Mass spectrometric analysis of novel phosphorylation sites in the TRPC4β channel

Authors


K.-S. Park, Department of Physiology, Kyung Hee University School of Medicine, Hoeki-dong, Dondaemun-gu, Seoul 130–701, South Korea.

E-mail: kspark@khu.ac.kr

Abstract

RATIONALE

The transient receptor potential canonical (TRPC) channel 4β is a non-selective cation channel that is regulated by intracellular Ca2+ 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.

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