Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo

Isabel Rodriguez-Escudero, Teresa Fernandez-Acero, Ignacio Bravo, Nicholas R. Leslie, Rafael Pulido, Maria Molina, Victor J. Cid

Research output: Contribution to journalArticle

8 Citations (Scopus)

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)P-2 in PtdIns(3,4,5)P-3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P-2 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. (C) 2014 Elsevier Inc. All rights reserved.

Original languageEnglish
Pages (from-to)172-179
Number of pages8
JournalMethods
Volume77-78
DOIs
Publication statusPublished - 1 May 2015

Keywords

  • PTEN
  • Lipid phosphatase
  • Saccharomyces cerevisiae
  • Humanized yeast
  • TUMOR-SUPPRESSOR PTEN
  • INTEGRITY SIGNALING PATHWAY
  • PROTEIN-KINASE B/AKT
  • SACCHAROMYCES-CEREVISIAE
  • FUNCTIONAL-ANALYSIS
  • CELLS
  • MUTATIONS
  • STRESS
  • MANNER
  • REPAIR

Cite this

Rodriguez-Escudero, I., Fernandez-Acero, T., Bravo, I., Leslie, N. R., Pulido, R., Molina, M., & Cid, V. J. (2015). Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo. Methods, 77-78, 172-179. https://doi.org/10.1016/j.ymeth.2014.10.020