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Research Article
In vivo Functional Analysis of the Counterbalance of Hyperactive
Phosphatidylinositol 3-Kinase p110 Catalytic Oncoproteins
by the Tumor Suppressor PTEN
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Amparo Andrés-Pons, Isabel Rodrı́guez-Escudero, Anabel Gil, Ana Blanco, Ana Vega,
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Marı́a Molina, Rafael Pulido, and Vı́ctor J. Cid
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Centro de Investigación Prı́ncipe Felipe, Valencia, Spain; 2Departamento de Microbiologı́a II, Facultad de Farmacia, Universidad
Complutense de Madrid, Madrid, Spain; and 3Unidad de Medicina Molecular, Fundación Pública Galega de Medicina
Xenómica-SERGAS, Grupo de Medicina Xenómica-CIBERER, Santiago de Compostela, Spain
Abstract
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 p110A and
p110B 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 p110A
or p110B 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
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 protooncogenic enzymes that synthesize the second messengers
Note: A. Andrés-Pons and I. Rodrı́guez-Escudero contributed equally to this work.
Requests for reprints: Rafael Pulido, Centro de Investigación Prı́ncipe Felipe,
Avda. Autopista del Saler 16-3, Valencia, Spain 46013. Phone: 34-96-3289680, ext. 2004;
Fax: 34-96-3289701; E-mail: [email protected] and Vı́ctor J. Cid, Departamento de
Microbiologı́a 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: [email protected].
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-1278
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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 (p110a,
p110h, p110y, or p110g) and a regulatory subunit (p85a, p85h,
p55g, 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 p110a 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 p110a (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
p110a kinase domain and the NH2-terminal region of the p110a
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 lipidbinding 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
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(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 p110a and p110h 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
Cells, media, growth conditions, transfection, and protein detection. The Saccharomyces cerevisiae strain used was YPH499 (MATa ade2101 trp1-63 leu2-1 ura3-52 his3-D200 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
A 600 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 30jC. 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 HAAkt1 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 proteincontaining 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-p110a and
myc-p110h 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-p110a-CAAX
and pYES2-PTEN, and pSG5 HA-Akt1/PKBa plasmids have been described
(19, 20). YCpLG-myc-p110a-wt was made eliminating the YCpLG-mycp110a-CAAX COOH-terminal signal from YCpLG-myc-p110a-CAAX and
restoring the original stop codon by PCR mutagenesis. Additional PTEN
and p110a mutations were made by PCR mutagenesis using a DpnI-based
strategy or a two-step PCR strategy. Cloning of PTEN mutations into
Cancer Res 2007; 67: (20). October 15, 2007
pYES2 and YCpUG was made from the corresponding mammalian
expression vectors pRK5-PTEN. YCpLG-myc-p110h-wt and YCpLG-mycp110h-CAAX were made by PCR from the plasmid pCR-TOPO p110h
(human sequence; Mammalian Gene Collection, IMAGE ID 40008544). All
constructs and mutations were checked by DNA sequencing. pYES3-GFPAkt1 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 Ag of DNA were
cotransformed by the standard procedures with 1 Ag of the pYES-PTEN
plasmid, digested previously with BglII-XbaI, into YPH499 yeast cells that
had been transformed previously with the YCpLG-myc-p110a-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 SGUL. 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-p110a-CAAX in YPH499 cells to
verify that PTEN had lost the ability to rescue the p110a-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, z150 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 antiPTEN mAb (21). Pellets were mixed with a reaction mixture (50 AL)
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 37jC. The reactions were stopped by
addition of 150 AL malachite green reagent followed by 25 AL of 34%
trisodium citrate, and absorbance was measured at 580 nm.
Results and Discussion
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 p110a containing the farnesylationpalmitoylation signal from H-Ras (p110a-CAAX), but not of wt
p110a, inhibits growth of S. cerevisiae (Fig. 1A; ref. 19).
Remarkably, expression in yeast of overactive p110a mutations
found in human tumors [mutations E545K (targeting the helical
domain) and H1047R (targeting the kinase domain; ref. 23)]
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PI3K/PTEN Mutational Functional Analyses in Yeast
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 p110a carrying a COOH-terminal prenylation signal (p110a-CAAX) or the
tumor-related H1047R mutation, but not by wt p110a or the E545K mutation. All versions of p110a 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 p110h when artificially bound to membranes
by a COOH-terminal prenylation signal (p110h-CAAX), but not by expression of wt p110h. Experimental conditions are identical to those in A. C, top, determination
of PIP3 levels in vivo on expression in yeast of different versions of p110a and p110h 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 p110a and p110h
proteins are tagged at their NH2 terminus with the myc epitope. Bottom, expression of myc-p110a and myc-p110h 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 p110a (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 p110a-CAAX (cotransformed
with pYES2-GFP-Akt1 and YCpLG-myc-p110a-CAAX) show fluorescence at the plasma membrane (bottom ) and were considered positive for the graph in C .
affected cell growth in a mutation-dependent manner. The p110a
H1047R mutation partially retarded yeast cell growth, whereas
p110a E545K effect in this assay was indistinguishable from
p110a wt (Fig. 1A). Ectopic expression of mammalian p110hCAAX also hampered yeast cell growth, although full growth
suppression was not achieved, whereas expression of wt p110h
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 p110a and p110h 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, p110a E545K enhanced the plasma
membrane relocation of GFP-Akt1 (60% of cells with relocation),
although to a lesser extent than p110a H1047R or p110a-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
p110a, but not wt p110h, produced a moderate increase in the
levels of PIP3 at the yeast plasma membrane (30% of relocation).
Finally, p110h-CAAX increased PIP3 plasma membrane levels with
a score of f70% of relocation (Fig. 1C). These results indicate
that S. cerevisiae is sensitive to hyperactivation of p110a and
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p110h, including tumor-related p110a activation, in a mutationdependent 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 p110a and p110h isoforms,
which render distinct quantitative lack-of-growth phenotypes.
This is consistent with the cell type–dependent distinct kinetic
and oncogenic properties of p110a and p110h 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.
In vivo counterbalance of p110a hyperactivity by PTEN
mutations. Overexpression of catalytically active human PTEN in
p110a-CAAX– or p110a 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
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Figure 2. Counterbalance of p110ainduced 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-p110a-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 p110a-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 p110a
H1047R. YPH499 cells were
cotransformed with LEU2-based
YCpLG-myc-p110a H1047R, TRP1 -based
pYES3-GFP-Akt1, and URA3 -based
empty pYES2 (vector) or pYES2-PTEN
expressing the indicated wt or tumorrelated 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.
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 p110a-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 p110a-CAAX–expressing
(Fig. 2B; Table 1) or p110a 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
Cancer Res 2007; 67: (20). October 15, 2007
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 p110a-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
p110h-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.
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PI3K/PTEN Mutational Functional Analyses in Yeast
Next, we assessed in p110a-CAAX–expressing yeast the functional properties of a panel of PTEN mutations targeting
functionally relevant PTEN regions (Table 1). The PTEN NH2terminal 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 Ca2, 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 p110a-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 lossof-function phenotype shown by the PTEN COOH-terminal
truncations is due to protein destabilization and degradation.
www.aacrjournals.org
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
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Table 1. Yeast functional analysis of tumor-associated
PTEN mutation and functionally relevant PTEN regions
Mutation
Wt
S10N
K13E
R15I
R15S
Y16C
I33S
A34D
M35R
G36R
L42P
N48K
H61D
P96Q
Y155C
S170R
D252Y
K289E
17-403
K6A
E7A
I8A
V9A
S10A
S10E
R11A
R11E
N12A
K13A
R14A
R15A
Y16A
Q17A
E18A
DLDL
TYY
1-301
1-350
RKK-PTP
RRK-C2
CBR3
Ca2
Tumor type/
disease
NHML
Glb
Glb
Glb
NHML
Glb
BRR
JPS, Glb
CD
Glb
CD
Vh
CD
CD
BRR
Glb
CD
Yeast in vivo
activity*
Yeast protein
c
expression level
+
+/
+/
+/
+/
+
+/
+
+/
+/
+/
+
+/
+
+
+
+
+
+
+/
+
Normal
Normal
Normal
Normal
Normal
Normal
Low
Normal
Normal
Normal
Normal
Normal
Low
Low
Low
Low
Low
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Low
Undetectable
Normal
Normal
Normal
Normal
Normal
Abbreviations: CD, Cowden syndrome; Glb, glioblastoma; BRR,
Bannayan-Riley-Ruvalcaba syndrome; NHML, non-Hodgkin’s malignant lymphoma; JPS, juvenile polyposis syndrome; Vh, VATERhydrocephalia; DLDL, D22A/L23A/D24A/L25A; TYY, T26A/Y27A/
Y29A; RKK-PTP, R161A/K163A/K164A; RRK-C2, R233A/R234A/K237A;
CBR3, K263A/M264A/L265G/K266A/K267A/K269A; Ca2, K327A/
N329G/K330A/K332A/R335A.
*The yeast in vivo activity indicates the reconstitution of the p110aCAAX–induced lack-of-growth phenotype (+, reconstitution; +/,
partial reconstitution; , no reconstitution), as shown in Fig. 2B.
cThe expression levels of PTEN proteins in yeast were determined by
immunoblot with anti-PTEN antibodies, as shown in Fig. 2.
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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 p110a-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-p110a-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).
of PTEN is frequently mutated in tumors (11, 30). Thus, we tested
in yeast the stability and functional properties of PTEN tumorrelated 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,
Cancer Res 2007; 67: (20). October 15, 2007
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 p110aCAAX 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
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PI3K/PTEN Mutational Functional Analyses in Yeast
versions of PTEN would have lost the capability to counteract the
lethality of p110a-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 p110aCAAX–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
Table 2. PTEN loss-of-function mutations isolated in this
work
Type of mutation
Nonsense
Missense (single)
Missense (double)
Mutation*
Amino acid change
T420!A
C734!T
G689!T
A881!G, C893!T
C1004!T
c
A1031!T
A276!G
A369!T
G386!A
G390!A
c
T402!C
T407!C
A476!G
T594!G
C216!T, G390!A
A476!G, T599!C
G519!A, T525!C
T533!C, A708!G
C738!T, T1041!C
Stop at 140
Stop at 245
Stop at 230
S292G, stop at 298
Stop at 335
Stop at 344
D92G
H123R
G129R
R130Q
M134T
C136R
R159G
M198R
A72V, R130Q
R159G, F200L
R173H, V175A
Y178H, D236G
P246L, F347S
*PTEN nucleotide sequence is according to Genbank accession no.
NM_000314.
cThese mutants were isolated twice.
www.aacrjournals.org
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
p110a-CAAX. Serial 10-fold dilutions of cultures from cotransformants bearing
YCpLG-myc-p110a-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
p110a-induced PIP3 levels in vivo by random-generated PTEN mutants.
YPH499 cells were cotransformed with LEU2 -based YCpLG-myc-p110a-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.
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Cancer Research
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 randomgenerated 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.
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 highthroughput possibilities of S. cerevisiae make this organism a
suitable model to monitor upstream alterations in the mammalian
PI3K/PTEN signaling pathway.
Acknowledgments
Received 4/9/2007; revised 7/24/2007; accepted 8/14/2007.
Grant support: Ministerio de Ciencia y Tecnologı́a and Ministerio de Educación y
Ciencia [Spain-Fondo Europeo de Desarrollo Regional (FEDER); grants BMC200302696 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. Rodrı́guezEscudero). 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. Garcı́a Sáez, T. Aparicio, and
R. Torremocha from the Unidad de Genómica y Proteómica (Parque Cientı́fico 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.
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In vivo Functional Analysis of the Counterbalance of
Hyperactive Phosphatidylinositol 3-Kinase p110 Catalytic
Oncoproteins by the Tumor Suppressor PTEN
Amparo Andrés-Pons, Isabel Rodríguez-Escudero, Anabel Gil, et al.
Cancer Res 2007;67:9731-9739.
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