title: Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.
abstract: In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α inSaccharomyces cerevisiaeleads to toxicity by conversion of essential PtdIns(4,5)P2into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, β, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), 28040 Madrid, Spain.|Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain.
authors.forename: María D
authors.initials: MD
authors.lastname: Oliver
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), 28040 Madrid, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Fernández-Acero
authors.affiliation: Biocruces Health Research Institute, 48903 Barakaldo, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Luna
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), 28040 Madrid, Spain.
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), 28040 Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Molina
authors.affiliation: Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain rpulidomurillo@gmail.com rafael.pulidomurillo@osakidetza.eus vicjcid@ucm.es.|Biocruces Health Research Institute, 48903 Barakaldo, Spain.|IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
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), 28040 Madrid, Spain rpulidomurillo@gmail.com rafael.pulidomurillo@osakidetza.eus vicjcid@ucm.es.
authors.forename: Víctor J
authors.initials: VJ
authors.lastname: Cid
mesh_terms: D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics* / Q000378:metabolism; D006130:Growth Disorders / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006801:Humans; D006934:Hypercalcemia / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D015151:Immunoblotting; D008659:Metabolic Diseases / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D008954:Models, Biological; D009154:Mutation; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D009397:Nephrocalcinosis / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D058543:Class Ia Phosphatidylinositol 3-Kinase
keywords: Phosphatidylinositol 3-kinase; SHORT disease; humanized yeast; oncogenic mutations; p110; p85