Browsing by Author "Pulido, Rafael"

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    A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.
    (2015-04-15) Gil, Anabel; Rodríguez Escudero, Isabel; Stumpf, Miriam; Molina Martín, María; Jiménez Cid, Víctor; Pulido, Rafael
    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.
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    A pathogenic role for germline PTEN variants which accumulate into the nucleus.
    (Springer Nature, 2018-04-30) Mingo, Janire; Rodríguez Escudero, Isabel; Luna, Sandra; Fernández Acero, Teresa; Amo, Laura; Jonasson, Amy R; Zori, Roberto T; López, José I; Molina, María; Cid, Víctor J.; Pulido, Rafael
    The PTEN gene encodes a master regulator protein that exerts essential functions both in the cytoplasm and in the nucleus. PTEN is mutated in the germline of both patients with heterogeneous tumor syndromic diseases, categorized as PTEN hamartoma tumor syndrome (PHTS), and a group affected with autism spectrum disorders (ASD). Previous studies have unveiled the functional heterogeneity of PTEN variants found in both patient cohorts, making functional studies necessary to provide mechanistic insights related to their pathogenicity. Here, we have functionally characterized a PTEN missense variant [c.49C>G; p.(Gln17Glu); Q17E] associated to both PHTS and ASD patients. The PTEN Q17E variant displayed partially reduced PIP3-catalytic activity and normal stability in cells, as shown using S. cerevisiae and mammalian cell experimental models. Remarkably, PTEN Q17E accumulated in the nucleus, in a process involving the PTEN N-terminal nuclear localization sequence. The analysis of additional germline-associated PTEN N-terminal variants illustrated the existence of a PTEN N-terminal region whose targeting in disease causes PTEN nuclear accumulation, in parallel with defects in PIP3-catalytic activity in cells. Our findings highlight the frequent occurrence of PTEN gene mutations targeting PTEN N-terminus whose pathogenicity may be related, at least in part, with the retention of PTEN in the nucleus. This could be important for the implementation of precision therapies for patients with alterations in the PTEN pathway.
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    A unified nomenclature and amino acid numbering for human PTEN.
    (American Association for the Advancement of Science, 2014-07-01) Pulido, Rafael; Baker, Suzanne J.; Barata, Joao T.; Carracedo, Arkaitz; Cid, Víctor J.; Chin-Sang, Ian D.; Davé, Vrushank; Hertog, Jeroen den; Devreotes, Peter; Eickholt, Britta J.; Eng, Charis; Furnari, Frank B.; Georgescu, María Magdalena; Gericke, Arne; Hopkins, Benjamin; Jiang, Xeujun; Lee, Seung Rock; Lösche, Mathias; Malaney, Prerna; Matias Guiu, Xavier; Molina, María; Pandolfi, Pier Paolo; Parsons, Ramon; Pinton, Paolo; Rivas, Carmen; Rocha, Rafael M.; Rodríguez, Manuel S.; Ross, Alonzo H.; Serrano, Manuel; Stambolic, Vuk; Stiles, Bangyan; Suzuki, Akira; Tan, Seong Seng; Tonks, Nicholas K.; Trotman, Lloyd C.; Wolff, Nicolas; Woscholski, Rudiger; Wu, Hong; Leslie, Nicholas R.
    The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.
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    Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L Subcellular Localization and Function
    (MDPI, 2019-11-26) Fernández Acero, Teresa; Bertalmio, Eleonora; Luna, Sandra; Mingo, Janire; Bravo Plaza, Ignacio; Rodríguez Escudero, Isabel; Molina Martín, María; Pulido, Rafael; Jiménez Cid, Víctor
    The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP3 counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP2-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation.
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    Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo
    (Elsevier, 2015-05-01) Rodríguez Escudero, Isabel; Fernández Acero, Teresa; Bravo, Ignacio; Leslie, Nicholas R.; Pulido, Rafael; Molina, María; Cid, Víctor J.
    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