کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8340640 | 1541247 | 2015 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo
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کلمات کلیدی
PI3KmTORPDK1CWIMAPK - MAPKPtdIns(4,5)P2 - PtdIns (4،5) P2phosphatidylinositol (4,5)-bisphosphate - فسفاتیدیلینواستول (4،5) -بیز فسفاتphosphoinositide 3-kinase - فسفینوزیتید 3-کینازmammalian target of rapamycin - هدف پستانداران رپامایسینPleckstrin Homology - همخوانی Pleckstrinmitogen-activated protein kinase - پروتئین کیناز فعال با mitogenphosphoinositide-dependent kinase - کیناز وابسته به فسفوئینوزیتهGal - گالGalactose - گالاکتوزمی cell wall integrity - یکپارچگی دیواره سلولی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volumes 77â78, 1 May 2015, Pages 172-179
Journal: Methods - Volumes 77â78, 1 May 2015, Pages 172-179
نویسندگان
Isabel RodrÃguez-Escudero, Teresa Fernández-Acero, Ignacio Bravo, Nicholas R. Leslie, Rafael Pulido, MarÃa Molina, VÃctor J. Cid,