title: Investigation of POPX2 phosphatase functions by comparative phosphoproteomic analysis.
abstract: Identifying the substrates and biochemical pathway regulated by phosphatases has always been more challenging than finding those regulated by kinases. Here, we report the use of phosphoproteomic methods to analyse the pathways regulated by POPX2 (partner of PIX 2) phosphatase. POPX2 is a serine/threonine phosphatase, found in many cancer types. The levels of the POPX2 have been found to be up-regulated in the more invasive breast cancer cells compared with non-invasive ones. Our observations also suggest that POPX2 level is positively correlated with cell motility. Thus, finding substrates or pathways regulated by POPX2 will help to elucidate the regulatory mechanism of cancer cell motility and invasiveness. We have also developed and validated a protocol using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to enrich the phosphopeptides followed by LC-MS/MS to allow comparison between the phosphoproteomes of control and POPX2 overexpressing cells. With this approach, we were able to identify a biochemical pathway through which POPX2 exerts its apparent cellular function: the regulation of activity of glycogen synthase kinase-3, which in turn modulates extracellular signal-regulated kinase and cell motility.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
authors.forename: Pritpal
authors.initials: P
authors.lastname: Singh
authors.forename: Chee Sian
authors.initials: CS
authors.lastname: Gan
authors.forename: Tiannan
authors.initials: T
authors.lastname: Guo
authors.forename: Hui-Qun
authors.initials: HQ
authors.lastname: Phang
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
authors.forename: Cheng Gee
authors.initials: CG
authors.lastname: Koh
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods*; D030562:Databases, Protein; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000201:enzymology / Q000473:pathology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D007477:Ions; D010750:Phosphoproteins; D020543:Proteome; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; C459194:PPM1F protein, human; D010749:Phosphoprotein Phosphatases