RT Journal Article
T1 Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo
A1 Rodríguez Escudero, María Isabel
A1 Fernández-Acero Bascones, Teresa
A1 Bravo, Ignacio
A1 Leslie, Nicholas R.
A1 Pulido, Rafael
A1 Molina, María
A1 Jiménez Cid, Víctor
AB 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
PB Elsevier
SN 1046-2023
YR 2015
FD 2015-05-01
LK https://hdl.handle.net/20.500.14352/24074
UL https://hdl.handle.net/20.500.14352/24074
LA eng
NO Rodríguez Escudero, I., Fernández-Acero Bascones, T., Bravo, I. et al. «Yeast-Based Methods to Assess PTEN Phosphoinositide Phosphatase Activity in Vivo». Methods, vol. 77-78, mayo de 2015, pp. 172-79. DOI.org (Crossref), https://doi.org/10.1016/j.ymeth.2014.10.020.
NO Comunidad de Madrid
NO Ministerio de Ciencia, Innovación y Universidades (España)
NO Ministerio de Educación, Formación Profesional y Deportes (España)
NO Ministerio de Economía, Comercio y Empresa (España)
DS Docta Complutense
RD 10 abr 2025