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2‐D Difference in gel electrophoresis combined with Pro‐Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets
Author(s) -
Orsatti Laura,
Forte Eleonora,
Tomei Licia,
Caterino Marianna,
Pessi Antonello,
Talamo Fabio
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800780
Subject(s) - ezrin , phosphorylation , phosphatase , difference gel electrophoresis , gel electrophoresis , protein tyrosine phosphatase , biology , protein phosphorylation , microbiology and biotechnology , kinase , chemistry , biochemistry , protein kinase a , proteomics , cytoskeleton , cell , gene
The protein tyrosine phosphatase PRL‐3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL‐3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over‐expressing PRL‐3. We used 2‐D difference gel electrophoresis combined with the fluorescence staining Pro‐Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL‐3 over‐expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL‐3‐induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [ Biochim. Biophys. Acta 2008, 1783, 334–344]. The combination of 2‐D difference in gel electrophoresis and Pro‐Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.