title: Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo.
abstract: The PTEN phosphoinositide 3-phosphatase is a tumor suppressor commonly targeted by pathologic missense mutations. Subject to multiple complex layers of regulation, its capital role in cancer relies on its counteracting function of class I phosphoinositide 3-kinase (PI3K), a key feature in oncogenic signaling pathways. Precise assessment of the involvement of PTEN mutations described in the clinics in loss of catalytic activity requires either tedious in vitro phosphatase assays or in vivo experiments involving transfection into mammalian cell lines. Taking advantage of the versatility of the model organism Saccharomyces cerevisiae, we have developed different functional assays by reconstitution of the mammalian PI3K-PTEN switch in this lower eukaryote. This methodology is based on the fact that regulated PI3K expression in yeast cells causes conversion of PtdIns(4,5)P2 in PtdIns(3,4,5)P3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P2 pools are essential for the yeast cell, or by using fluorescent reporters amenable for microscopy or flow cytometry. Here we describe the methodology and review its application to evaluate the functionality of PTEN mutations. We show that the technique is amenable to both directed and systematic structure-function relationship studies, and present an example of its use for the study of the recently discovered PTEN-L variant.
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Pza. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: isabelre@farm.ucm.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Rodríguez-Escudero
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Pza. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: teresafe@farm.ucm.es.
authors.forename: Teresa
authors.initials: T
authors.lastname: Fernández-Acero
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Pza. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: igbravo93@gmail.com.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Bravo
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh EH14 4AS, UK. Electronic address: n.r.leslie@hw.ac.uk.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Leslie
authors.affiliation: Biocruces Health Research Institute, Hospital Universitario de Cruces, Plaza Cruces s/n, Barakaldo 48903, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48011, Spain. Electronic address: rafael.pulidomurillo@osakidetza.net.
authors.forename: Rafael
authors.initials: R
authors.lastname: Pulido
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Pza. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: molmifa@ucm.es.
authors.forename: María
authors.initials: M
authors.lastname: Molina
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Pza. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: vicjcid@ucm.es.
authors.forename: Víctor J
authors.initials: VJ
authors.lastname: Cid
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D051059:PTEN Phosphohydrolase / Q000032:analysis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015398:Signal Transduction / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D025521:Tumor Suppressor Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Humanized yeast; Lipid phosphatase; PTEN; Saccharomyces cerevisiae