies of TRPC4β Ser/Thr phosphorylation are lacking. METHODS To investigate the phosphorylation sites involved in regulating the diverse functions of TRPC4β in mammalian cells, we used nano‐liquid chromatography/tandem mass spectrometry to identify key phosphorylation sites in TRPC4β that was immunopurified from HEK293 cells with monoclonal anti‐TRPC4β antibody. RESULTS We identified four phosphorylation sites in the C‐terminus of TRPC4β, none of which had been previously reported. Our data show that TRPC4β in mammalian cells is highly phosphorylated under basal conditions at multiple sites, and that a mass spectrometric proteomic technique combined with antibody‐based affinity purification is an effective approach to define the phosphorylation sites of TRPC4β channels in mammalian cells. CONCLUSIONS These novel phosphorylation sites on TRPC4β may play a potential role in the phosphorylation‐mediated regulation of TRPC4β channel activity and function in mammalian cells. Copyright © 2012 John Wiley & Sons, Ltd.