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Cancer Research 67, 9731, October 15, 2007. doi: 10.1158/0008-5472.CAN-07-1278
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

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

Amparo Andrés-Pons1, Isabel Rodríguez-Escudero2, Anabel Gil1, Ana Blanco3, Ana Vega3, María Molina2, Rafael Pulido1 and Víctor J. Cid2

1 Centro de Investigación Príncipe Felipe, Valencia, Spain; 2 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; and 3 Unidad de Medicina Molecular, Fundación Pública Galega de Medicina Xenómica-SERGAS, Grupo de Medicina Xenómica-CIBERER, Santiago de Compostela, Spain

Requests for reprints: Rafael Pulido, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16-3, Valencia, Spain 46013. Phone: 34-96-3289680, ext. 2004; Fax: 34-96-3289701; E-mail: rpulido{at}cipf.es and Víctor J. Cid, Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Pza. de Ramón y Cajal s/n, Madrid 28040, Spain. Phone: 34-91-3941888; Fax: 34-91-3941745; E-mail: vicjcid{at}farm.ucm.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
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 p110{alpha} and p110ß 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 p110{alpha} or p110ß 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. [Cancer Res 2007;67(20):9731–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Alterations in the phosphatidylinositol 3-kinase (PI3K)/PTEN signal transduction pathway account for the etiology of a large number of tumors in mammals, making this route a major target for intervention in human cancer (1–3). Class I PI3Ks are proto-oncogenic enzymes that synthesize the second messengers PI(3,4)P2 and PI(3,4,5)P3 [hereafter phosphatidylinositol-(3,4,5)-trisphosphate (PIP3)] from PI(4)P and PI(4,5)P2 [hereafter phosphatidylinositol 4,5-bisphosphate (PIP2)] phosphoinositides. On the other hand, the tumor suppressor phosphatase PTEN counteracts the activity of class I PI3Ks, dephosphorylating the 3-position of the PI3K-synthesized phosphoinositides. The transient accumulation of PIP3 at specific plasma membrane compartments triggers the recruitment and activation of protein kinase effectors, such as the product of the proto-oncogene product protein kinase B (PKB)/Akt, which ultimately leads to proliferative and survival cell responses (4, 5). Thus, a coordinated regulation of PI3K and PTEN activities must exist in cells that tightly controls the synthesis and degradation of PIP3 during cell adaptive responses. Class I PI3Ks are heterodimeric enzymes containing a catalytic subunit (p110{alpha}, p110ß, p110{delta}, or p110{gamma}) and a regulatory subunit (p85{alpha}, p85ß, p55{gamma}, or p101). Activation of PI3K p110 catalytic subunits takes place by growth factor and hormone binding to tyrosine kinase– and G protein–coupled receptors and involves the recruitment of the PI3K regulatory subunits to tyrosine-phosphorylated receptors and scaffolding signaling proteins, as well as binding to Ras (6, 7). The modular structure of p110 proteins includes a COOH-terminal kinase catalytic domain, preceded by a helical domain, a C2 domain, a Ras-binding domain, and an NH2-terminal regulatory subunit-binding domain (8). Forced localization of p110{alpha} at the plasma membrane increases the cellular levels of PIP3 and favor cell survival and transformation (9). Moreover, gain-of-function mutations that target the gene encoding p110{alpha} (PIK3CA), as well as genomic amplification of the PIK3CA gene, are oncogenic and frequently found in a variety of human tumors. Hotspots for PIK3CA tumor mutations include the COOH-terminal region of the p110{alpha} kinase domain and the NH2-terminal region of the p110{alpha} helical domain (2, 10). PTEN is encoded by a unique gene, which is frequently targeted in tumors by loss-of-function mutations and genomic deletions, as well as in the germ line of patients with hereditary neoplastic syndromes [PTEN hamartoma tumor syndrome (PHTS); ref. 11]. PTEN is composed of an NH2-terminal phosphatase catalytic domain and a COOH-terminal C2 lipid-binding domain, and both domains are required for optimal catalysis (12). In addition, PTEN possesses NH2-terminal and COOH-terminal tails that control PTEN function in cells. The regulation of PTEN function is complex and not fully understood and involves post-translational modifications, protein-protein interactions, and the control of PTEN protein stability and subcellular compartmentation. Of relevance, PTEN binding to membranes is essential for its tumor suppressor activity, and tumor mutations found in the PTEN gene negatively affect not only enzyme catalysis but also PTEN recruitment to cell membranes (13–16). In addition, the steady-state levels of PTEN seem to be important in the control of oncogenesis because decreased PTEN protein expression due to diminished protein stability or to haploinsufficiency is frequent in some tumor types (17, 18). Here, we have investigated the enzymatic activity of mammalian hyperactive forms of PI3K p110{alpha} and p110ß catalytic subunits and its counterbalance by the tumor suppressor PTEN, using an in vivo yeast-based reconstitution system (19). We show that this heterologous system provides a reliable and sensitive assay to monitor in vivo the activity of PI3K and PTEN mutations found in human tumors and to identify novel mutations and/or protein regions relevant for the function of these enzymes in a cellular context. Furthermore, our findings suggest that specific combinations of gain-of-function PI3K mutations and loss-of-function PTEN mutations, including those reducing the PTEN protein steady-state levels, may cooperate to keep the cellular PIP3 levels above the oncogenic threshold required for transformed cells to survive and proliferate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells, media, growth conditions, transfection, and protein detection. The Saccharomyces cerevisiae strain used was YPH499 (MATa ade2-101 trp1-63 leu2-1 ura3-52 his3-{Delta}200 lys2-801). YPD (1% yeast extract, 2% peptone, and 2% glucose) broth or agar was the general nonselective yeast growth medium. Synthetic minimal medium (SM) contained 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, and 2% glucose and was supplemented with appropriate amino acids and nucleic acid bases. SG and SR were SM with 2% galactose or raffinose, respectively, instead of glucose. Yeast was transformed by standard procedures. Growth of yeast on plates was tested by spotting transformant cells onto SM or SG plates lacking the corresponding auxotrophic markers. Transformants were grown overnight in SM lacking uracil, leucine, or both (SM-U, SM-L, or SM-UL) as required and adjusted to an A600 of 0.15. Five microliters of aliquots of each sample plus three serial 1:10 dilutions were deposited on the surfaces of solid media SG-U, SG-L, or SG-UL. Growth was monitored after 2 to 3 days at 30°C. To overexpress PTEN mutations in mammalian cells, COS-7 cells (simian kidney) were transfected by the DEAE-dextran method and processed after 48 h, and MCF-7 cells (human breast carcinoma) were transfected with LipofectAMINE (Invitrogen) and processed after 24 h. Stability of PTEN mutations in MCF-7 cells was analyzed by incubation of cells in the presence of the protein synthesis inhibitor cycloheximide (0.1 mg/mL) for 6 h, followed by immunoblot analysis. Phospho-HA-Akt1 content in COS-7 cells in the presence of PTEN mutations was tested by immunoprecipitation of HA-Akt1 with the anti-HA 12CA5 monoclonal antibody (mAb), followed by immunoblot using an anti–phospho-Ser473-Akt antibody (Cell Signaling Technologies), as described (20). Standard procedures were used for cell harvesting and cell breakage, as well as for preparation of protein-containing cell-free extracts, fractionation by SDS-PAGE, and transfer to nitrocellulose membranes. 421B or 425A anti-PTEN mAb (21) followed by horseradish peroxidase (HRP)–conjugated antimouse (Calbiochem) was used to detect PTEN or its mutant versions. To verify myc-p110{alpha} and myc-p110ß expression on yeast lysates by immunoblot, anti-myc antibody (clone 4A6; Millipore) was used at a 1:2,000 dilution, followed by secondary HRP-conjugated antimouse antibodies. Monoclonal anti-actin C4 antibodies (MP Biomedicals) were used at a 1:2,000 dilution in immunoblots as a loading control.

Plasmid construction and mutagenesis. YCpLG-myc-p110{alpha}-CAAX and pYES2-PTEN, and pSG5 HA-Akt1/PKB{alpha} plasmids have been described (19, 20). YCpLG-myc-p110{alpha}-wt was made eliminating the YCpLG-myc-p110{alpha}-CAAX COOH-terminal signal from YCpLG-myc-p110{alpha}-CAAX and restoring the original stop codon by PCR mutagenesis. Additional PTEN and p110{alpha} mutations were made by PCR mutagenesis using a DpnI-based strategy or a two-step PCR strategy. Cloning of PTEN mutations into pYES2 and YCpUG was made from the corresponding mammalian expression vectors pRK5-PTEN. YCpLG-myc-p110ß-wt and YCpLG-myc-p110ß-CAAX were made by PCR from the plasmid pCR-TOPO p110ß (human sequence; Mammalian Gene Collection, IMAGE ID 40008544). All constructs and mutations were checked by DNA sequencing. pYES3-GFP-Akt1 was made by subcloning from pYES2-GFP-Akt1 (19). The sequences of oligonucleotides used for cloning and mutagenesis are available on request.

Random mutagenesis of PTEN, isolation of mutants, and germ-line mutations. The region of PTEN from nucleotide 63 to the end of the coding region was randomly mutagenized by PCR using Taq DNA Polymerase (Biotools) under standard conditions. The PCR was purified with a QIAquick Gel Extraction kit (250) kit (Qiagen) and 5 µg of DNA were cotransformed by the standard procedures with 1 µg of the pYES-PTEN plasmid, digested previously with BglII-XbaI, into YPH499 yeast cells that had been transformed previously with the YCpLG-myc-p110{alpha}-CAAX plasmid. BglII-XbaI digestion of pYES-PTEN produces a gap that expands from nucleotide 318 of the PTEN open reading frame (ORF) to the linker of pYES2, so that the PTEN gene can only be reconstructed on recombination with the amplicon by in vivo gap repair (22). Recombinants were recovered by plating the transformation mixture onto SM-UL plates. A total of 1,200 clones thus obtained were grown and plated in parallel in SM-UL and SG-UL. Those clones growing on glucose (SM) but not on galactose-based (SG) plates were selected. The pYES-PTEN plasmid was isolated from such clones, amplified in Escherichia coli, verified by restriction analysis, and cotransformed again with YCpLG-myc-p110{alpha}-CAAX in YPH499 cells to verify that PTEN had lost the ability to rescue the p110{alpha}-CAAX–induced toxicity. Mutations were identified on the positive clones by bidirectional DNA sequencing. G36R and Y155C PTEN mutations were identified in the germ line of patients with clinical features of Cowden disease, as described (20), and will be described elsewhere.

Microscopy techniques. To measure green fluorescent protein (GFP)-Akt1 plasma membrane localization, as an indirect indicator of cellular PIP3 levels, transformant cells were grown to log phase in liquid SR medium lacking the corresponding auxotrophic markers, and then 2% galactose was added for 6 to 8 h. GFP-Akt1 was visualized by fluorescence microscopy. To obtain reliable and statistically significant date, ≥150 cells were examined for each condition or experiment for either cytoplasmic or membrane-associated localization. Cells were examined under an Eclipse TE2000U microscope (Nikon) and digital images were acquired with Orca C4742-95-12ER charge-coupled device camera (Hamamatsu) and Aquacosmos Imaging Systems software.

In vitro phosphatase assays. Phosphoinositide phosphatase activity was measured using a chromogenic assay based on the malachite green method. Pellets containing wild-type (wt) PTEN or mutations were obtained by immunoprecipitation from cell lysates from COS-7 cells transfected with the appropriate pRK5-PTEN construct, using a mixture of 421B+425A anti-PTEN mAb (21). Pellets were mixed with a reaction mixture (50 µL) consisting of 100 mmol/L Tris-HCl (pH 8), 10 mmol/L DTT, and 0.1 mmol/L diC8-PIP3 (Echelon) for 40 min at 37°C. The reactions were stopped by addition of 150 µL malachite green reagent followed by 25 µL of 34% trisodium citrate, and absorbance was measured at 580 nm.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Tumor-related and gain-of-function mutations of mammalian hyperactive PI3K p110 catalytic subunits are traceable in vivo by expression in S. cerevisiae. The yeast S. cerevisiae lacks the orthologue of mammalian PI3K type I catalytic subunits (p110). Ectopic expression from the galactose-inducible GAL1 promoter of mammalian p110{alpha} containing the farnesylation-palmitoylation signal from H-Ras (p110{alpha}-CAAX), but not of wt p110{alpha}, inhibits growth of S. cerevisiae (Fig. 1A ; ref. 19). Remarkably, expression in yeast of overactive p110{alpha} mutations found in human tumors [mutations E545K (targeting the helical domain) and H1047R (targeting the kinase domain; ref. 23)] affected cell growth in a mutation-dependent manner. The p110{alpha} H1047R mutation partially retarded yeast cell growth, whereas p110{alpha} E545K effect in this assay was indistinguishable from p110{alpha} wt (Fig. 1A). Ectopic expression of mammalian p110ß-CAAX also hampered yeast cell growth, although full growth suppression was not achieved, whereas expression of wt p110ß did not affect cell growth (Fig. 1B). The different p110 proteins were expressed in equivalent amounts in yeast, as determined by immunoblot (Fig. 1C, bottom). The effect of p110{alpha} and p110ß in the plasma membrane levels of PIP3 in yeast was also indirectly measured by the recruitment of ectopically expressed mammalian Akt (GFP-Akt1; Fig. 1C and D). Quantification of the proportion of yeast cells in the population that relocated GFP-Akt1 to the plasma membrane provided a highly sensitive indicator of PI3K p110 activity in vivo. Thus, p110{alpha} E545K enhanced the plasma membrane relocation of GFP-Akt1 (60% of cells with relocation), although to a lesser extent than p110{alpha} H1047R or p110{alpha}-CAAX (90% of relocation; Fig. 1C). This suggests that the mutation H1047R generates a more active enzyme in yeast than the mutation E545K, in agreement with its stronger oncogenic properties found in chicken embryo fibroblasts (24–26). Wt p110{alpha}, but not wt p110ß, produced a moderate increase in the levels of PIP3 at the yeast plasma membrane (30% of relocation). Finally, p110ß-CAAX increased PIP3 plasma membrane levels with a score of ~70% of relocation (Fig. 1C). These results indicate that S. cerevisiae is sensitive to hyperactivation of p110{alpha} and p110ß, including tumor-related p110{alpha} activation, in a mutation-dependent manner. Our data also show quantitative differences in the amount of PIP3 (as measured indirectly by the relocation of GFP-Akt1) generated in yeast by p110{alpha} and p110ß isoforms, which render distinct quantitative lack-of-growth phenotypes. This is consistent with the cell type–dependent distinct kinetic and oncogenic properties of p110{alpha} and p110ß kinases (27–29). The comparison of the growth inhibition (Fig. 1A and B) and the PIP3 accumulation results (Fig. 1C) suggests that yeast growth is compromised above a threshold of PIP3 generation by hyperactive p110 enzymes. This may resemble the situation in mammalian cells, which above a threshold of PIP3 levels trigger proliferative and survival responses that may lead to cell transformation.


Figure 1
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  		Figure 1. Gain-of-function p110 mutants negatively affect yeast viability and relocate coexpressed heterologous GFP-Akt1 to cellular membranes. A, growth of a yeast wt strain (YPH499) is impaired by expression of the catalytic subunit of PI3K p110{alpha} carrying a COOH-terminal prenylation signal (p110{alpha}-CAAX) or the tumor-related H1047R mutation, but not by wt p110{alpha} or the E545K mutation. All versions of p110{alpha} were expressed from the LEU2-marked vector (YCpLG) under the control of the galactose-inducible GAL1 promoter. Serial 10-fold dilutions of cultures of representative transformants were spotted on synthetic medium lacking leucine under repressing (glucose) or inducing (galactose) conditions. B, yeast growth is impaired by expression of the p110ß when artificially bound to membranes by a COOH-terminal prenylation signal (p110ß-CAAX), but not by expression of wt p110ß. Experimental conditions are identical to those in A. C, top, determination of PIP3 levels in vivo on expression in yeast of different versions of p110{alpha} and p110ß by monitoring GFP-Akt1 localization. Yeast clones as in A and B were cotransformed with a vector bearing a GFP fusion of murine c-Akt1 cDNA under the control of the GAL1 promoter (pYES2-GFP-Akt1; ref. 19). Transformants were grown in SR-UL to log phase, and then galactose was added to a final concentration of 2% and cells were incubated for 6 h and processed for fluorescence microscopy. Cells with GFP signal concentrated at the plasma membrane [see microscope images in D for a visual reference] were counted as positive for PIP3-dependent Akt1 relocation. Columns, average of three different experiments; bars, SD. At least 150 cells were counted for each sample in each experiment. All p110{alpha} and p110ß proteins are tagged at their NH2 terminus with the myc epitope. Bottom, expression of myc-p110{alpha} and myc-p110ß in the same transformants. Cell-free lysates were subjected to immunoblot using either anti-myc antibodies or anti-actin antibodies as a loading control, as indicated. D, example of cells considered negative and positive for association of GFP-Akt1 to membranes. Cells expressing GFP-Akt1 in the absence of p110{alpha} (cotransformed with pYES2-GFP-Akt1 and the YCpLG empty vector) show a diffuse cytoplasmic GFP fluorescence (top; dark spots correspond to vacuoles); cells coexpressing GFP-Akt1 and p110{alpha}-CAAX (cotransformed with pYES2-GFP-Akt1 and YCpLG-myc-p110{alpha}-CAAX) show fluorescence at the plasma membrane (bottom) and were considered positive for the graph in C.

 
In vivo counterbalance of p110{alpha} hyperactivity by PTEN mutations. Overexpression of catalytically active human PTEN in p110{alpha}-CAAX– or p110{alpha} H1047R–expressing yeast rescues cell growth inhibition (Fig. 2B ; data not shown). We sought to use this heterologous system to assess the activity of tumor-derived PTEN mutations in a cellular context. A panel of PTEN mutations found in tumor samples and in the germ line of patients with PHTS (11, 30) was chosen, mapping at the PTEN NH2 terminus, or the PTP and C2 domains. PTEN mutations found in PHTS patients in our laboratory (G36R and Y155C) were also included (Table 1 ). The functional properties of most of the analyzed mutations were unknown, although for some of the mutations included in the study, in vitro catalytic function data were available (20, 31–33). As shown, the majority of the PTEN mutations tested displayed no activity in vivo, as monitored by the lack of cell growth rescue of p110{alpha}-CAAX–expressing yeast (Table 1). The diminished activity of some of the mutations (S10N, K13E, R15I, R15S, Y16C, A34D, M35R, G36R, L42P, and N48K) was likely due to defects in catalysis in vivo because their levels of expression were comparable with PTEN wt. However, other PTEN mutations that displayed no activity in vivo (I33S, H61D, P96Q, Y155C, S170R, and D252Y) were expressed at low levels in yeast, suggesting that these mutations could also affect the stability of PTEN protein (see Fig. 3D ; Table 1). Interestingly, some mutations that were expressed at normal levels (S10N and Y16C; see Fig. 3D; Table 1) displayed partial loss of activity in this system, rescuing the phenotype of either p110{alpha}-CAAX–expressing (Fig. 2B; Table 1) or p110{alpha} H1047R–expressing yeast (Fig. 2C). The intrinsic in vitro phosphatase activity of the S10N and Y16C mutations toward water-soluble PIP3 was not compromised compared with mutations targeting other PTEN NH2-terminal or catalytic residues (Fig. 2D), suggesting that these mutations could affect PTEN activation in vivo. It is also possible that the yeast system could not account for all aspects of PTEN regulation that may be relevant in disease. This could be the case of the K289E mutation, which has been found in the germ line of a Cowden disease family, whereas in yeast, as well as in vitro phosphatase assays, PTEN K289E was active. Interestingly, ubiquitination at Lys289 residue regulates PTEN nuclear import and tumor suppression (31, 34). Diminishing the steady-state levels of PTEN protein may confer advantages for tumor growth (35). To investigate the importance of PTEN expression levels in yeast for the functional properties of the partially inactive S10N and Y16C PTEN mutations, experiments were designed using yeast expression plasmids that empirically achieved distinct PTEN protein expression levels (pYES2, low expression; YCpUG, high expression; Fig. 2A). As shown, increasing the expression levels of the S10N or the Y16C PTEN mutations resulted in an increased reversion of the lack of cell growth phenotype of the p110{alpha}-CAAX–expressing cells. (Fig. 2B). In addition, the mutations S10N and Y16C, even at low expression levels, fully reverted the weaker phenotype of yeast expressing p110ß-CAAX (data not shown). These results show that PTEN activity in S. cerevisiae depends on both its mutational status and its steady-state expression levels and indicate that the pathogenicity of mutations at the PTEN gene found in tumors may be influenced by the levels of PTEN protein expression, as well as by additional alterations of PIK3C genes. In this regard, the frequency of coexistent alterations at the PIK3CA and PTEN genes seems to be tumor type dependent (36–38). We speculate that raising the steady-state levels of PTEN could alleviate tumor incidence and/or tumor progression in patients harboring particular pathogenic PTEN or PIK3C gene alterations.


Figure 2
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  		Figure 2. Counterbalance of p110{alpha}-induced growth defect and high PIP3 levels by PTEN is dependent on the PTEN alleles expressed and their expression levels. A, wt and mutant PTEN are expressed at different levels from the pYES2 and YCpUG vectors. Yeast strain YPH499 was cotransformed with YCpLG-myc-p110{alpha}-CAAX (19) and pYES2 or YCpUG (as indicated) URA3-marked empty vectors or YCpUG containing the cDNA of wt or tumor-related PTEN alleles. Cell-free lysates were subjected to immunoblot with either 425A anti-PTEN antibodies (21) or anti-actin antibodies as a loading control. B, efficiency of different PTEN alleles expressed from two different GAL1-based vectors on rescuing growth in yeast cells expressing p110{alpha}-CAAX. Serial 10-fold dilutions of cultures of representative transformants were spotted on synthetic medium lacking leucine and uracil under repressing (glucose) or inducing (galactose) conditions. C, different efficiency in the reduction by distinct PTEN alleles of PIP3 levels induced in vivo by hyperactive p110{alpha} H1047R. YPH499 cells were cotransformed with LEU2-based YCpLG-myc-p110{alpha} H1047R, TRP1-based pYES3-GFP-Akt1, and URA3-based empty pYES2 (vector) or pYES2-PTEN expressing the indicated wt or tumor-related alleles. Transformants were grown in SR-ULT and processed as in Fig. 1C. Columns, average of three different experiments; bars, SD. At least 150 cells were counted for each sample in each experiment. D, in vitro phosphatase activity of recombinant PTEN proteins purified from COS-7 cell line transfectants, as determined by the malachite green assay, using diC8-PIP3 as the substrate. Data are normalized with respect to wt PTEN (100% activity). Columns, average of three independent experiments; bars, SD.

 

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  		Table 1. Yeast functional analysis of tumor-associated PTEN mutation and functionally relevant PTEN regions

 

Figure 3
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  		Figure 3. PTEN protein destabilization is a frequent cause of loss-of-function. A, ability of COOH-terminal PTEN truncations (top) and single mutations (bottom) to rescue growth of YPH499 yeast cells expressing p110{alpha}-CAAX. PTEN 1 to 347 truncation was partially inactive, whereas PTEN 1 to 346 and larger truncations were fully inactive. Serial 10-fold dilutions of cultures from cotransformants bearing YCpLG-myc-p110{alpha}-CAAX and pYES2-based PTEN-expressing vectors were spotted on synthetic medium lacking leucine and uracil under repressing (glucose) or inducing (galactose) conditions. B, expression of PTEN COOH-terminal truncations (top) and PTEN single mutants (bottom) in yeast compared with wt PTEN. Arrows, migration of full-length PTEN (1–403; top arrow) and PTEN truncations (bottom arrow). Cotransformants as in A were grown in SR-UL and processed for immunoblot as in Fig. 2A, using 425A anti-PTEN antibodies (21) or anti-actin antibodies, as indicated. C, stability of PTEN mutations in mammalian cells. PTEN wt or mutations were transiently expressed in MCF-7 cells, and cultures were grown under normal conditions or incubated in the presence of cycloheximide for 6 h. Cell-free lysates were obtained and equal amounts of protein were analyzed by immunoblot using anti-PTEN antibodies (421B mAb). PTEN bands were quantified using ImageQuant TL (Amersham Biosciences). Results are percentage of PTEN protein after 6 h of cycloheximide cell treatment with respect to the untreated cells (t = 0 h). D, steady-state expression levels of PTEN wt and mutations in yeast. Cell-free lysates were processed for immunoblot using 425A anti-PTEN mAbs (21) or anti-actin antibodies, as indicated. Mutation procedence: V343E, Cowden disease; L345Q, glioblastoma multiforme; and T348I, atypical endometrial hyperplasia (11, 30).

 
Next, we assessed in p110{alpha}-CAAX–expressing yeast the functional properties of a panel of PTEN mutations targeting functionally relevant PTEN regions (Table 1). The PTEN NH2-terminal tail, which contains a PIP2-binding motif and a nucleus/cytoplasm–targeting domain, was necessary for PTEN function because deletion of the residues 1 to 17 resulted in lack of activity in yeast. A detailed mutational analysis of this region was done, and residues Arg15 and Tyr16 were found to be essential for PTEN activity. Residues Lys6, Ile8, Val9, Ser10, and Arg11 were also found to contribute to PTEN function in yeast, and an important PTEN functional region was identified from residues Asp22 to Tyr29 (mutations DLDL and TYY; Table 1). It is remarkable the overlapping in this PTEN NH2-terminal region of distinct functional and subcellular localization motifs, which might be the target of tight regulation in vivo (13, 39, 40). Interestingly, mutations at the PTEN NH2-terminal region have been found in human tumors (see Table 1; refs. 11, 30), and the involvement of NH2-terminal residues in the control of PTEN subcellular localization and function in both Dyctiostelium and mammalian cell systems has been documented (32, 33, 41–43). Positively charged clusters of residues at both the PTP and the C2 domain, which have also been involved in PTEN binding to membranes, subcellular localization, and function (39), were analyzed. Among the positive-charge motifs tested, the C{alpha}2, CBR3, and RKK-PTP motifs were found to be required for PTEN function. This agrees with the importance of these motifs in PTEN binding to membranes (12, 44). In contrast, mutation of the RRK-C2 motif did not affect PTEN function in yeast. The PTEN COOH-terminal tail (last 50 residues), which contains several serine and threonine phosphorylation sites and a PDZ domain binding motif, was dispensable for PTEN function in this system, as the PTEN 1 to 350 truncation was fully active. On the other hand, the PTEN 1 to 301 truncation, lacking the COOH-terminal portion of the C2 domain, displayed impaired activity, as a result of its elevated instability (see Fig. 3; Table 1). Together, these findings highlight the functional importance of regions at the NH2 terminus of PTEN and at the COOH terminus of its C2 domain in the control of cellular PIP3 levels.

Elucidation of the minimum length of PTEN required for stability and function. PTEN COOH-terminal truncation 1 to 350 was fully functional in yeast (Table 1). Because premature stop codon mutations that render truncated proteins, including mutations between residues 340 to 350, are frequently found in tumors (11, 30), we investigated more in detail the minimal COOH-terminal truncation of PTEN resulting in loss-of-function in yeast. Single amino acid truncation mutants were generated from the 1 to 350 COOH-terminal truncation, and their function was assessed in the yeast strain expressing p110{alpha}-CAAX (Fig. 3A). Full PTEN activity was present in truncations 1 to 350, 1 to 349, and 1 to 348. Truncation 1 to 347 was partially inactive, whereas truncations 1 to 346, 1 to 345, and 1 to 344 were inactive. Thus, the minimal PTEN COOH-terminal truncation that abrogates PTEN function in yeast introduces a premature stop codon in position 347. The COOH-terminal portion of PTEN has been reported to be important for PTEN protein stability, and PTEN COOH-terminal truncations show decreased half-lives (35, 45, 46). Next, we tested the steady-state levels of PTEN COOH-terminal truncations in the yeast. Remarkably, a correlation was found between decreased expression levels of PTEN truncations and lack of PTEN activity in yeast (Fig. 3B), indicating that the loss-of-function phenotype shown by the PTEN COOH-terminal truncations is due to protein destabilization and degradation. These results also suggest that critical determinants of PTEN protein stability exist in the COOH-terminal region of the C2 domain. In this regard, the region flanked by residues 341 to 348 of PTEN is frequently mutated in tumors (11, 30). Thus, we tested in yeast the stability and functional properties of PTEN tumor-related mutations (V343E, L345Q, and T348I) within this region (Fig. 3A and B). Mutations V343E and L345Q were inactive in yeast, likely as a result of low stability, as suggested by the low steady-state levels of expression of these mutations. On the other hand, the mutation T348I was expressed at normal levels and was active in yeast. These results corroborate recent findings showing a key role of this region of PTEN in protein stabilization in mammalian cells (47). Next, we tested whether the steady-state levels of PTEN mutations in yeast correlate with their stability in mammalian cells. A panel of PTEN mutations that included mutations expressed at normal levels (S10N, K13E, and Y16C) and mutations expressed at low levels in yeast (I33S, H61D, P96Q, S170R, D252Y, and L345Q; Fig. 3C; Table 1) were transiently expressed in human breast carcinoma MCF-7 cells, and their stability was evaluated by their decreased expression after cycloheximide treatment. As shown, the stability of S10N, K13E, and Y16C PTEN mutations in MCF-7 cells was comparable with that displayed by PTEN wt. On the other hand, mutations I33S, H61D, P96Q, S170R, D252Y, and L345Q were strongly destabilized in MCF-7 cells, in correlation with their lower steady-state levels in yeast (Fig. 3D). These results outline the importance of protein stability in the tumor suppressor function of PTEN and indicate that the yeast system used in our study is a suitable method to test not only PTEN catalytic activity but also PTEN stability in cells. Because the proteasome mediates PTEN degradation, it is conceivable that some of the antitumoral effects of proteasome inhibition therapy may be due to increasing the steady-state expression levels of PTEN (45, 48, 49).

Isolation of novel loss-of-function PTEN mutations and validation in mammalian cells. S. cerevisiae expressing p110{alpha}-CAAX on galactose induction was used to isolate novel PTEN loss-of-function mutations, by means of an indirect mutational screening that covered nucleotides 63 to 1212 from the PTEN ORF (amino acids 21–403). Recombinants that bore inactive versions of PTEN would have lost the capability to counteract the lethality of p110{alpha}-CAAX and such yeast clones would be unable to grow in galactose. After two rounds of selection, 21 clones of these characteristics were obtained and sequenced. The nucleotide changes present in the random-generated PTEN cDNA mutations isolated and the resulting amino acid substitutions in the PTEN protein are shown in Table 2 . Figure 4 shows the phenotype of some of the mutations isolated. Nineteen of the 21 clones obtained corresponded to different mutations. Almost one third (7 of 21) of the clones corresponded to truncation mutants that generated stop termination codons ranging from residue 140 to residue 344, as discussed above. Notably, most of the single amino acid substitutions (eight of nine) targeted the PTEN PTP domain, with a clear hotspot at the catalytic site and mapped mostly to residues that are frequently mutated in tumors. Undescribed single PTEN mutations (R159G and M198R) were also found. All missense mutant proteins were expressed in yeast at similar levels (Fig. 4B, top). Also of interest, clones bearing double point mutations resulting in the substitution of two residues were selected (5 of 21), suggesting the additive effect of some mutations in PTEN loss-of-function. To test this possibility, individual mutations (P246L and F347S) from one of the double mutants obtained (P246L/F347S) were generated and tested for function (Fig. 4A and B). As shown, PTEN bearing the P246L and F347S individual mutations rescued yeast cells from p110{alpha}-CAAX–induced growth inhibition and released GFP-Akt1 from cellular membranes, although, for F347S, not as efficiently as wt PTEN. However, the compound P246L/F347S mutant did neither restore growth nor decrease GFP-Akt1 accumulation at the cell membrane (Fig. 4A and B, bottom). Remarkably, a P246L mutation and a related compound mutation (V343E/F347L) have been found in PHTS patients (50, 51), suggesting the existence of patient-dependent loss-of-function effects on certain PTEN tumor-related mutations. Next, we tested in a mammalian cell system the functional properties of some of the random-generated PTEN mutations in yeast. COS-7 cells were cotransfected with PTEN wt or mutations and the downstream PI3K effector Akt1, and the activity of Akt1 was analyzed by immunoblot using anti–phospho-active Akt antibodies (Fig. 4C). As shown, coexpression of wt PTEN diminished the phosphorylation of Akt1, whereas coexpression of phosphatase-dead C124S or the random PTEN mutations G129R, R159G, and M198R generated in yeast did not abrogate Akt1 phosphorylation. These results prove that the activity of PTEN mutations in yeast can reflect their performance in mammalian cells and indicate that random mutagenesis coupled to the yeast functional assay could be used as a high-throughput system to generate a comprehensive map of pathologic human PTEN mutations.


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  		Table 2. PTEN loss-of-function mutations isolated in this work

 

Figure 4
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  		Figure 4. Functional activity of the random-generated PTEN mutations in yeast and in mammalian cells. A, ability of single and double random-generated PTEN mutants to rescue growth of YPH499 yeast cells expressing p110{alpha}-CAAX. Serial 10-fold dilutions of cultures from cotransformants bearing YCpLG-myc-p110{alpha}-CAAX and pYES2-based PTEN-expressing vectors were spotted on synthetic medium lacking leucine and uracil under repressing (glucose) or inducing (galactose) conditions. B, bottom, reduction of p110{alpha}-induced PIP3 levels in vivo by random-generated PTEN mutants. YPH499 cells were cotransformed with LEU2-based YCpLG-myc-p110{alpha}-CAAX, TRP-based pYES3-GFP-Akt1, and URA3-based pYES2 empty (vector) or containing the indicated human PTEN alleles. Transformants were grown in SR-ULT and processed as in Fig. 1C. Columns, average of three different experiments; bars, SD. At least 150 cells were counted for each sample in each experiment. Top, the relative expression of the different mutations in yeast is shown. Cotransformants were grown in SR-UL and processed for immunoblot using 425A anti-PTEN mAbs (21) or anti-actin antibodies. C, ability of random-generated PTEN mutants to abolish HA-Akt1 activation in mammalian cells. PTEN wt or the indicated mutations were coexpressed with HA-Akt1 in COS-7 cells. Cell-free lysates were subjected to immunoprecipitation with the anti-HA 12CA5 mAb, and HA-Akt1 activation was monitored by immunoblot using an anti–phospho-active Akt (anti-P-Ser473) antibody (bottom). The content of PTEN and HA-Akt1 in the lysates was monitored using anti-PTEN (421B mAb) and anti-HA (12CA5) antibodies, respectively (top and middle). Protein bands were quantified using ImageQuant TL. The numbers indicate the relative phospho-Akt1 content, normalized to the HA-Akt1 content. The experiment was repeated thrice with similar results, and a representative experiment is shown.

 
In summary, our results show that the phenotype found in S. cerevisiae on ectopic expression of tumor-associated alterations of mammalian p110 and PTEN proteins may recapitulate the pathologic changes in phosphoinositide levels that occur in human carcinogenesis and validate the S. cerevisiae heterologous system as a powerful biological tool to monitor the tumor suppressor function of mammalian PTEN. The simplicity, sensitivity, and high-throughput possibilities of S. cerevisiae make this organism a suitable model to monitor upstream alterations in the mammalian PI3K/PTEN signaling pathway.


   Acknowledgments
 
Grant support: Ministerio de Ciencia y Tecnología and Ministerio de Educación y Ciencia [Spain-Fondo Europeo de Desarrollo Regional (FEDER); grants BMC2003-02696 and SAF2006-08319 (R. Pulido) and grant BIO2004-02019 (M. Molina and V.J. Cid)], Fundación de Investigación Mutua Madrileña grant (Spain; R. Pulido and A. Vega), and Instituto de Salud Carlos III [Spain-FEDER; grant CP04/00318 (A. Gil) and grant ISCIII-RETIC RD06/0020]. Grant BIO2004-02019 (I. Rodríguez-Escudero). A. Andrés-Pons has been the recipient of predoctoral fellowships from Ministerio de Educación y Ciencia and from Ayuntamiento de Valencia (Spain), and A. Blanco has been the recipient of a fellowship from Instituto de Salud Carlos III (Spain).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Geneservice and A. Carnero (Centro Nacional de Investigaciones Oncológicas, Madrid, Spain) for providing plasmids; M.I. García Sáez, T. Aparicio, and R. Torremocha from the Unidad de Genómica y Proteómica (Parque Científico de Madrid/Universidad Complutense de Madrid, Madrid, Spain) for DNA sequencing; I. Roglá for expert technical assistance; and J. Thorner and C. Nombela for their continuous support and encouragement.


   Footnotes
 
Note: A. Andrés-Pons and I. Rodríguez-Escudero contributed equally to this work.

Received 4/ 9/07. Revised 7/24/07. Accepted 8/14/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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