Phosphorylation by polo-like kinase 1 induces the tumor-suppressing
activity of FADD
M-S Jang1, S-J Lee1, C-J Kim2, C-W Lee3and E Kim1,4
1College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, Korea;2College of Veterinary Medicine,
Chungnam National University, Daejeon, Korea;3Department of Molecular Cell Biology, School of Medicine, Sungkyunkwan
University, Suwon, Korea and4BK21 Daedeok R&D Innopolis Bio Brain Center, Chungnam National University, Daejeon, Korea
Phosphorylation of the Fas-associated death domain
(FADD) protein sensitizes cancer cells to various chemo-
therapeutics. However, the molecular mechanism under-
(P-FADD) is poorly understood. In this study, we describe
the physical interactions and functional interplay between
Polo-like kinase 1 (Plk1) and FADD. Plk1 phosphory-
lates FADD at Ser-194 in response to treatment with
taxol. Overexpression of a phosphorylation-mimicking
mutant, FADD S194D, caused degradation of Plk1 in an
ubiquitin-independent manner, and delayed cytokinesis,
consistent with the expected cellular phenotype of Plk1
deficiency. This demonstrates that Plk1 is regulated via
a negative feedback loop by its substrate, FADD.
Overexpression of FADD S194D sensitized HeLa cells
to a low dose of taxol independently of caspase activation,
whereas overexpression of FADD S194D resulted in
caspase activation in response to a high dose of taxol.
Therefore, we examined whether the death potential of
P-FADD affected Plk1-mediated tumorigenesis. Trans-
fection of FADD S194D inhibited colony formation by
Plk1-overexpressing HeLa cells (HeLa-Plk1). Moreover,
overexpression of FADD S194D suppressed tumorigenesis
in nude mice xenografted with HeLa-Plk1. Therefore, this
study reports the first in vivo validation of tumor-suppressing
activity of P-FADD. Collectively, our data demonstrate that
in response to taxol, Plk1 endows death-promoting and
tumor-suppressor functions to its substrate, FADD.
Oncogene (2011) 30, 471–481; doi:10.1038/onc.2010.423;
published online 4 October 2010
Keywords: FADD; Plk1; phosphorylation; tumor sup-
pressor; negative feedback
Polo-like kinase 1 (Plk1) is a well-characterized oncopro-
tein that is highly expressed in various cancer tissues
(Eckerdt et al., 2005). Overexpression of Plk1 in murine
fibroblasts (that is, NIH3T3 cells) yielded a transformed
phenotype in culture, with large cells having multiple
fragmented nuclei, and caused tumor formation in nude
mice (Smith et al., 1997). However, Plk1 heterozygous
mice also developed spontaneous tumors (Lu et al., 2008),
implying that regulation of Plk1 expression is important in
preventing tumorigenesis. Many studies have shown
correlations between Plk1 expression and poor prognosis
in various cancers (Takai et al., 2005). Therefore, elevated
expression of Plk1 is considered as a novel prognostic
marker in many types of cancer. Several Plk1 inhibitors
are currently under investigation, including ON01910,
BI2536, GSK461364A, DH166 and BI6727 (Gumireddy
et al., 2005; Steegmaier et al., 2007; Gilmartin et al., 2009;
Rudolph et al., 2009; Zhang et al., 2009).
Plk1 is a 67kDa serine/threonine protein kinase that
has pivotal roles in several G2- and M-phase-related
events, namely centrosome maturation, a-tubulin asso-
ciation with centrosomes, bipolar spindle formation,
chromosome segregation and cytokinesis (Barr et al.,
2004; Strebhardt and Ullrich, 2006). Plk1 has conserved
sequence motifs in two domains: the N-terminal serine/
threonine catalytic domain and the C-terminal non-
catalytic domain, the latter of which is termed the polo-
box domain (PBD). The PBD has an important role in
regulating interactions with substrates (Cheng et al.,
2003). Consistent with the prominent role of Plk1 in G2/
M phase, Plk1 expression and activity remain low
throughout G0, G1 and S phases, rise in G2 phase
and peak during M phase (Strebhardt and Ullrich,
2006). Elevated expression of Plk1 during G2/M phase
promotes the degradation of Plk1 by anaphase-promot-
ing complex/cyclosome via the ubiquitin–proteasome
pathway, as cells exit mitosis (Lindon and Pines, 2004).
(FADD) was first identified as an adapter molecule
involved in formation of a death-inducing signaling
complex upon Fas stimulation (Peter et al., 1999). The
FADD protein mediates two types of cell death path-
ways (Vanden Berghe et al., 2004). The FADD protein
is recruited to the death receptor via a death domain
(DD) and interacts with the death effector domain of
caspase-8 through a FADD-intrinsic death effector
domain. The FADD/caspase-8 interaction provides a
platform for caspase-8 activation and subsequently
leads to activation of caspase-3, a hallmark of apoptosis.
Received 23 December 2009; revised and accepted 23 July 2010;
published online 4 October 2010
Correspondence: Dr E Kim, College of Biological Sciences and
Biotechnology, Chungnam National University, 220, Gung-Dong,
Yuseong-Gu, Deajeon 305764, Korea.
Oncogene (2011) 30, 471–481
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
In addition, FADD has an important role in necrosis.
Necrotic cell death was stimulated by oligomerization
of FADD-death domain in caspase-8-inactivated Jurkat
cells (Kawahara et al., 1998). Furthermore, FADD
binds to RIP1 (receptor-interacting protein 1) kinase, an
important necrotic marker (Vanden Berghe et al., 2004).
Consistent with these characteristics, overexpression of
FADD-death domain sensitized TNF-resistant U937
and NIH3T3 cells, leading to necrotic cell death
(Khwaja and Tatton, 1999).
The phosphorylation status of FADD serves as a
valuable marker for human cancer progression (Shima-
da et al., 2005). Expression of phosphorylated FADD
(P-FADD) is lower in cancer cells than in normal
epithelial cells. Expression of P-FADD increases in
response to chemotherapeutic treatments, and over-
expression of P-FADD sensitizes cancer cells to such
materials. However, expression of a phosphorylation-
deficient FADD mutant (FADD S194A) abolished
sensitization to chemotherapeutics (Shimada et al.,
2002, 2004, 2005), prompting investigators to identify
the kinase(s) responsible for the phosphorylation of
FADD. Casein kinase 1a (CK1a) was shown to
phosphorylate serine 194 of FADD (Alappat et al.,
2005), and an unknown kinase of B70kDa was thought
to phosphorylate FADD (Scaffidi et al., 2000).
This study demonstrates that Plk1 phosphorylates
FADD upon taxol treatment and thereby promotes
degradation of Plk1, resulting in negative feedback regu-
lation. Moreover, phosphorylation of FADD confers
death-promoting potential and tumor-suppressing ac-
tivity. In this study, we describe a cell cycle kinase that
performs an antiproliferative role in response to the
anticancer drug, taxol.
FADD interacts with Plk1 upon taxol treatment
Expression of FADD is often reduced in cancerous
tissues, including gastric cancer (Yang et al., 2005;
Matsuyoshi et al., 2006; Yoo et al., 2007; Xu et al.,
2009). To elucidate the molecular mechanisms under-
lying this phenomenon, we searched for FADD inter-
actors among complementary DNAs that displayed
differential expression patterns in healthy and cancerous
gastric tissues from Korean patients. Briefly, genes dis-
playing elevated or reduced expression in gastric
cancer tissues were translated in vitro, and glutathione
S-transferase (GST) pull-down assays were performed
on GST-tagged FADD and the in vitro-translated genes.
Several proteins interacted with FADD, including Plk1.
The FADD–Plk1 interactions were further examined
in HeLa cells (Figures 1a and b). Immunoprecipitation
and subsequent immunoblotting with the anti-FADD
antibody revealed that FADD and Plk1 did not interact
in unstimulated HeLa cells. However, FADD and Plk1
formed complexes in taxol-treated HeLa cells in a time-
and dose-dependent manner. Moreover, FADD and
Plk1 did not colocalize in untreated cells; however, they
Figure 1, upper panel). In addition, taxol treatment
significantly increased the expressions of P-FADD and
Plk1, demonstrating their colocalization upon taxol
treatment (Supplementary Figure 1, bottom panel).
To further investigate the formation of these com-
plexes by Plk1 and FADD, we performed gel-filtration
analysis using Superose 6 column (GE Healthcare,
Piscataway, NJ, USA). The majority of endogenous
FADD appeared in low-molecular-weight (B43kDa)
fractions, and Plk1 was found in fractions that ranged in
molecular weight from B43 to 42000kDa. However, a
significant amount of Plk1 and FADD formed high-
molecular-weight (42000kDa) complexes in taxol-treated
HeLa cells (Figure 1c). This raised the possibility that
FADD and Plk1 form complexes with a molecular weight
of 42000kDa in taxol-treated HeLa cells.
To identify the Plk1 domain responsible for binding
to FADD, the GST pull-down assay was performed
using GST-tagged truncated mutants of Plk1. These
mutants encompassed the kinase domain (that is, Plk1-
K, amino acid residues 1–308) or the PBD (that is, Plk1-
PBD, amino acid residues 306–604), together with
in vitro-translated FADD. The FADD protein bound
specifically to the Plk1-PBD, the known binding domain
to its substrate proteins, but not to the Plk1-K domain
Plk1 phosphorylates FADD at Ser-194
Next, we determined if FADD–Plk1 interactions led to
the phosphorylation of FADD. Western blot analysis
with an anti-P-FADD antibody revealed a band that
formed in a time- and dose-dependent manner in taxol-
treated HeLa cells, indicating the presence of P-FADD
(Figures 1a and b). Next, we examined whether Plk1 was
responsible for phosphorylation of FADD in taxol-
treated HeLa cells. Cells were transfected with Plk1-
specific small interfering RNA to promote the transient
depletion of Plk1. Phosphorylation of FADD was
clearly inhibited in Plk1-depleted, taxol-treated HeLa cells
(Figure 2a). The Plk1 kinase assay was next performed
in vitro using GST-FADD as a substrate. At 36h after
transfection with FLAG-tagged Plk1 wild type or kinase
dead, cells were treated with 25nM taxol for 12h. After
taxol stimulation, Plk1 was immunoprecipitated using an
anti-FLAG antibody. The immunoprecipitates were used
to perform an in vitro kinase assay. Plk1 wild type, but not
Plk1 kinase dead, phosphorylated GST-FADD in vitro,
(Figure 2b). Moreover, P-FADD was increased by forced
expression of Plk1 T210D, a constitutively activated
mutant form of Plk1, when compared with cells trans-
fected with Plk1 T210A, a kinase-defective mutant form of
Plk1 (Supplementary Figure 2).
We reasoned that if Plk1 was the binding partner of
FADD, then GST-FADD-mediated depletion of en-
dogenous FADD from taxol-treated HeLa cells should
result in the concordant loss of Plk1 expression and
kinase activity. To test this idea, we depleted cell lysates
via sequential pull-downs with GST-FADD (Figure 2c).
Tumor suppression by phosphorylated FADD
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Tumor suppression by phosphorylated FADD
M-S Jang et al