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

Abstract

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.

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Biochem. J. (2005) 390, 613–623 (Printed in Great Britain)doi:10.1042/BJ20050574
613
Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast
Isabel RODR´IGUEZ-ESCUDERO*, Franc ¸oise M. ROELANTS†, Jeremy THORNER†, C´ esar NOMBELA*, Mar´ ıa MOLINA*1and
V´ ıctor J. CID*
*Departamento de Microbiolog´ ıa II, Facultad de Farmacia, Universidad Complutense de Madrid, Pza. de Ram´ on y Cajal s/n, 28040 Madrid, Spain, and †Department of Molecular and
Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, U.S.A.
ThemammaliansignallingpathwayinvolvingclassIPI3K(phos-
phoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphat-
ase) and PKB (protein kinase B)/c-Akt has roles in multiple pro-
cesses, including cell proliferation and apoptosis. To facilitate
novelapproachesforgenetic,molecularandpharmacologicalana-
lyses of these proteins, we have reconstituted this signalling path-
way 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-bisphos-
phate)anditsconversionintoPIP3(phosphatidylinositol3,4,5-tri-
sphosphate).PIP2depletioncausessevererearrangementsofactin
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 phospho-
rylated at Thr308and Ser473. Thr308phosphorylation, 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 Ser473phos-
phorylation, 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 simpli-
fied context, establishes a new tool to screen for novel agonists
and antagonists and provides a method to deplete PIP2uniquely
in the yeast cell.
Key words: c-Akt, phosphorylation, phosphoinositide 3-kinase
(PI3K), protein kinase B (PKB), PTEN, Saccharomyces cer-
evisiae.
INTRODUCTION
Generation of PIP3(phosphatidylinositol 3,4,5-trisphosphate) at
the plasma membrane is key to the regulation of mammalian cell
proliferation and survival by growth-promoting factors [1]. Many
humantumours[2]havealterationsinthePI3K(phosphoinositide
3-kinase) that generates this lipid [3], in the PTEN (phosphat-
idylinositol 3-phosphatase) that degrades this lipid [4] and in a
protein kinase [PKB (protein kinase B)/c-Akt] that requires this
lipid for its activation [5].
ClassIAPI3K,thebeststudiedandmostrelatedtooncogenesis,
comprises a regulatory/adaptor subunit (p85) and a catalytic sub-
unit (p110). In humans, seven regulatory subunits, generated by
alternative splicing from three genes (p85α, p85β and p55γ)
and three p110 catalytic subunit isoforms (α, β and γ), have been
described.p85containsanSH2domain(Srchomology2domain)
that binds phosphotyrosine residues on receptor-tyrosine kinases,
simultaneouslyrecruitingp110,stimulatingitsactivityandgener-
ating locally PIP3from PIP2(phosphatidylinositol 4,5-bisphos-
phate) in the plasma membrane.
The reverse reaction – dephosphorylation of PIP3to PIP2– is
catalysed by PTEN, which is considered a tumour suppressor
becauseloss-of-functionmutationsarefoundinovert tumours[6]
and in the germline in diseases marked by hereditary predis-
position to cancer [7]. PIP3 is also metabolized to PI(3,4)P2
(phosphatidylinositol 3,4-bisphosphate) by 5-phosphoinositide
phosphatases, like SHIP1 and SHIP2 (SH2-containing inositol
phosphatase-1 and -2); but PIP3and PI(3,4)P2engage the same
effectors, for the most part.
Thethreeisoforms(α,β andγ)ofPKB/c-Aktaremajortargets
of PIP3, which binds to the N-terminal PH domain (pleckstrin
homology domain), thereby anchoring the enzyme to the plasma
membrane and inducing a conformational change that exposes its
catalytic domain to its upstream activators. Full activation of Akt
requires phosphorylation of Thr308within the activation loop (or
T-loop) and also at a more distal site, Ser473. Phosphorylation of
Akt1 at Thr308is mediated by PDK1 (3-phosphoinositide-depen-
dent protein kinase-1), which has a C-terminal PIP3-specific PH
domain that facilitates its recruitment to membranes. Hence,
Thr308is called the PDK1 site. The enzyme responsible for phos-
phorylation of Akt1 at Ser473(termed the PDK2 site) is contro-
versial. Over time, various investigators have implicated several
different classes of protein kinase in PDK2 site phosphorylation
[8], whereas others suggest that autophosphorylation is the cause
[9]. Recently, it has been reported that members of the PIKK
(phosphatidylinositol kinase-related kinase) family of atypical
protein kinases are responsible for PDK2 site phosphorylation of
Akt1, including DNA-PKcs (the catalytic subunit of DNA-depen-
dent protein kinase) [10],the product ofthe genemutated in ATM
(ataxia telangiectasia) [11], and the mTOR (mammalian target of
rapamycin) orthologue [12]. Once activated, Akt phosphorylates
substrates that promote cell survival and stimulate cell growth.
Thus hyperactivation of Akt is considered a prerequisite for
oncogenesis. Consequently, the PI3K/Akt pathway has become
Abbreviations used: ATM, ataxia telangiectasia; CPY, carboxypeptidase Y; DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase; ERK,
extracellular-signal-regulated kinase; HA, haemagglutinin; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PDK1, 3-
phosphoinositide-dependent protein kinase-1; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PIKK, phosphatidylinositol
kinase-related kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-
trisphosphate; PKB, protein kinase B; PLCδ1, phospholipase δ1; PTEN, phosphatidylinositol 3-phosphatase; SH2 domain, Src homology 2 domain.
1To whom correspondence should be addressed (email molmifa@farm.ucm.es).
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I. Rodr´ ıguez-Escudero and others
Table 1 Sacch. cerevisiae strains used in the present study
StrainGenotypeReference or source
YPH499
YFR105
YFR106
YPT40
DLY1
INA106
SH100
SH221
DL454
YFR55
BY4741
Y03114
RCY307
RCY426
YCB655
MATa ade2-101ochhis3-?200 leu2-?1 lys2-801ambtrp1-?1 ura3-52
YPH499 pkh1-?1::TRP1
YPH499 pkh2-?1::HIS3
YPH499 ypk1-1ts::HIS3 ykr2-?1::TRP1
MATa ade1 his2 leu2-3,112 trp1-1 ura3?
DLY1 pkh1D398G pkh2::LEU2
MATa leu2-3,112 ura3-52 trp1 his3 ade2?
SH100 tor1::HIS3-3 tor2::ADE2-3 YCplac111(LEU2)-tor2-21
MATa ura3-52 his4 trp1-1 leu2-3,112 canR slt2?::TRP1
YPH499 sch9?::HIS3
MATa; his3?1; leu2?; met15?; ura3?
BY4741 tel1::kanMX4
MATa HO::hisG esr1(mec1)?::LEU2, lys2, ura3, leu2::hisG, ade2::LK, his4 arg4N::ESR1-kanMX4
MATa HO::hisG esr1(mec1)?::LEU2, lys2, ura3, leu2::hisG, ade2::LK, his4 arg4N::esr1-40 kanMX4
MATa leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,25 tra1-2
*EUROpean Saccharomyces Cerevisiae ARchive for Functional analysis, Johann Wolfgang Goethe-University, Frankfurt, Germany.
[30]
[17]
[17]
[16]
Daniel Lew
Kunihiro Matsumoto
Michael Hall
Michael Hall
David Levin
[57]
EUROSCARF*
EUROSCARF
Rita Cha
Rita Cha
Jerry Workman
an important target in attempts to develop more efficacious anti-
tumour agents [13].
Althoughithasbeenreportedthatfissionyeast(Schizosacchar-
omycespombe)cangeneratePIP3[14],PIP3seemstohavenorole
in the physiology of budding yeast (Saccharomyces cerevisiae)
[15]. Nonetheless, the Sacch. cerevisiae genome encodes: (i) two
functional PDK1 orthologues (Pkh1 and Pkh2) involved in
cell integrity and endocytosis [16,17]; (ii) an apparent PTEN
orthologue (Tep1) of uncharacterized biological function [18,19];
(iii) an Akt-like protein kinase (Sch9), which lacks an apparent
PH domain, involved in nutrient sensing, ribosome biogenesis,
lifespan and cell-size control [20]; and (iv) clear-cut homologues
of the PIKK family, specifically Tor1 and Tor2 (mTOR) [21],
Tel1 (ATM) [22], Mec1 (ATR) [23] and Tra1 (most resembles
DNA-PKcs) [24].
To address central questions in the biology of PIP3-dependent
signalling and to establish a readily accessible and versatile
tool to screen for pharmacological agents that influence this
critically important pathway, we devised methods to successfully
reconstitute the mammalian PI3K/PTEN/Akt pathway in yeast
cells, which is described here. In vivo conversion of the essential
PIP2pool into PIP3by expression of PI3K impaired yeast growth
by altering morphogenesis and vesicular trafficking. The function
of PTEN could be readily assessed by its ability to reverse the
growth inhibition caused by PI3K. PIP3generation led to mem-
brane translocation and activation of Akt, enhancing its phos-
phorylation at both Thr308and Ser473. The yeast PDK1 ortho-
logues are required for PDK1 site phosphorylation, whereas
none of the yeast PIKK family members seems necessary for
PDK2 site phosphorylation, implicating some other endogenous
enzyme.
EXPERIMENTAL
Strains, media and growth conditions
The Sacch. cerevisiae strains used in the present study are listed
in Table 1. Escherichia coli DH5α F?[K12?(lacZYA-argF)U169
deoR supE44 thi-1 recA1 endA1 hsdR17 gyrA96 relA1
(Φ80lacZ?M15)F?] was used for routine molecular biology tech-
niques.YPD[1%(w/v)yeastextract,2%(w/v)peptoneand2%
(w/v) dextrose/glucose] broth or agar was the general non-
selective medium used for yeast cell growth. Synthetic minimal
medium (SGlc) contained 0.17% yeast nitrogen base without
amino acids, 0.5% ammonium sulphate and 2% glucose and
lackedappropriateaminoacidsandnucleicacidbasestomaintain
selection for plasmids. In SGal (synthetic galactose) and SRaf
(synthetic raffinose) media, glucose was replaced with 2% (w/v)
galactoseor2%(w/v)raffinoserespectively.Galactoseinduction
in liquid medium was performed by growing cells in SRaf to
mid-exponential phase and then adding galactose to 2% for
6–8 h. Growth assays on plates were performed, typically, by
growing transformants overnight in SGlc lacking uracil, leucine
orboth,adjustingtheculturetoanabsorbanceA6000.5andspotting
samples (5 µl) of the cell suspension and three serial 10-fold
dilutions on to the surface of SGlc or SGal plates lacking the
appropriate nutrients to maintain selection for plasmids, followed
byincubationat28◦Cfor2–3 days.Whenyeastcellsweretreated
with PI3K inhibitors, transformants were grown overnight in
selective medium, adjusted to an A600of 0.1, and then samples
(5 µl)ofthecellsuspensionwereusedtoseedthewellsof96-well
microtitre plates containing serial dilutions of either wortmannin
(Sigma) or LY294002 (Biomol, Hamburg, Germany) suspended
in100 µlofliquidSGalmediumlackingtheappropriatenutrients
to maintain selection for plasmids.
Plasmid construction
Transformation of E. coli and yeast and other basic molecular
biology methods were carried out using standard procedures.
To generate plasmid YCpLG-PI3K, the cDNA encoding PI3K-
CAAXwasexcisedfromplasmidPsg5/5MycTp110XCAAX[25]
(agiftfromM.Collado,SpanishNationalCancerCentre,Madrid,
Spain) with BamHI and cloned into the same site in yeast vector
YCpLG [26]. To produce plasmid YCpLG-PI3KK802R(where
K802R stands for Lys802→Arg), bearing a catalytically inactive
(‘kinase-dead’) allele of PI3K-CAAX, site-directed mutagenesis
was carried out using a DpnI-based strategy [27] with Turbo PfuI
DNA polymerase (Stratagene) and the primers 5?-CAGAACAA-
TGAGATCATCTTTCGAAATGGGGATGATTTACGGC-3?and
5?-GCCGTAAATCATCCCCATTTCGAAAGATGATCTCATT-
GTTCTG-3?. Cassettes in which cDNAs encoding either c-Akt,
c-AktK179Mor an N-myristoylated c-Akt were fused in frame to an
HA (haemagglutinin) epitope-tagged version of eGFP (enhanced
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PIP3-dependent PKB/c-Akt activation in yeast
615
green fluorescence protein) [HA–eGFP-Akt, HA–eGFP-AktK179M
andmyr-HA–eGFP-Aktrespectively]
HindIII and BamHI from the original Pcefl(X)-derived plasmids
that were constructed for expression in mammalian cells [28] (a
gift from M. Lorenzo, Universidad Complutense, Madrid, Spain)
and cloned into the corresponding sites in yeast vector pYES2
(Invitrogen), yielding plasmids pYES-GFP-c-Akt, pYES-GFP-
c-AktK179Mand pYES-myr-GFP-c-Akt respectively. The cDNA
encoding PTEN was excised with EcoRI from plasmid Pcmv-
pten [29] [a gift from J.M. Paramio, CIEMAT (Centro de Inves-
tigaciones Energ´ eticas, Medioambientales y Tecnol´ ogicas,
Madrid, Spain)] and inserted into the same site in pYES2, gen-
erating pYES-PTEN. Plasmid pYES-PTENG129D, expressing a
catalytically inactive (‘phosphatase-dead’) allele, was generated
bysite-directedmutagenesis,asabove,butusingprimers5?-CAC-
TGTAAAGCTGGAAAGGAACGAACTGGTGTAATG-3?(up-
per) and 5?-CATTACACCAGTTCGTTCCTTTCCAGCTTTAC-
AGTG-3?(lower).ToconstructplasmidpYES-Tep1-Myc,firstthe
TEP1codingsequencewasamplifiedbyPCRfromyeastgenomic
DNA using the primers 5?-CGGATCCATGAGAGAGGAGGGG-
AGTG-3?(upper) and 5?-GGGATCCTATAATTTCCCATTCCA-
AT-3?(lower) and cloned into the BamHI site in a yeast vector,
pRS306-Myc6,whichhadbeenpreviouslygeneratedbyinserting
a Myc6epitope into the polylinker in the integrative URA3 vector,
pRS306 [30]. Secondly, the resulting TEP1–myc6 fusion was
excised using EcoRI and cloned into the same site in pYES2.
To construct a PTEN-TEP1 chimaera, we used a now-standard
PCR-based method [31] in which PTEN was amplified using the
‘upper’ primer indicated above, TEP1 was amplified with
the ‘lower’ primer indicated above, with the following primers to
generate the desired junction: 5?-GCCTCATTAGAAATTCCT-
GGTCTATAATCCAGAT-3?
and 5?-ATCTGGATTATAGACC-
AGGAATTTCTAATGAGGC-3?. To construct a plasmid express-
ing a Tep1–GFP fusion, the TEP1 gene was amplified by PCR
using the same oligonucleotides described above, which provide
BamHI sites at both ends of the amplicon, and cloned in frame
into the BamHI site of the polylinker in pGFP-C-FUS [32],
yielding pTep1-GFP. All constructs were verified by direct DNA
sequencing. Other plasmids used in the present study were
pLA10H [33] to express GFP-tagged septin and pRS426-GFP-
2xPH(PLCδ) to visualize PIP2in vivo [34].
wereexcisedwith
Protein detection by immunoblotting
Standard procedures, as described previously [35], were used for
yeast growth, cell harvesting and cell breakage, as well as for the
preparation of protein-containing cell-free extracts, fractionation
by SDS/PAGE and transfer on to nitrocellulose membranes.
Polyclonal anti-PTEN antibodies (Upstate Biotechnology, Lake
Placid, NY, U.S.A.) were used to detect PTEN or the PTEN-
Tep1 chimaera in yeast extracts. Yeast MAPK (mitogen-activ-
ated protein kinase) Slt2 [orthologue of mammalian ERK5
(extracellular-signal-regulated kinase 5)] was detected with the
polyclonal anti-Slt2 antibodies described previously [36]. Rabbit
anti-phospho-p44/p42 MAPK (anti-P-Thr202-P-Tyr204) antibodies
(New England Biolabs, Hitchin, Herts., U.K.) were used to detect
dually phosphorylated forms of Slt2 and of two other yeast
MAPKs, Kss1 and Fus3 (orthologues of mammalian ERK1 and
ERK2). Anti-phospho-p38 (Cell Signaling Technology, Beverly,
MA, U.S.A.) was used to detect dual phosphorylation of yeast
MAPK Hog1 [orthologue of mammalian SAPKα (stress-activ-
ated protein kinase α)]. Rabbit anti-phospho-Akt(P-Thr308) and
rabbit anti-phospho-Akt(P-Ser474) (Cell Signaling Technology)
wereusedtodetectthephosphorylationofthecorrespondingsites
in PKB/c-Akt. Yeast CPY (carboxypeptidase Y) was detected
with anti-CPY antiserum (Molecular Probes). Yeast actin was
detected by cross-reaction with a mouse anti-actin monoclonal
antibody (clone C4; MP Biomedicals, Irvine, CA, U.S.A.). GFP
fusion proteins were detected using a mouse anti-GFP antibody
(JL-8; BD Biosciences, San Jose, CA, U.S.A.). After washing,
bound primary antibodies were revealed using horseradish
peroxidase-conjugated anti-rabbit or anti-mouse secondary anti-
bodies,asappropriate,andachemiluminescencedetectionsystem
(ECL®; Amersham Biosciences). For quantitative measurements
on immunoblots, densitometry [in a GS-800 densitometer, model
Power Look 2100xL (Bio-Rad), using the software Quantity
One] was applied to films and the extent of Thr308and Ser473
phosphorylation was normalized to the amount of protein
recovered in the extract as follows. Ratios of c-AktK179M/c-Akt
were calculated by dividing the percentages of the phospho-
rylation signal of Thr308or Ser473of GFP–c-AktK179Mrelative
to GFP–c-Akt by the corresponding percentages of the GFP
signal of GFP–c-AktK179Msignal relative to GFP–c-Akt. To obtain
these ratios, duplicate measurements on at least three different
experiments were performed.
Microscopy techniques and immunofluorescence
Forfluorescencemicroscopyofliveyeastcells(tovisualizeGFP),
exponentially growing cultures were harvested by brief centri-
fugation, washed once with sterile water and viewed directly.
To observe actin, yeast cells were treated with FITC-conjugated
phalloidin (Sigma) as described previously [37]. To monitor
vacuolar morphology and bulk fluid-phase endocytosis into
the vacuole, staining with FM4-64 [38] and Lucifer Yellow [39]
respectively was performed. To obtain reliable and statistically
significant data, ?200 cells were examined for each condition or
experiment. Indirect immunofluorescence with monoclonal anti-
PIP3antibodies (Echelon, Salt Lake City, UT, U.S.A.) at a 1:200
dilution was performed, essentially as described for animal cells
[40], using Cy3-conjugated monoclonal anti-mouse IgG anti-
bodies (Chemicon, Temecula, CA, U.S.A.) at a 1:500 dilution as
the secondary antibodies. Cells were examined under an Eclipse
TE2000U microscope (Nikon, Tokyo, Japan) and digital images
were acquired with an Orca C4742-95-12ER charge-coupled-
device camera (Hamamatsu Photonics, Hamamatsu City, Japan)
and Aquacosmos Imaging Systems software.
RESULTS
Expression of PI3K inhibits yeast cell growth and generates PIP3
For expression in yeast, the cDNA encoding p110α was cloned
under the control of the galactose-inducible GAL1 promoter. To
target the enzyme to the plasma membrane in the absence of
the p85 subunit, the -CAAX box of H-ras was fused in frame
to the C-terminus of p110α. Wild-type Sacch. cerevisiae cells
(YPH499) transformed with this construct grew normally on
glucose medium, but failed to grow on galactose medium (Fig-
ure 1A). The growth inhibition caused by PI3K expression was
alleviatedbyapointmutation(K802R,Lys802→Arg)inaresidue
essential for enzyme activity. Hence, the growth inhibition was
duetothecatalyticfunctionofPI3Kandnottheresultofanynon-
specific toxic effect of producing this heterologous membrane-
targeted polypeptide in yeast.
To determine whether the presence of active PI3K generates
PIP3at the expense of PIP2, both phosphoinositides were visual-
ized in the cells. To detect PIP2, cells were co-transformed with
a plasmid expressing a fluorescent PIP2-specific reporter [GFP
fused to two tandem copies of the PH domain of mammalian
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Figure 1Expression of active PI3K in yeast inhibits growth, depletes PIP2and generates PIP3at the plasma membrane
(A)Awild-typeyeaststrain(YPH499)carryingaURA3-markedvector(pYES2)wastransformedwithanotheremptyLEU2-markedvector(YCpLG)orthesamevectorharbouringaconstructtoexpress,
under the control of a galactose-inducible promoter (GAL1), either membrane-targeted mammalian PI3K, namely p110α-CAAX (YCpLG-PI3K), or a catalytically inactive mutant (YCpLG-PI3KK802R).
Serial 10-fold dilutions of cultures of representative transformants were spotted on minimal synthetic (S) medium lacking Ura and Leu under repressing (glucose) or inducing (galactose) conditions.
(B)Wild-typecells(YPH499)carryinganemptyLEU2-markedvector(YCpLG)orthesamevectorexpressingPI3K(YCpLG-PI3K)weretransformedwithaURA3-markedvectorexpressingtwotandem
copies of the PIP2-specific PH domain of mammalian PLCδ1 fused to GFP [pRS426-GFP-2xPH(PLCδ)]. Representative transformants were propagated on SGlc-Leu-Ura, shifted to SGal-Leu-Ura
for 6h, and then viewed under the fluorescence microscope. (C) Wild-type cells (YPH499) were transformed with empty vector (YCpLG) or the same vector expressing PI3K (YCpLG-PI3K).
Representative transformants were propagated on SGlc-Leu, shifted to SGal-Leu for 6h, fixed, permeabilized, stained with specific anti-PIP3antibodies (Echelon), counterstained with fluorescently
labelled secondary antibodies and then viewed under the fluorescence microscope.
PLCδ1 (phospholipase δ1)]. In yeast, this reporter specifically
decorates the plasma membrane [34]. Indeed, in our control cells,
the reporter clearly stained the plasma membrane prominently.
In contrast, in the cells where catalytically active PI3K was ex-
pressed, only cytoplasmic fluorescence was seen, indicating a
dramatic depletion of PIP2 from the plasma membrane (Fig-
ure 1B). To confirm that PIP3was produced at the expense of
PIP2, the same cells were fixed and stained with an anti-PIP3
antibody. Similar to other methods that failed to detect PIP3in
Sacch. cerevisiae [14,15], no staining was seen in the control
cells. In contrast, large patches of fluorescent staining with the
anti-PIP3antibodies were found around the perimeter of the cells
expressing functional PI3K (Figure 1C). Thus the PI3K produced
was catalytically active, and the most likely cause of its observed
inhibitory effect on yeast cell growth was efficient conversion of
the PIP2pool into PIP3.
Expression of PTEN antagonizes the effect of PI3K in yeast
If the growth inhibitory action of PI3K is due to conversion of
PIP2into PIP3, then reversal of this reaction by PTEN should
ameliorate growth arrest. Therefore PTEN cDNA was cloned
under the control of the GAL1 promoter in a vector compatible
with that used to express PI3K. Expression of PTEN alone at a
highlevel,confirmedbyimmunoblottingwithspecificanti-PTEN
antibodies(resultsnotshown),hadnodiscernibleeffectongrowth
or morphology (Figure 2A). However, in cells expressing PI3K,
co-expressionofPTENpreventedgrowtharrest.Apointmutation
(G219E) reported to abrogate the lipid phosphatase activity of
PTEN [41] blocked its ability to reverse PI3K-induced growth
inhibition (Figure 2A). Thus the counteracting effect of PTEN on
PI3K-initiated signalling was reproduced in yeast cells.
Other authors have suggested that the Sacch. cerevisiae TEP1
gene encodes a PTEN orthologue [18,19]. Tep1 is similar to
Figure 2
expression of active mammalian PTEN, but not by Sacch. cerevisiae Tep1
Growth inhibition caused by PI3K expression is relieved by co-
(A) Wild-type cells (YPH499) were co-transformed with the following sets of URA3-
and LEU2-marked vectors: pYES2 and YCpLG-PI3K (top row); pYES-PTEN and YCpLG
(second row); pYES-PTEN and YCpLG-PI3K (third row); pYES-PTENG129E(catalytically
inactive) and YCpLG-PI3K (fourth row); pYES-TEP1-Myc and YCpLG-PI3KA (fifth row); and
pYES-PTEN-TEP1 and YCpLG-PI3K (bottom row). Serial 10-fold dilutions of cultures of
representativetransformantswerespottedonminimalsynthetic(S)mediumlackingUraandLeu
under repressing (glucose) or inducing (galactose) conditions. (B) Wild-type cells (YPH499)
carrying a URA3-marked plasmid (pTep1-GFP) expressing a Tep1–GFP fusion under the
control of the methionine-repressible MET25 promoter were grown on complete SGlc-Ura-Met
medium for 8h, and observed by transmitted light (left panel) or by fluorescence (right panel)
microscopy.
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PTENthroughitsN-terminalcatalyticregion(27%identityfrom
residues1–260),butcontainsalargeinsert(residues104–160)and
also differs completely from PTEN over its C-terminal segment.
Although Ptn1, the apparent PTEN homologue in fission yeast,
hydrolyses 3-phosphoinositides in vitro [14], no such activity for
budding yeast Tep1 has been detected [42]. Normally, TEP1 is
expressed only in MATa/MATα diploids undergoing meiosis and
sporulation [19]. Therefore TEP1 (fused in frame at its C-ter-
minus to six tandem c-Myc epitopes for immunodetection) was
cloned in a vector under GAL1 promoter control. This Tep1
derivativerescuesthephenotypesoftep1?/tep1?cellsandhasno
detectably deleterious effect on haploid cells (results not shown).
However, Tep1 expression was unable to alleviate PI3K-induced
growth inhibition (Figure 2A), even though immunoblotting
with specific anti-c-Myc antibodies confirmed that Tep1 was
expressed at a high level (results not shown). Thus, as observed
invitro,Tep1maybeincapableofdephosphorylatingPIP3invivo.
However,aTep1–GFPfusionwaslocalizedexclusivelyasdiscrete
cytoplasmicspots(Figure2B),reminiscentofthecytosolicpuncta
reportedforSchizo.pombePtn1[19],andwasneverdetectedatthe
cell periphery (or even as a diffuse cytoplasmic signal) in any of
the ?200 cells examined. Therefore, even if Tep1 is a functional
3-phosphoinositide phosphatase, it may be incapable of gaining
access to the plasma membrane-localized pool of PIP3generated
by PI3K. In contrast, PTEN displayed a diffuse cytoplasmic
pattern in yeast, revealed by indirect imunofluorescence using
anti-PTEN antibodies (results not shown) and, operationally,
must gain access to the cytosolic face of the plasma membrane.
Unlike PTEN itself, a PTEN1-190-Tep1261-434chimaera, in which
the unique C-terminal extension of Tep1 was fused in frame
totheN-terminal catalyticdomainofPTEN,wasunabletorescue
the growth inhibition caused by PI3K (Figure 2A), even though
expression of the chimaera in yeast lysates (detected with anti-
PTEN antibodies) was as efficient as that of PTEN itself (results
not shown). This result suggests that a localization signal in the
C-terminal extension of Tep1 prevents the protein from reaching
the plasma membrane.
Sublethal concentrations of LY294002 partially alleviate
PI3K-induced growth arrest
Wortmannin and LY294002 are two relatively specific inhibitors
of mammalian PI3K, effective over the nanomolar range [1]. The
sole PI3K in Sacch. cerevisiae, Vps34, converts phosphatidyl-
inositol into phosphatidylinositol 3-phosphate, but cannot use
PIP2; at the nanomolar level, neither wortmannin nor LY294002
has any effect on Vps34 or on protein trafficking or cell growth,
largely due to their poor penetration through the yeast cell wall
[43]. At much higher doses, wortmannin shows some toxicity
againstyeast[44].Likewise,overthemillimolarrange,LY294002
was inhibitory to the growth of our strain (IC50∼1.5 mM).
However, at this concentration, LY294002 actually stimulated
the growth of cells expressing mammalian PI3K by 3–5-fold
(Figure 3). Therefore compounds that inhibit human PI3K, but
are generally non-toxic, could be selected from chemical libraries
by screening for those that fully restore growth to yeast cells
expressing p110α. Moreover, any compound that acts on yeast
will surely enter an animal cell.
PI3K expression disrupts the cytoskeleton and alters secretory and
endocytic trafficking
Because p110α expression efficiently converted plasma-mem-
brane PIP2into PIP3and prevented growth, it provided a novel
means to examine the physiological consequences of this pertur-
bation and thereby shed light on the essential functions of PIP2
Figure 3
imposed growth inhibition
Sublethal doses of a specific PI3K inhibitor partially relieve PI3K-
Wild-type cells (YPH499) were transformed with an empty vector (YCpLG; white bars) or with
the same vector expressing PI3K (YCpLG-PI3K; black bars). Representative transformants were
propagated in SGlc-Leu medium, then shifted to SGal-Leu medium containing the indicated
concentrations of LY294002 (Biomol), and cell growth after 24 h in the SGal-Leu medium was
determinedbymeasuringA at600nm.Valuesshownarethemeansforthreeindependenttrials
(error bars represent S.D. values).
in yeast. Upon shift to galactose, asynchronous cultures of
PI3K-expressing cells did not arrest with a uniform cellular or
nuclear morphology, assessed by microscopy and flow cytometry
(resultsnotshown),indicatingthatlossofplasmamembranePIP2
does not block cell-division cycle progression at a specific stage.
PIP2hasbeenimplicatedasaregulatorofboththeactin[45]and
septin [46]cytoskeletons. Oursystem provideda uniquemeansto
confirm and extend those conclusions. In control cells, the actin
cytoskeleton showed a highly polarized distribution, with
actinfilamentsinthemothercellandactinpatchesrestrictedtothe
bud (Figure 4A, left panel). In the same strain expressing PI3K,
this asymmetry was lost because actin patches and diffuse actin
staining were found in more than 80% of the mother cells (Fig-
ure 4A, right). Similarly, in control cells, a fluorescently tagged
septin was incorporated exclusively into the collar-like structure
found at the mother-bud neck (Figure 4B, left panel), whereas
in the same strain expressing PI3K, abnormal septin-containing
structures were seen in >50% of cells (Figure 4B, right panel).
Thus PIP2in the plasma membrane is important for maintaining
proper architecture of both the actin and septin cytoskeletons.
Since the functions of both structures are essential for yeast
cell viability, their disruption is sufficient to explain the growth
inhibitory effect of PI3K expression.
Specific phosphoinositides have been implicated in both
secretory and endocytic transport in yeast [47]. To examine se-
cretion, we monitored the precursor forms of vacuolar CPY
because their productive processing and maturation depends on
their efficient delivery by Golgi-derived vesicles. In control cells,
both the p1 and p2 precursor forms, and also the mature (m)
form, were observed by immunoblotting (Figure 4C, left panel),
whereas in cells expressing PI3K, the ER (endoplasmic reti-
culum)-specific precursor (p1) accumulated at the expense of the
p2form(Figure4C,right),althoughtheeffectwasrathermildand
the amount of mature CPY generated was little affected. Using
similarmeans,maturationofothervacuolar(alkalinephosphatase
Pho8) and exocytic (cell-wall protein Gas1) marker proteins was
unaffected (results not shown).
Endocytic transfer of plasma-membrane material to the yeast
vacuole membrane can be monitored by staining with a lipophilic
c ?2005 Biochemical Society