Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE
Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.
Key wordsProteomics Phosphoproteomics Immobilized metal affinity chromatography 2D-DIGE LC-MS/MS Phos-tag Phosphorylation ERK MAP kinase
We thank Megumi Kawano, Mayumi Kajimoto, and Junya Yabuno for experimental assistance, Mayumi Iwata for secretarial assistance, Dr. Maria Tsoumpra for helpful advice, and Dr. Naoki Tani for mass spectrometry analysis. This work was supported by JSPS KAKENHI Grant Numbers 23570231 and 26440101, and the program of the Joint Usage/Research Center for Developmental Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University to H.K.
- 11.Carlson SM, Chouinard CR, Labadorf A et al (2011) Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3. Sci Signal 4:rs11Google Scholar