Download Interaction with PI3-kinase contributes to the cytotoxic activity ofApoptin S Maddika

Oncogene (2008) 27, 3060–3065
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SHORT COMMUNICATION
Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin
S Maddika1,2,10,11, E Wiechec1,3,11, SR Ande1,2,11, IK Poon4, U Fischer5, S Wesselborg6, DA Jans4,
K Schulze-Osthoff5 and M Los1,2,7,8,9
1
Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada; 2Department of
Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada; 3Institute of Human Genetics, University
of Aarhus, Aarhus, Denmark; 4Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash
University, Clayton, Victoria, Australia; 5Institute of Molecular Medicine, University of Düsseldorf, Düsseldorf, Germany;
6
Department of Internal Medicine I, University of Tübingen, Tübingen, Germany; 7Manitoba Institute of Child Health, University of
Manitoba, Winnipeg, Manitoba, Canada; 8Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg,
Manitoba, Canada and 9BioApplications Enterprises, Winnipeg, Manitoba, Canada
Apoptin, a small protein from the chicken anemia virus,
has attracted attention because of its specificity in killing
tumor cells. Localization of apoptin in the nucleus of
tumor cells has been shown to be vital for proapoptotic
activity, however, targeted expression of apoptin in the
nucleus of normal cells does not harm the cells, indicating
that nuclear localization of apoptin is insufficient for its
cytotoxicity. Here, we demonstrate for the first time that
apoptin interacts with the SH3 domain of p85, the
regulatory subunit of phosphoinositide 3-kinase (PI3-K),
through its proline-rich region. Apoptin derivatives devoid
of this proline-rich region do not interact with p85, are
unable to activate PI3-K, and show impaired apoptosis
induction. Moreover, apoptin mutants containing the
proline-rich domain are sufficient to elevate PI3-K activity
and to induce apoptosis in cancer cells. Downregulation of
p85 leads to nuclear exclusion of apoptin and impairs cell
death induction, indicating that interaction with the p85
PI3-K subunit essentially contributes to the cytotoxic
activity of apoptin.
Oncogene (2008) 27, 3060–3065; doi:10.1038/sj.onc.1210958;
published online 3 December 2007
Keywords: anticancer drugs; apoptin; apoptosis;
PI3-kinase
Apoptin, a 14 kDa protein encoded by VP3 gene of
chicken anemia virus, represents a novel potential
anticancer therapeutic, because it induces apoptotic
death specifically in tumor but not normal cells (Fischer
and Schulze-Osthoff, 2005; Alvisi et al., 2006). The
mechanism by which apoptin is able to distinguish
between tumor and normal cells remains to be
Correspondence: Dr M Los, BioApplications Enterprises, Winnipeg,
Manitoba, Canada R2V 2N6.
E-mail: [email protected]
10
Current address: Department of Therapeutic Radiology, Yale School
of Medicine, New Haven, CT, USA.
11
These authors contributed equally to this work
Received 17 September 2007; revised 23 October 2007; accepted 29
October 2007; published online 3 December 2007
elucidated. The subcellular localization of apoptin
appears to be important for the selective toxicity
towards transformed and cancer cells. In cells resistant
to apoptin-mediated cell death, the protein has a
cytoplasmic localization, whereas in sensitive cells,
apoptin is found in the nucleus (Danen-Van Oorschot
et al., 2003). Apoptin was described to be specifically
phosphorylated in transformed cells by an unknown
kinase, which was proposed to be important for the
nuclear localization and apoptotic activity of apoptin
(Rohn et al., 2002). However, it was also shown that a
C-terminally truncated apoptin mutant, in which the
phosphorylation site was deleted, was still able to
translocate to the nucleus and induce apoptosis (Guelen
et al., 2004). Thus, the interaction with other molecules
or additional modifications of apoptin that are not
present in nontransformed cells may be required for the
selective cytotoxicity of apoptin.
Employing a mass spectrometric approach, we
previously found that apoptin interacts with the
p85 regulatory subunit of phosphoinositide 3-kinase
(PI3-K) (Maddika et al., 2007). Class I PI3-Ks play a
central role in various regulatory processes, such as cell
growth, survival and differentiation, by converting
phosphatidylinositol-4,5-phosphate to phosphatidylinositol-3,4,5-phosphate, leading to the activation of AKT
kinase and other downstream effectors (Fruman et al.,
1998; Vanhaesebroeck and Waterfield, 1999). To investigate the relevance of an interaction of apoptin with
the p85 subunit of PI3-K, we performed glutathione Stransferase (GST)–apoptin pull-down assays using total
cell extracts of different tumor cell lines, including PC-3
prostate cancer cells, L929 mouse fibrosarcoma cells,
293 transformed human embryonic kidney cells and
MCF-7 human breast cancer cells. We detected that
apoptin interacts with the p85 subunit in all the cell lines
tested (Figure 1a), indicating that interaction of apoptin
with p85 is neither cell type- nor species-specific.
To identify and map the sites on apoptin responsible
for interaction with PI3-K, PC-3 cells were transfected
to express either full-length green fluorescent protein
(GFP)-apoptin or various deletion mutants tagged
with an N-terminal GFP (Figure 1b). Apoptin was
Interaction of apoptin with PI3-kinase
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Figure 1 In vivo association of apoptin with the p85 subunit of PI3-K. (a) GST pull-down assay performed with PC-3, MCF-7, 293
and L929 cell lysates with either GST or GST–apoptin. The proteins cloned in PGEX-2T (Amersham Biosciences, Mississauga, ON,
Canada) were along with the different cell lysates immobilized on glutathione sepharose beads overnight at 4 1C. The beads were
washed thrice with ice-cold lysis buffer and bound proteins were separated on sodium dodecyl sulphate–PAGE. The presence of the
p85 subunit of PI3-K in apoptin complexes was determined by immunoblotting using anti-p85 and horseradish peroxidase (HRP)
coupled anti-mouse IgG (Upstate Cell Signaling, Lake Placid, NY, USA). (b) Schematic representation of apoptin deletion mutants
tagged with N-terminal GFP. The deletion mutants (Poon et al., 2005) and wild type apoptin (1–121) were transfected into PC-3 cells,
immunoprecipitated with anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 18 h post-transfection, and the
association of p85 was detected by immunoblotting. (c) Induction of apoptosis by the different apoptin mutants. The percentage of
apoptosis was assessed in PC-3 cells by flow cytometric detection of hypodiploid DNA (left panel) and in MCF-7 cells by the MTT
assay (right panel) 24 h post-transfection essentially as described previously (Maddika et al., 2005; Burek et al., 2006).
immunoprecipitated with anti-GFP antibodies at 24 h
post-transfection, and the immune complexes were
analysed for interaction with PI3-K by immunoblotting
using anti-p85 antibodies. PI3-K was found in the
immunoprecipitates of full-length apoptin and apoptin
derivatives that harbored amino acids 74–100 (a prolinerich sequence), implying that this region of apoptin is
important for interaction with PI3-K (Figure 1b). We
then tested whether interaction of PI3-K with apoptin is
essential for apoptin-induced cell death by assessing the
percentage of apoptosis induced by the different
mutants. In agreement with the interaction data, only
those mutants that had the intact interaction site
for PI3-K were able to induce apoptosis both in PC-3
and MCF-7 cells (Figure 1c). Interestingly, the mutant
Ala-108, which was reported to be noncytotoxic due the
substitution of a Thr-phosphorylation site (Rohn et al.,
2002), was still able to induce significant apoptosis, as
long as its interaction site with the PI3-K remained intact.
Next, we investigated the apoptin interaction site on
p85, which is attached to the p110 catalytic subunit and,
depending on its conformation, can act as a repressor or
inducer of kinase activity. The p85 subunit contains an
N-terminal SH3 domain, two proline-rich domains
and a breakpoint cluster region homology domain
(Figure 2a). p85 also has two SH2 domains (nSH2,
cSH2) that are separated by a coiled-coil region, called
the inter (i) SH2 domain, which mediates the stable
dimerization with the p110 catalytic subunit. Under
resting conditions, p85 inhibits PI3-K activity. The
binding of tyrosine-phosphorylated proteins to the
nSH2 domain of p85 inhibits PI3-K and is most
frequently responsible for kinase activation, however,
alternate mechanisms have been reported. For example,
the conformational switch between p85 and p110
holoenzyme can also occur upon the interaction of
signaling mediators with the SH3 or breakpoint cluster
region homology domain (Liu et al., 1993; Prasad et al.,
1993; Pleiman et al., 1994; Zheng et al., 1994). To
investigate the apoptin interaction site of the p85
subunit, we cotransfected PC-3 cells with a vector
encoding full-length apoptin, together with full-length
p85 or various deletion mutants. Both full-length
p85 and the mutant lacking the iSH2 domain were
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Interaction of apoptin with PI3-kinase
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Figure 2 Apoptin interacts with SH3 domain of p85 and activates PI3-K activity. (a) Schematic representation of the PI3-K p85
deletion mutants highlighting the structural domains. The p85 mutants (Jascur et al., 1997) were cotransfected together with apoptin
into PC-3 cells. A 28 h post-transfection, cells were lysed, and p85 was immunoprecipitated using either anti-p85 (for wild type and
DiSH2 mutants) or anti-HA antibodies (Upstate) for the HA-tagged mutants. Apoptin interaction with the mutants was detected using
antiapoptin antibodies. Expression of the deletion mutants was verified by anti-p85 or anti-HA antibodies. (b) PI3-K activity was
measured 24 h after transfecting PC-3 cells with the indicated apoptin mutants using an ELISA (Echleon Biosciences). (c) The indicated
p85 PI3-K mutants were expressed after transfection in PC-3 cells. A 24 h post-transfection, p85 was immunoprecipitated from cell
lysates and assessed for PI3-K activity as described above. (d) PC-3 cells were either transfected with apoptin alone, pretreated with
wortmannin (5 nM) or LY294002 (1.5 mM) followed by apoptin transfection, or transfected to coexpress apoptin and a dominantnegative (DN) PI3-K mutant (Kauffmann-Zeh et al., 1997).
immunoprecipitated by the anti-p85 antibody, while
other mutants were immunoprecipitated using antihemagglutinin antibodies. Immunodetection of apoptin
in the immune complexes revealed that apoptin interaction
required an intact SH3 domain of p85 (Figure 2a).
To determine the functional significance of the
interaction of apoptin with the p85-regulatory subunit,
we measured the PI3-K activity by using a nonradioactive ELISA-based method. We tested whether the
activation of PI3-K is a direct consequence of apoptin’s
interaction by observing the PI3-K activity in cells
transfected with different apoptin deletion mutants
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(Figure 2b). Full-length apoptin induced six-fold activation, while the 1–89 mutant, which may contain only a
partially preserved interaction site, weakly activated
PI3-K. The mutants 1–111, 74–121, Ala-108 and Glu-108
were able to activate PI3-K similar to the levels of
activation by the full-length apoptin. In contrast, the
1–73 and 104–121 mutants were unable to activate
beyond basal levels, indicating that the intact p85
interaction site on apoptin is crucial for PI3-K activation
(Figure 2b).
On the other hand, the observed PI3-K activity in
cells cotransfected with full-length apoptin and p85
Interaction of apoptin with PI3-kinase
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deletion mutants confirmed that the interaction of
apoptin at the SH3 domain of p85 is essential for PI3-K
activity (Figure 2c). The p85 mutants with both the SH3
and iSH2 domains had constitutive PI3-K activity in the
presence of apoptin, whereas others lacking these
domains were severely impaired in their activity
(Figure 2c). Interestingly, the mutant N þ I þ C þ iSH2,
containing only the SH2 domains but lacking the SH3
domain, conferred relatively higher PI3-K activity
compared to other deletion mutants and the control,
indicating that the intact SH3 domain in the wild-type
p85 protein might have an inhibitory action on its
activity (Figure 2c). In control experiments, different
PI3-K inhibitors, including wortmannin and LY294002,
and expression of a dominant-negative PI3-K mutant
reduced the apoptin-induced PI3-K activation to the
basal levels (Figure 2d). Thus, the interaction of apoptin
with the SH3 domain might lead to a conformational
change of p85, converting it from an inhibitory to an
active state.
To investigate the functional significance and the
requirement of PI3-K activity towards apoptin-induced
cell death, we used RNA interference and tested whether
knocking down endogenous p85 would affect resistance
to apoptin-induced cell death. In PC-3 and MCF-7 cells,
the expression of p85 was completely abrogated by p85specific small interfering (si) RNA but not by a control
siRNA (Figure 3a), as assayed 30 h post-transfection.
The assay revealed that cells lacking p85 expression
were strongly resistant towards apoptin-induced cell
death compared to control siRNA-expressing cells
(Figure 3b). Similar observations were made in 293 cells
(data not shown).
We also tested whether PI3-K inhibition affects
subcellular localization of apoptin. Apoptin was mainly
localized in the nucleus of PC-3 and MCF-7 cells, but in
the presence of wortmannin, apoptin was found mainly
distributed in the cytoplasm, as shown either by
immunostaining and confocal laser microscopic imaging
(Figure 4a) or by subcellular fractionation and western
blotting (Figure 4c). The effect of PI3-K inhibition on
the localization of apoptin was further studied using p85
siRNA. Upon inhibition of p85 expression, apoptin was
almost completely excluded from the nucleus
(Figure 4b), indicating that PI3-K activity is necessary
for not only apoptin-induced cell death, but also for the
nuclear localization of the protein.
In summary, we demonstrate that apoptin interacts
with the p85 subunit of PI3-K in various cell types.
Apoptin specifically associates with the SH3 domain
of p85, leading to the activation of PI3-K presumably
by a conformational change. Importantly, downregulation of the p85 subunit by RNA interference or
PI3-K inhibition severely impairs apoptin-induced cell
death.
p85 specifically interacts with the proline-rich domain
of apoptin localized between amino acids 80 and 90. The
PKPPSK sequence (amino acids 81–86) of apoptin is a
perfect consensus motif (PxxPxR/K) recognized by SH3
domains. Mutants lacking this particular proline-rich
region were unable to interact with the p85, were not
able to activate PI3-K pathway and were nontoxic. This
clearly implies that apoptin interacts with SH3 domain
of p85 and activates PI3-K. Previously, it has been
shown that phosphorylation of Thr-108 is specific for
tumor cells and is required for apoptin-induced cell
Figure 3 Downregulation of p85 impairs apoptin-induced apoptosis. (a) PC-3 cells (left panels) and MCF-7 cells (right panels) were
transfected with a p85 siRNA plasmid (Upstate Cell Signaling) or control siRNA for 48 h using Lipofectamine (Invitrogen, Burlington,
ON, Canada). Downregulation of p85 was verified by immunoblotting; tubulin was used as loading control. (b) PC-3 cells (left panel)
and MCF-7 cells (right panel) were transfected with p85 siRNA or control siRNA vectors, followed by transfection after 48 h to
express apoptin. After further 24 and 48 h, cell death was measured as described in Figure 1c.
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Interaction of apoptin with PI3-kinase
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Figure 4 Downregulation of p85 affects apoptin localization. (a) The localization of GFP-apoptin in MCF-7 and PC-3 cells was
detected by confocal microscopy after transfection in the absence or presence of wortmannin (5 nM). (b) The effect of p85 knockdown
on apoptin localization was determined by confocal microscopy, 24 h post-transfection with GFP-apoptin with or without a
cotransfected p85 siRNA plasmid. The expression of p85 was detected by immunohistochemistry as described (Janssen et al., 2007)
using anti-p85 and a Cy-3-conjugated secondary antibody. (c) The nuclear/cytoplasmic distribution of apoptin in the presence of
wortmannin or p85-inhibitory siRNA in both MCF-7 and PC-3 cells was investigated by subcellular fractionation, followed by western
blotting as described previously (Maddika et al., 2005). Scale bars: 10 mm.
death (Rohn et al., 2002). However, phosphorylation of
apoptin was severely impaired in a mutant lacking
amino acids 80–90, even though the residues within the
phosphorylation site remained intact (Rohn et al., 2002).
Our results explain this interesting observation by
showing that amino acids 80–90 are vital for the
interaction with PI3-K, and this interaction may
be required for further activation of downstream molecules.
Mutants of Thr-108, that is Ala-108 and Glu-108,
were still cytotoxic, indicating that phosphorylation of
apoptin is not of crucial importance.
Our data further suggest that the PI3-K pathway is
involved in localization of apoptin. Inhibition of the
PI3-K pathway using wortmannin or RNA interference
retained apoptin in the cytoplasm and led to its nuclear
exclusion, hence impairing cell death. PI3-K has been
mainly implicated in lipid signaling at the cell
membrane, but increasing evidence indicates that all
components of the PI3-K pathway also reside in the
nucleus (Neri et al., 2002). The functions regulated
by nuclear PI3-K are still rather unclear. Although PI3K is important for apoptin localization, it may not be a
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direct regulator, as we did not observe any direct
phosphorylation of apoptin by PI3-K in in vitro kinase
assays (data not shown). Apoptin phosphorylation
may, however, be regulated by downstream effectors
of the PI3-K pathway, a possibility that we are
currently investigating. In conclusion, the data presented here not only advance our knowledge on the
mechanisms that are activated by apoptin to kill
cancer cells, but they also demonstrate that the
prosurvival PI3-K/Akt pathway, frequently aberrantly
active in cancer, may have additional functions in cell
death propagation.
Acknowledgements
We thank J Downward and T Mustelin for valuable reagents.
SM was funded by a studentship from the Manitoba Health
Research Council (MHRC) and the CancerCare Manitoba
Foundation (CCMF). EW was supported through ITMHRT.
ML thankfully acknowledges the support by the CFI-Canada
Research Chair program, MICH-, CIHR, CIHR-IPM-,
CCMF- and MHRC-financed programs.
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