Isabel Rodríguez-Escudero

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast

The mammalian signalling pathway involving class I PI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tep1). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidylinositol 4,5-bisphosphate) and its conversion into PIP3 (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr308 and Ser473. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.

Modulation of host cytoskeleton function by the enteropathogenic Escherichia coli and Citrobacter rodentium effector protein EspG.

ABSTRACT EspG is a conserved protein encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli and Citrobacter rodentium. EspG is delivered into infected host cells by a type III secretion system. The role of EspG in virulence has not yet been defined. Here we describe experiments that probe the virulence characteristics and biological activities of EspG in vitro and in vivo. A C. rodentium espG mutant displayed a significantly reduced ability to colonize C57BL/6 mice and to cause colonic hyperplasia. Epitope-tagged EspG was detected in the apical regions of infected colonic epithelial cells in infected mice, partially localizing with another LEE-encoded effector protein, Tir. EspG was found to interact with mammalian tubulin in both genetic screens and gel overlay assays. Binding to tubulin by EspG caused localized microtubule depolymerization, resulting in actin stress fiber formation through an undefined mechanism. Heterologous expression of EspG in yeast resulted in loss of cytoplasmic microtubule structure and function, preventing coordination between bud development and nuclear division. Yeast expressing EspG were also unable to control cortical actin polarity. We suggest that EspG contributes to the ability of A/E pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the Shigella flexneri VirA protein.

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells.

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C‐terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5‐bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118–142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N‐terminal region and independent of its phosphatase activity.

Interaction of the Salmonella Typhimurium effector protein SopB with host cell Cdc42 is involved in intracellular replication

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non‐activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42‐interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella‐containing vacuole was reduced compared with wild‐type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB–Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment.

In vivo Functional Analysis of the Counterbalance of Hyperactive Phosphatidylinositol 3-Kinase p110 Catalytic Oncoproteins by the Tumor Suppressor PTEN

The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110alpha and p110beta in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110alpha or p110beta mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.

Addressing the effects of Salmonella internalization in host cell signaling on a reverse‐phase protein array

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island‐1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re‐programming host cell pathways, we used ZeptoMARK reverse‐phase protein array technology, which allows printing 32‐sample lysate arrays that can be analyzed with phospho‐specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS‐deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen‐activated protein kinases, mainly dependent on SopE/E2, as well as SigD‐dependent phosphorylation of PKB/Akt and its targets GSK‐3β and FKHR/FoxO. This is the first time that reverse‐phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.

A yeast-based in vivo bioassay to screen for class I phosphatidylinositol 3-kinase specific inhibitors.

The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS.

Phosphatidylinositol 3-Kinase-dependent Activation of Mammalian Protein Kinase B/Akt in Saccharomyces cerevisiae, an in Vivo Model for the Functional Study of Akt Mutations

In animal cells, Akt (also called protein kinase B) is activated by stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is a major effector for eliciting responses that support cell growth and survival. We have shown previously that co-expression of Akt1 in budding yeast (Saccharomyces cerevisiae) along with hyperactive p110α, the catalytic subunit of mammalian phosphatidylinositol 3-kinase, results in Akt1 relocalization to cellular membranes and activation. In the present study, we show that activation of all three mammalian Akt isoforms by wild-type p110α causes deleterious effects on yeast cell growth. Toxicity of Akt in S. cerevisiae required its catalytic activity, its pleckstrin homology domain, and phosphorylation of its activation loop, but not phosphorylation of its hydrophobic motif. We demonstrate that expression in yeast of the only purported oncogenic allele, Akt1(E17K), leads to enhanced phenotypes. Ala-scanning mutagenesis of the VL1 region within the phosphatidylinositol 3,4,5-trisphosphate-interacting pocket of the Akt1 pleckstrin homology domain revealed that most residues in this region are essential for Akt1 activity. We found that active Akt leads to enhanced signaling through the yeast cell wall integrity pathway. This effect requires the upstream Rho1 activator Rom2 and involves both phosphorylation of the MAPK Slt2 and expression of its transcriptional targets, thus providing a quantitative reporter system for heterologous Akt activity in vivo. Collectively, our results disclose a heterologous yeast system that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.

A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity

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.