• YAKHAK HOEJI
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• v.52 no.5
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• pp.407-411
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• 2008

Phosphorylation plays the most important role in cell signaling mechanism. Various methods to identify the phosphorylation sites of proteins using tandem mass spectrometry (MS/MS) have been reported recently. Furthermore, the enrichment strategy such as Titanium dioxide ($TiO_2$) method should be combined with MS/MS analysis to effectively identify phosphorylation sites. It is necessary to optimize phosphopeptide-enrichment strategy, $TiO_2$ method in this study, due to the low amount of phosphorylated form followed by analyzing them by MS/MS. To evaluate the several conditions to enrich phosphopeptides using $TiO_2$ method, we used ${\apha}$-casein as a standard phosphoprotein and analyzed a representative phosphopeptide (VPQLEIVPNpSAEER) peak of MS spectrum. Batch is better than column method for binding and 300 g/l DHB in loading buffer is better than lower concentration of DHB. 3% TFA and pH 10.5 shows high efficiency of phosphopeptide-enrichment for washing and elution steps, respectively. Finally we identified various efficient conditions of phosphopeptide-enrichment method using $TiO_2$. This optimized method would assist in reliable identifying thousands of phosphorylation sites existed in low abundance from various complex proteins.

• BMB Reports
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• v.34 no.6
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• The Plant Pathology Journal
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• v.26 no.1
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• pp.1-7
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• 2010

Phosphorylation is a major post-translational modification of proteins that regulate diverse signal transduction pathways in eukaryotic cells. 14-3-3 proteins are regulatory proteins that bind to target proteins in a phosphorylation-dependent manner and have been shown to play an important role in plant growth and development, primary metabolism, and signal transduction. Because phosphorylation plays a critical role in signal transduction pathways to trigger plant immunity, involvement of 14-3-3 proteins in plant immunity has been suggested for a long time. Recent studies have provided new evidence to support a role for 14-3-3 proteins in plant immunity. This review will briefly discuss general characteristics of 14-3-3 proteins and their involvement in plant immunity.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.544-546
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• 2000

Grb2 is an important adaptor protein in the mitogenic Ras signaling pathway of receptor tyrosine kinases, and contains one SH2 domain and two SH3 domains. The SH2 domain binds to specific phosphotyrosine motifs on receptors or adaptor proteins such as Shc. The SH2 domain antagonists may lead to blocking of the oncogenic Ras signals and to developing new antitumor agents. In the course of screening SH2 antagonists from natural sources, cslerotiorin (1) and isochromophilone IV (2) were isolated from a strain, Penicillium multicolor F1753, and their structures were established by NMR spectral data. The metabolites significantly inhibited the binding between the Grb2-SH2 domain and phosphopeptide derived from the Shc protein, with $IC_{50}$ values of $22{\;}\mu\textrm{M}{\;}and{\;}48{\;}\mu\textrm{M}$ for (1) and (2), respectively. The compounds are the first nonpeptidic inhibitors of the SH2 domain from a natural source.