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

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dc.contributor.authorRodríguez Escudero, Isabel
dc.contributor.authorFernández Acero, Teresa
dc.contributor.authorBravo, Ignacio
dc.contributor.authorLeslie, Nicholas R.
dc.contributor.authorPulido, Rafael
dc.contributor.authorMolina, María
dc.contributor.authorCid, Víctor J.
dc.date.accessioned2023-06-18T06:45:45Z
dc.date.available2023-06-18T06:45:45Z
dc.date.issued2015-05-01
dc.description.abstractThe 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
dc.description.departmentDepto. de Microbiología y Parasitología
dc.description.facultyFac. de Farmacia
dc.description.refereedTRUE
dc.description.sponsorshipComunidad de Madrid
dc.description.sponsorshipMinisterio de Ciencia e Innovación (MICINN)
dc.description.sponsorshipMinisterio de Educación y Ciencia
dc.description.sponsorshipMinisterio de Economía y Competitividad (MINECO)
dc.description.statusinpress
dc.eprint.idhttps://eprints.ucm.es/id/eprint/31271
dc.identifier.doi10.1016/j.ymeth.2014.10.020
dc.identifier.issn1046-2023
dc.identifier.officialurlhttp://dx.doi.org/10.1016/j.ymeth.2014.10.020
dc.identifier.relatedurlhttp://www.elsevier.com/locate/ymeth
dc.identifier.urihttps://hdl.handle.net/20.500.14352/24074
dc.journal.titleMethods
dc.language.isoeng
dc.page.final179
dc.page.initial172
dc.publisherElsevier
dc.relation.projectIDPROMPT (S2010/BMD-2414)
dc.relation.projectIDBIO2013-44112-P
dc.relation.projectIDSAF2009-10226
dc.relation.projectIDSAF2013-48812-R
dc.rights.accessRightsrestricted access
dc.subject.cdu579
dc.subject.keywordHumanized yeast
dc.subject.keywordLipid phosphatase
dc.subject.keywordPTEN
dc.subject.keywordSaccharomyces cerevisiae
dc.subject.ucmMicrobiología (Farmacia)
dc.subject.unesco3302.03 Microbiología Industrial
dc.titleYeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo
dc.typejournal article
dc.volume.number77-78
dspace.entity.typePublication
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