2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets

Laura Orsatti*, Eleonora Forte, Licia Tomei, Marianna Caterino, Antonello Pessi, Fabio Talamo

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

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 overexpression 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 identifi-cation of kinase/phosphatase targets.

Original languageEnglish (US)
Pages (from-to)2469-2476
Number of pages8
JournalELECTROPHORESIS
Volume30
Issue number14
DOIs
StatePublished - Jul 1 2009

Keywords

  • 2-D difference in gel electrophoresis
  • Elongation factor 2
  • Ezrin
  • PRL-3
  • Phosphorylation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Fingerprint Dive into the research topics of '2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets'. Together they form a unique fingerprint.

Cite this