title: 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.
abstract: 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.
authors.affiliation: IRBM P. Angeletti, Pomezia, Italy. laura_orsatti@merck.com
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.forename: Eleonora
authors.initials: E
authors.lastname: Forte
authors.forename: Licia
authors.initials: L
authors.lastname: Tomei
authors.forename: Marianna
authors.initials: M
authors.lastname: Caterino
authors.forename: Antonello
authors.initials: A
authors.lastname: Pessi
authors.forename: Fabio
authors.initials: F
authors.lastname: Talamo
mesh_terms: D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D009942:Organometallic Compounds; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D009363:Neoplasm Proteins; D009942:Organometallic Compounds; D020652:Peptide Elongation Factor 2; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D011494:Protein Kinases; D017027:Protein Tyrosine Phosphatases; D000099149:Ezrin; C515601:Sypro Ruby; C484730:PTP4A3 protein, human