Biochem. J. (2012) 447, 93–102 (Printed in Great Britain)
93
doi:10.1042/BJ20120751
Phospho-regulation of KIBRA by CDK1 and CDC14 phosphatase controls
cell-cycle progression
Ming JI, Shuping YANG, Yuanhong CHEN, Ling XIAO, Lin ZHANG and Jixin DONG1
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A.
KIBRA (kidney- and brain-expressed protein) is a novel regulator
of the Hippo pathway, which controls tissue growth and
tumorigenesis by regulating both cell proliferation and apoptosis.
In mammals, KIBRA is associated with memory performance.
The physiological function and regulation of KIBRA in nonneuronal cells remain largely unclear. We reported recently that
KIBRA is phosphorylated by the mitotic kinases Aurora-A and B. In the present study, we have expanded our analysis of KIBRA’s
role in cell-cycle progression. We show that KIBRA is also
phosphorylated by CDK1 (cyclin-dependent kinase 1) in response
to spindle damage stress. We have identified KIBRA Ser542 and
Ser931 as main phosphorylation sites for CDK1 both in vitro and
in vivo. Moreover, we found that the CDC (cell division cycle)
14A/B phosphatases associate with KIBRA, and CDK1-nonphosphorylatable KIBRA has greatly reduced interaction with
CDC14B. CDC14A/B dephosphorylate CDK1-phosphorylated
KIBRA in vitro and in cells. By using inducible-expression cell
lines, we show further that phospho-regulation of KIBRA by
CDK1 and CDC14 is involved in mitotic exit under spindle
stress. Our results reveal a new mechanism through which KIBRA
regulates cell-cycle progression.
INTRODUCTION
leukaemia has not been determined. We demonstrated recently
that KIBRA is also a negative regulator of the Hippo pathway in
mammalian cells [20].
Mitosis is a critical step to ensure the genome integrity during
cell-cycle progression. Thus aberration of mitosis is often seen
in human malignancy. Interestingly, several components of the
Hippo pathway have been shown to be important regulators of
mitosis [21–23], implicating a new mechanism through which the
Hippo pathway exerts its tumour-suppressive function in cancer.
Microtubules play pivotal roles in mitosis and cell division and
are targets of anti-mitotic chemotherapeutics [24,25]. Spindle
poisons, such as paclitaxel (Taxol) and nocodazole, interfere with
microtubule dynamics, thus precluding the normal function of
the mitotic spindle and resulting in extensive mitotic arrest and
cell death [26]. Upon treatment with spindle poisons, several
kinases including CDK (cyclin-dependent kinase) 1 are activated
[26–28]. CDK1 controls critical cell-cycle events by
phosphorylating various substrates [29].
In budding yeast, the protein phosphatase Cdc (cell division
cycle) 14 controls mitotic exit by antagonizing Cdk function
[30]. Mammals encode two Cdc14 orthologues, CDC14A and
CDC14B [31]. The cellular function of CDC14A/B in higher
eukaryotes has just begun to be elucidated [32]. CDC14A
localizes at the centrosome in interphase and its deregulation
disrupts centrosome separation and chromosome segregation [33].
Knockdown of CDC14B induces centriole amplification [34], but
it is dispensable for chromosome segregation and mitotic exit
[35]. Interestingly, CDC14B has also been implicated in the DNAdamage response [36,37] and DNA repair [38].
We reported previously that KIBRA phosphorylation is regulated by the mitotic kinase Aurora and PP1 (protein phosphatase 1)
KIBRA, a WW domain-containing protein, is highly expressed
in kidney and brain [1]. KIBRA was identified as a memory
performance-associated protein [2–6]. This function is linked
to a C>T single nucleotide polymorphism in the ninth intron
of the KIBRA gene [2]. A recent report using a knockout
mouse model confirmed further that KIBRA plays a role in
learning and memory [7]. Besides the function in neurons,
KIBRA is also involved in diverse processes in non-neuronal
cells. For example, KIBRA has been shown to interact with
the polarity protein PATJ [PALS1 (protein associated with Lin7
1)-associated tight junction] and synaptopodin, and it regulates
podocyte migration [8]. Furthermore, KIBRA mediates the
aPKC (atypical protein kinase C)–exocyst interaction and is
also required for NRK (normal rat kidney) cell migration [9].
It was demonstrated recently that KIBRA regulates epithelial cell
polarity by suppressing apical exocytosis through inhibition of
aPKC activity in the PAR (partitioning-defective) 3–aPKC–PAR6
tight junction complex [10]. KIBRA also modulates the collageninduced ERK (extracellular-signal-regulated kinase) signalling
via interactions with discoidin domain receptor tyrosine kinase
1 [11]. Genetic studies in Drosophila have identified KIBRA as a
potential tumour suppressor acting through the Hippo signalling
pathway [12–14], which is a central player in controlling organ
size, tumorigenesis and cell contact inhibition by inhibiting
cell proliferation and promoting apoptosis [15–17]. Interestingly,
KIBRA expression is frequently down-regulated by promoter
methylation in B-cell acute lymphocytic leukaemia [18] and
chronic lymphocytic leukaemia [19]; however, the molecular
function of methylation/demethylation at the KIBRA locus in
Key words: cell division cycle 14A/B (CDC14A/B),
cyclin-dependent kinase 1 (CDK1), Hippo pathway, kidney- and
brain-expressed protein (KIBRA), mitosis, phosphorylation.
Abbreviations used: aPKC, atypical protein kinase C; CDC, cell division cycle; CDK, cyclin-dependent kinase; DN, dominant-negative; ERK, extracellularsignal-regulated kinase; GFP, green fluorescent protein; GST, glutathione transferase; HA, haemagglutin; HEK, human embryonic kidney; JNK, c-Jun Nterminal kinase; KIBRA, kidney- and brain-expressed protein; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NF2, neurofibromatosis
type 2; PAR, partitioning-defective; PP1, protein phosphatase 1; siRNA, small interfering RNA; WT, wild-type; YAP, Yes-associated protein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
94
M. Ji and others
[39]. KIBRA is hyperphosphorylated at Ser539 in spindle poisoninduced mitosis [39]. In the present study, we have shown that,
besides Ser539 phosphorylation, phospho-regulation of KIBRA
is also mediated by the mitotic kinase CDK1 and protein
phosphatases CDC14A/B upon treatment with spindle poisons.
Our results suggest further a potential role for KIBRA in mitosisrelated cell-cycle events, especially under microtubule stress.
antibodies were from Santa Cruz Biotechnology. Anti-AuroraA, anti-GST (glutathione transferase) and anti-His6 antibodies
were from Bethyl Laboratories. Anti-(phospho-Thr288 AuroraA), anti-(phospho-Ser10 histone H3), anti-(phospho-Thr202 /Tyr204
ERK1/2), anti-[phospho-Ser127 YAP (Yes-associated protein)],
anti-(phospho-Thr180 /Tyr182 p38), anti-phospho-threonine, antip38 and anti-Cdc2 antibodies were from Cell Signaling
Technology. Anti-CDC14B antibody was purchased from
Invitrogen.
EXPERIMENTAL
Expression constructs
λ-Phosphatase treatment
The human KIBRA constructs have been described previously
[20]. Myc-tagged human CDC14A and CDC14B expression
constructs have been described previously [33]. Point mutations
were generated by the QuikChangeTM Site-Directed PCR
mutagenesis kit (Stratagene) and verified by sequencing.
Cells were lysed in Nonidet P-40 buffer and treated with λphosphatase (New England Biolabs) as described in [39].
Cell culture and transfection
HEK (human embryonic kidney)-293T, HeLa and MCF-7 cell
lines were maintained as described in [39]. All transient
overexpression transfections were performed using Attractene
(Qiagen) following the manufacturer’s instructions. All siRNA
(small interfering RNA) transfections were performed using
HiPerFect (Qiagen). Nocodazole (100 ng/ml for 16–20 h) and
Taxol (1 μM for 16 h) (Sigma) were used to arrest cells in
mitosis. VX680 (Selleck Chemicals) was used at 1 μM as an
Aurora kinase inhibitor. U0126 {a MEK [MAPK (mitogenactivated protein kinase)/ERK kinase]/ERK inhibitor}, SB203580
(p38 inhibitor) and SP600125 [JNK (c-Jun N-terminal kinase)
inhibitor] were from LC Laboratories. RO-3306 (CDK1 inhibitor)
and roscovitine (CDK inhibitor) were from ENZO Life Sciences.
CDC14B siRNA (SMARTpool) was purchased from Dharmacon.
All other chemicals were from either Sigma or Thermo Fisher.
Establishment of Tet-On-inducible cell lines
The parental MCF-7-rtTA cell line was purchased from Clontech
Laboratories. The cell line expressing WT (wild-type) KIBRA
has been described previously [39]. The MCF-Tet-On-inducible
cell line expressing the KIBRA-4SA (KIBRA with four serine
residues changed to alanine) mutant was established similarly.
Cells were maintained in medium containing Tet system-approved
fetal bovine serum (Clontech Laboratories).
Immunoprecipitation, Western blot analysis and metabolic
labelling
Immunoprecipitation, Western blotting and metabolic labelling
assays were carried out as described previously [39]. [32 P]Pi was
purchased from MP Biomedicals.
Antibodies
Rabbit polyclonal and mouse monoclonal antibodies against
human KIBRA have been described previously [39]. Rabbit
polyclonal phospho-specific antibodies against KIBRA Ser542 ,
Ser548 and Ser931 were generated and purified by AbMart.
The anti-(phospho-Ser539 KIBRA) antibody has been described
previously [39]. Anti-FLAG, anti-HA (haemagglutin) and
anti-Myc antibodies were from Sigma. Anti-β-actin, antiERK, anti-cyclin B and anti-GFP (green fluorescent protein)
c The Authors Journal compilation c 2012 Biochemical Society
Recombinant protein purification
The GST-tagged proteins were bacterially expressed and
purified on GSTrap FF affinity columns (GE Healthcare)
following the manufacturer’s instructions. To make His6 tagged human CDC14A/B and their corresponding catalytically
inactive phosphatases, full-length CDC14A/B and CDC14AC278S/CDC14B-C314S cDNAs were subcloned into the pET21c vector (Novagen/EMD Chemicals). The proteins were
expressed and purified on HisPurTM Cobalt spin columns (Thermo
Scientific/Pierce) following the manufacturer’s instructions. His6 –
YAP was expressed and purified similarly. The Aurora-A kinase
has been described previously [39].
In vitro kinase assay
GST–KIBRA (1–2 μg) was incubated with 10 units of
recombinant CDK1–cyclin B complex (New England Biolabs) or
100 ng of CDK1–cyclin B (SignalChem) or HeLa cell total lysate
(treated with DMSO or Taxol) in kinase buffer [39] in the presence
of 5 μCi of [γ -32 P]ATP (3000 Ci/mmol) (PerkinElmer). MEK1,
p38α, JNK1, JNK2 and CDK5 active kinases were purchased
from SignalChem. The samples were resolved by SDS/PAGE (8
or 4–20 % gels), transferred on to PVDF membranes (Millipore)
and visualized by autoradiography followed by Western blotting.
Flow cytometry
Alexa Fluor® 488–annexin V/Dead cell apoptosis kit was from
Invitrogen. Propidium iodide was purchased from Sigma. The
cell-cycle profile was analysed using a standard protocol [40].
In vitro dephosphorylation/phosphatase assay
GST–KIBRA-M or GST–KIBRA-C was phosphorylated by
CDK1–cyclin B or Aurora-A in vitro as described in [39].
Phosphorylated GST–KIBRA was pulled down by glutathione–
agarose (Santa Cruz Biotechnology) and the dephosphorylation
assay was performed as described previously [39]. The reaction
mixture was incubated at 30 ◦ C for 45 min. The relative amounts
of 32 P released into the supernatant, as well as the 32 P bound to
GST–KIBRA, were quantified using a liquid-scintillation counter
(Beckman LS6500).
Statistical analysis
Statistical significance was performed using a two-tailed unpaired
Student’s t test.
CDK1 and CDC14 regulate KIBRA phosphorylation
Figure 1
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Phosphorylation of KIBRA is stimulated by spindle poisons in a CDK1-dependent manner
(A) FLAG-tagged KIBRA-S539A was transfected into HEK-293T cells. At 30 h after transfection, cells were treated with DMSO or nocodazole or Taxol for 12 h and metabolically labelled in the presence
of 32 P for an additional 2 h as described in [39]. Immunoprecipitated products were separated by SDS/PAGE and transferred on to PVDF membranes, followed by autoradiography and Western blot
analysis. (B) GST–KIBRA-M and -C proteins were used as substrates for in vitro kinase assays with asynchronized ( − ) or Taxol-treated ( + ) HeLa cell lysates in the presence of kinase inhibitors
as indicated. The Aurora inhibitor VX680 was used at 1 μM and roscovitine was used at 10 μM for inhibiting CDK1. 32 P incorporation was revealed by autoradiography. The Western blot shows the
substrate loading. The relative band intensity (normalized to the amount of protein) was quantified by Alphaview 1.3.0.7 software (Alpha Innotech). (C) GST–KIBRA-M and -C proteins were used as
substrates for in vitro kinase assays with mock- or CDK1-depleted Taxol-treated cell lysates [27,43]. GST–KIBRA was pulled down by glutathione–agarose and visualized by autoradiography and
Western blot analysis. Total cell lysates were subjected to Western blot analysis with the indicated antibodies. The relative band intensity (normalized to the amount of protein) was quantified as in
(B). (D) GST–KIBRA-N, -M and -C proteins were used as substrates for in vitro kinase assays with purified CDK1–cyclin B complex. Molecular masses (M) are indicated in kDa. IP, immunoprecipitation;
WB, Western blot.
RESULTS AND DISCUSSION
KIBRA is phosphorylated by CDK1
We previously identified Ser539 of KIBRA as a major
phosphorylation site for Aurora kinases in mitosis [39]. To
investigate further whether additional phosphorylation of KIBRA
occurs during spindle-stress-induced mitosis, we performed
metabolic labelling on a transfected KIBRA S539A mutant. As
shown in Figure 1(A), Taxol or nocodazole treatment greatly
increased 32 P incorporation into KIBRA, indicating that additional
KIBRA phosphorylation, besides that of Ser539 , occurs in cells in
response to spindle damage (Figure 1A, compare lanes 2 and 3
with lane 1).
By using mitotic lysates from Taxol-treated cells, we
demonstrated further that both GST–KIBRA-M (amino acids
428–835) and -C (amino acids 832–1119) were phosphorylated
by mitotic kinases (Figure 1B, compare lanes 2 and 1, and
lanes 7 and 6). GST–KIBRA-N (amino acids 1–453) was not
phosphorylated by the mitotic lysate in this setting (results
not shown). Although KIBRA is phosphorylated on Ser539 by
Aurora kinases during Taxol-induced mitosis, Aurora kinases do
not phosphorylate KIBRA-C [39] and inhibition of Aurora kinases
only partially decreased the 32 P incorporation into KIBRA-M
(Figure 1B, compare lanes 3 and 2), indicating that there is
an additional kinase(s) responsible for KIBRA phosphorylation
during Taxol treatment and that this kinase can phosphorylate
both the central part and the C-terminus of KIBRA. Using
small-molecule inhibitors, we found that roscovitine inhibited
32
P incorporation into KIBRA-M and -C (Figure 1B, compare
lanes 4 and 2, and lanes 9 and 7). Roscovitine inhibits CDK1,
CDK2 and CDK5 [41,42]. CDK1 is a well-known mitotic
kinase and is activated during spindle poison-arrested mitosis
[27,28,39,43]. CDK2 regulates G1 –S-phase transition [44,45]
and is not activated by Taxol treatment [46]. CDK5 failed to
phosphorylate KIBRA in vitro (Supplementary Figure S1 at
http://www.BiochemJ.org/bj/447/bj4470093add.htm). Thus our
results identify CDK1 as a likely candidate. Addition of both
VX680 (an Aurora kinase inhibitor) and roscovitine inhibitors
abolished the phosphorylation of KIBRA (Figure 1B, lanes 5
and 10), suggesting that CDK1 and Aurora are the major kinases
responsible for Taxol-induced KIBRA phosphorylation.
To confirm further that KIBRA phosphorylation is CDK1dependent, we depleted CDK1 in Taxol-treated mitotic cell lysates
[27,43] and used them for in vitro kinase assays. Figure 1(C)
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Figure 2
M. Ji and others
MAPKs are not responsible for KIBRA phosphorylation during spindle stress
(A) GST–KIBRA-M and -C and His6 –YAP proteins were used as substrates for in vitro kinase assays with purified JNK1 or JNK2. The products were separated by SDS/PAGE and transferred on to
PVDF membranes, followed by autoradiography and Western blot analysis. * and 䊊 mark GST–JNK1 and GST–JNK2 respectively. (B) HeLa cells were treated with DMSO or nocodazole (100 ng/ml
for 16 h) or Taxol (1 μM for 16 h) and total cell lysates were probed with the indicated antibodies. (C) GST–KIBRA-M and -C and MBP (myelin basic protein, positive control) were used as substrates
for in vitro kinase assays with purified MEK1. (D) HeLa cells were treated as in (B) and total cell lysates were probed with the indicated antibodies. (E) GST–KIBRA-M and -C and MBP (positive
control) proteins were used as substrates for in vitro kinase assays with active p38α kinase. Molecular masses (M) are indicated in kDa.
shows that CDK1 depletion reduced phosphorylation of GST–
KIBRA-M and -C by 50 % and 40 % respectively (top row,
compare lanes 2 and 1, and lanes 4 and 3). Indeed, purified CDK1–
cyclin B complex phosphorylated both GST–KIBRA-M and -C
(Figure 1D). Taken together, these results indicate that CDK1
phosphorylates KIBRA directly in vitro.
Taxol-treated cell lysates failed to inhibit 32 P incorporation
into GST–KIBRA-M and -C (Supplementary Figure S2
at http://www.BiochemJ.org/bj/447/bj4470093add.htm). These
data suggest that Taxol-induced KIBRA phosphorylation is
independent of MEK, JNK and p38 kinase.
MAPKs are not responsible for Taxol-induced phosphorylation
of KIBRA
CDK1–cyclin B complex phosphorylates multiple sites in
KIBRA in vitro
Since MAPKs, including MEK/ERK, JNK and p38, and
CDK1 are all proline-directed kinases, we tested whether
these kinases phosphorylate KIBRA. JNK1 or JNK2 could
not phosphorylate KIBRA, whereas the same amounts of
these kinases robustly phosphorylated their known substrate
YAP [47] (Figure 2A). Consistent with a previous study
[48], Taxol or nocodazole treatment strongly inhibited the
ERK and p38 kinase activity in HeLa cells (Figures 2B and
2D). Thus we considered MEK/ERK and p38 unlikely to
be the kinases responsible for KIBRA phosphorylation under
spindle stress. Indeed, in vitro kinase assays demonstrated that
KIBRA is not a suitable substrate for MEK1 (Figure 2C) or
p38α (Figure 2E). In line with these observations, addition
of U0126 (a MEK/ERK inhibitor), SP600125 (a JNK1/2
inhibitor) or SB203580 (a MAPK p38 inhibitor) to the
CDK1 recognizes a minimal Ser/Thr-Pro consensus site [49].
Sequence analysis showed that KIBRA-M contains a highly
conserved region encompassing multiple proline-directed serine
residues including Ser535 , Ser542 , Ser544 and Ser548 (SP cluster)
around the Aurora-site Ser539 (Figure 3A). To test whether this
SP cluster is phosphorylated by CDK1, we mutated all four
serine residues to non-phosphorylatable alanine residues and
performed in vitro kinase assays. As shown in Figure 3(B),
the CDK1-dependent phosphorylation of GST–KIBRA-M is
largely abolished when all four serine residues are changed
to alanine (KIBRA-M-4SA), suggesting that CDK1 potentially
phosphorylates this well conserved SP cluster (Figure 3B). GST–
KIBRA and GST–KIBRA-M-4SA are similarly phosphorylated
by Aurora-A, suggesting that mutating Ser535 , Ser542 , Ser544 and
Ser548 to alanine does not affect the Ser539 phosphorylation
c The Authors Journal compilation c 2012 Biochemical Society
CDK1 and CDC14 regulate KIBRA phosphorylation
97
demonstrated that CDK1 phosphorylated Ser542 (Figure 3D) and
Ser931 (Figure 3E), but not Ser548 (results not shown) of KIBRA
in vitro.
CDK1 phosphorylates KIBRA at Ser542 and Ser931 in vivo
To explore whether Ser542 , Ser548 and Ser931 are also
phosphorylated within cells in response to spindle damage,
we transfected KIBRA or corresponding non-phosphorylatable
mutants into cells, treated the cells with Taxol and determined
levels of phosphorylation by phospho-antibodies. Taxol treatment
significantly increased the phospho-signal of Ser542 and Ser931 ,
but not Ser548 , and the signal was abolished by mutating
the relevant serine residue to alanine and by λ-phosphatase
treatment (Figures 4A–4C). Taxol or nocodazole treatment also
significantly increased the phospho-signal of Ser542 and Ser931
in immunoprecipitated endogenous KIBRA (Figure 4D). Using
an inhibitor for CDK1, we demonstrated that phosphorylation
of KIBRA Ser542 and Ser931 was CDK1-dependent (Figure 4D).
Taken together, these results support the notion that Ser542 and
Ser931 of KIBRA are phosphorylated during spindle damage stress
in a CDK1-dependent manner. We reported recently that KIBRA
phosphorylation peaked and coincided with increased cyclin B
levels in mitosis [39], supporting further that CDK1-dependent
phosphorylation of KIBRA occurs in cells.
CDC14 phosphatases dephosphorylate KIBRA in vitro
Figure 3
CDK1 phosphorylates KIBRA at Ser542 and Ser931 in vitro
(A) Sequence alignment of a highly conserved region encompassing an SP cluster in KIBRA.
(B) GST–KIBRA-M and GST–KIBRA-M-4SA were used as substrates for in vitro kinase assays
with the CDK1–cyclin B complex. Autoradiography shows 32 P incorporation, Western blot
shows the substrate loading. (C) In vitro kinase assays using the CDK1–cyclin B complex
to phosphorylate GST–KIBRA-C with or without mutations as indicated. 2A, T895A/T912A.
(D) In vitro kinase assays with the CDK1–cyclin B complex without 32 P. The samples were
probed with a phospho-specific antibody against KIBRA Ser542 . (E) In vitro kinase assays with
the CDK1–cyclin B complex without 32 P. The samples were probed with a phospho-specific
antibody against KIBRA Ser931 . Roscovitine (10 μM) was used to inhibit CDK1 activity. Molecular
masses (M) are indicated in kDa.
mediated by Aurora-A kinase (Supplementary Figure S3 at
http://www.BiochemJ.org/bj/447/bj4470093add.htm).
A previous proteomic study has identified several potential
CDK1 sites in the C-terminus of KIBRA, including Thr895 ,
Thr912 and Ser931 [50]. Mutating Thr895 and Thr912 to
alanine (KIBRA-C-2A) or Ser931 to alanine (KIBRA-C-S931A)
reduced 32 P incorporation mediated by CDK1 (Figure 3C).
Combined mutations (KIBRA-C-2A/931A) decreased further
the phosphorylation of KIBRA-C stimulated by CDK1 in vitro
(Figure 3C, lane 4). Ser931 is conserved from Drosophila to
humans and we did not observe any increase in phospho-threonine
of KIBRA after Taxol treatment (results not shown). Therefore
Ser931 was chosen for further study.
To facilitate our studies, we have generated phospho-specific
antibodies against Ser542 , Ser548 and Ser931 . We also attempted
to generate phospho-specific antibodies against Ser535 and
Ser544 , but failed. Using these phospho-specific antibodies, we
To analyse the kinetic changes in KIBRA phosphorylation,
we collected mitotic cells by nocodazole treatment, released
the cells into normal medium and then determined KIBRA’s
phospho-level using phospho-antibodies. Phosphorylation of
KIBRA Ser542 and Ser931 is clearly reduced during mitotic exit
(Figure 5A), suggesting the presence of a potential phosphatase
responsible for the dephosphorylation. The Cdc14 phosphatase controls mitotic exit by antagonizing/dephosphorylating the
CDK1-phosphorylated substrates in yeast [30]. Furthermore,
human CDC14 phosphatases have also been implicated in
dephosphorylating proline-directed phosphorylation [31,51–57].
Accordingly, co-transfection of WT CDC14A or CDC14B, but
not their catalytically inactive (CS) mutants (C278S or C314S
respectively), with KIBRA greatly enhanced the mobility of
KIBRA, suggesting that KIBRA is dephosphorylated when
CDC14 is overexpressed (Figure 5B). Expression of another
cell-cycle phosphatase, CDC25A, or its DN (dominant-negative)
mutant had no effects on KIBRA’s mobility (Figure 5B, lanes 6
and 7).
To test whether KIBRA is a direct substrate for CDC14
phosphatases, we purified CDC14A/B phosphatases (Figure 5C)
and performed in vitro dephosphorylation assays using
CDK1-phosphorylated GST–KIBRA-M or GST–KIBRA-C as
substrates. Figures 5(D) and 5(E) show that CDK1-mediated
phosphorylation of KIBRA was greatly reduced by purified WT
CDC14A or CDC14B, and the catalytically inactive (CS mutant)
phosphatases failed to dephosphorylate CDK1-phosphorylated
KIBRA. Whereas PP1 efficiently dephosphorylated Auroramediated KIBRA phosphorylation [39], it only moderately
dephosphorylated CDK1-phosphorylated KIBRA (Figures 5D
and 5E). As controls, we also included Aurora-A-phosphorylated
KIBRA as a substrate for dephosphorylation assays. Our data
show that neither CDC14A nor CDC14B dephosphorylated
Aurora-A-mediated phosphorylation on KIBRA (Figure 5D,
columns marked by CDC14A* and CDC14B*), confirming their
specificity.
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 4
M. Ji and others
CDK1 phosphorylates KIBRA at Ser542 and Ser931 in cells
(A) HEK-293T cells were transfected with HA–KIBRA or HA–KIBRA-S542A. At 30 h after transfection, the cells were treated with Taxol for 12 h. The cells were lysed (treated with λ-phosphatase
as needed) and immunoprecipitated with anti-HA antibody. The immunoprecipitates were probed with anti-(phospho-Ser542 KIBRA) and subsequent anti-HA antibodies. (B) HEK-293T cells were
transfected with FLAG–KIBRA or FLAG–KIBRA S931A. Taxol treatment and immunoprecipitation were carried out as in (A). The immunoprecipitates were probed with anti-(phospho-Ser931 KIBRA)
and subsequent anti-FLAG antibodies. (C) Transfection and immunoprecipitation were carried out as in (A) except that anti-(phospho-Ser548 KIBRA) was used. (D) HeLa cells were treated with
nocodazole or Taxol for 12 h and RO-3306 (5 μM) was added to cells 2 h before harvesting. Proteasome inhibitor MG132 was also added (together with RO-3306) to prevent cyclin B from degradation
and cells exiting mitosis. Endogenous KIBRA was immunoprecipitated and probed with the indicated antibodies. Total cell lysates before immunoprecipitation were also analysed. Molecular masses
(M) are indicated in kDa. IP, immunoprecipitation.
CDC14 dephosphorylates KIBRA in cells
Next, we explored further whether KIBRA could be
dephosphorylated by CDC14 in cells. Consistent with the
mobility-shift assays in Figure 5(B), co-transfection of WT
CDC14A or CDC14B, but not the CS mutants, almost completely
dephosphorylated KIBRA at Ser931 , Ser548 and Ser542 (Figure 6A).
As expected, the Aurora-mediated Ser539 phosphorylation of
KIBRA was not affected (Figure 6A, second bottom row),
which aligns perfectly with our in vitro dephosphorylation data
(Figure 5D). Importantly, knockdown of CDC14B alone was
sufficient to increase the phosphorylation levels of KIBRA Ser542
and Ser931 , but not Ser539 (Figure 6B). Taken together, these results
indicate that KIBRA is a physiological substrate for CDC14
phosphatase.
CDC14 phosphatases associate with KIBRA
The CDC14A/B activity towards KIBRA prompted us to explore
whether KIBRA forms a complex with CDC14A or CDC14B.
To do so, we performed immunoprecipitation with transfected
plasmids and found that the CDC14A-CS mutant, but not
WT CDC14A, associated with KIBRA, suggesting that the
catalytically inactive CDC14A functions as a binding trap for
c The Authors Journal compilation c 2012 Biochemical Society
KIBRA (Figure 6C). The CDC14B-CS mutant also showed
much stronger binding affinity to KIBRA than WT CDC14B
(Figure 6D). To test whether phosphorylation of KIBRA Ser542 ,
Ser544 , Ser548 and Ser931 is involved in KIBRA’s interaction with
CDC14, we used the KIBRA-4SA mutant and performed coimmunoprecipitation. The KIBRA-4SA mutant showed similar
binding affinity with CDC14-CS when compared with WT
KIBRA in transfected cells (Figure 6E, compare lane 8 with
lane 7). However, whereas WT KIBRA bound to CDC14B or
CDC14B-CS efficiently, the interaction between KIBRA-4SA
and CDC14B was significantly weakened (for WT CDC14B)
or abolished (for CDC14B-CS), indicating that CDK1-mediated
phosphorylation of KIBRA is required for association with
CDC14B (Figure 6F). However, our results cannot exclude the
possibility that mutating the CDK1 phosphorylation sites of
KIBRA affects KIBRA’s conformation, leading to disassociation
from CDC14B.
Results supporting the well-established role of Cdc14 in
controlling mitotic exit were largely obtained from studies in
yeast. The cellular function of CDC14A/B is not well understood
in higher eukaryotes, probably due to the fact that very few
substrates have been identified so far for CDC14 phosphatases
[32]. The present study has identified KIBRA as a novel substrate
for CDC14 phosphatase, which may advance our understanding
CDK1 and CDC14 regulate KIBRA phosphorylation
Figure 5
99
CDC14A/B phosphatases dephosphorylate KIBRA in vitro
(A) HeLa cells were treated with nocodazole. Mitotic cells were collected by mechanic shake-off, released into fresh medium and harvested at the indicated time. KIBRA was immunoprecipitaed
and analysed by Western blot analysis with the indicated antibodies. (B) HEK-293T cells were transfected with various DNAs as indicated. At 48 h post-transfection, total cell lysates were
analysed with the indicated antibodies. (C) The purified His6 –CDC14 proteins were stained with Coomassie Blue. (A–C) Molecular masses (M) are indicated in kDa. IP, immunoprecipitation.
(D) In vitro dephosphorylation assays using CDC14A/B and their catalytically inactive (CS) phosphatases. GST–KIBRA-M proteins were first phosphorylated by the CDK1–cyclin B complex or Aurora-A
kinase and used as substrates for dephosphorylation assays. Results are mean +
− S.E.M. percentages of dephosphorylation from three independent experiments. ***P < 0.001 (Student’s t test).
(E) GST–KIBRA-C proteins were first phosphorylated by the CDK1–cyclin B complex and used as substrates for in vitro dephosphorylation assays with phosphatases as indicated. Results are
mean +
− S.E.M. percentages of dephosphorylation from three independent experiments. ***P < 0.001 (Student’s t test).
towards the functions of CDC14. It is important to investigate
whether the Hippo pathway and/or KIBRA regulates CDC14
activity during mitotic exit.
Furthermore, the detailed temporal and spatial dephosphorylation of KIBRA also remains to be explored; the phosphoantibodies we have generated provide important tools to dissect
the regulation of KIBRA under both physiological and spindle
stress conditions.
542
Phosphorylation of KIBRA on Ser , Ser
not affect the Hippo signalling activity
544
, Ser
548
and Ser
931
does
KIBRA interacts with the tumour suppressor NF2 (neurofibromatosis type 2) in both Drosophila and mammalian
cells [12–14,39,58] and we demonstrated further that Aurora
phosphorylation of KIBRA on Ser539 weakened the interaction
between KIBRA and NF2 [39]. Mutating Ser542 , Ser544 ,
Ser548 and Ser931 to alanine (4SA) did not alter the
association between KIBRA and NF2 (Supplementary Figure
S4A at http://www.BiochemJ.org/bj/447/bj4470093add.htm). As
expected, KIBRA and KIBRA-4SA have similar abilities to
stimulate Lats1/2 phosphorylation (results not shown).
KIBRA promotes YAP Ser127 phosphorylation (through Lats1/2
kinases) [20], so we also tested whether CDK1-mediated
phosphorylation of KIBRA changes its ability to promote
YAP phosphorylation. No significant change was detected
towards YAP Ser127 phosphorylation (with or without Taxol
treatment), regardless of whether KIBRA or KIBRA-4SA had
been transfected into the cells (Supplementary Figure S4B). These
results suggest that phosphorylation of KIBRA on Ser542 , Ser544 ,
Ser548 and Ser931 does not significantly affect Hippo–YAP activity.
Phospho-regulation of KIBRA by CDK1 and CDC14 affects cell-cycle
progression
CDK1 and CDC14 are well-established regulators of the cell
cycle, and so we examined further the functional significance
of KIBRA phosphorylation mediated by CDK1 and CDC14 in
cell-cycle progression. To do so, we established an inducible
KIBRA-expressing system (Figure 7A). Under normal culture
conditions, expressing KIBRA or KIBRA-4SA had no discernible
effect on the cell-cycle distribution (Figure 7B). Taxol treatment
for 24 h greatly increased the fraction of cells in G2 /M-phase
(72.39 % and 71.80 % for control and KIBRA-expressing cells
respectively) and a co-ordinated decrease of the fraction of
cells in G1 -phase (8.72 % and 9.34 % for control and KIBRAexpressing cells respectively). In contrast, expression of
KIBRA-4SA greatly decreased the fraction of cells in G2 /Mphase (59.45 % compared with 71.80 % when compared KIBRAexpressing cells) and increased the fraction of cells in G1 -phase
(20.17 % compared with 9.34 % in KIBRA-expressing cells)
(Figure 7C). A longer treatment with Taxol resulted in even more
significant difference among the fraction of the cells in G2 /Mphase (53.38 % compared with 70.26 % for cells expressing
c The Authors Journal compilation c 2012 Biochemical Society
100
Figure 6
M. Ji and others
CDC14A/B associate with and dephosphorylate KIBRA in cells
(A) HEK-293T cells were transfected with various DNAs as indicated. At 48 h post-transfection, cells were lysed and immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates were
probed with the indicated antibodies. Total cell lysates before immunoprecipitation were used to check the expression of CDC14A (A) or CDC14B (B). (B) HeLa cells were transfected with control
(lane 1) or siRNAs for human CDC14B (lane 2). At 48 h after transfection, endogenous KIBRA was immunoprecipitated. The immunoprecipitates and total protein lysates were subjected to Western
blot analysis with the indicated antibodies. (C) HEK-293T cells were transfected with various DNAs as indicated. At 48 h after transfection, cells were lysed and immunoprecipitated with an anti-FLAG
antibody. The immunoprecipitates were probed with an anti-Myc antibody to check the presence of CDC14A. Total cell lysates were subjected to Western blot analysis with antibodies as indicated.
(D) HEK-293T cells were transfected with various DNAs as indicated. Immunoprecipitation and Western blots were carried out as in (C). HC, heavy chain. (E) HEK-293T cells were transfected with
various DNAs as indicated. Immunoprecipitation and Western blots were carried out as in (C). (F) HEK-293T cells were transfected with various DNAs as indicated. Immunoprecipitation and Western
blots were carried out as in (C). Molecular masses (M) are indicated in kDa. IP, immunoprecipitation.
KIBRA-4SA and KIBRA respectively) or G1 -phase (23.24 %
compared with 7.89 % for cells expressing KIBRA-4SA and
KIBRA respectively) (Figure 7C). The fraction of cells in Sphase remained unaffected. These results suggest that elimination
of KIBRA phosphorylation by CDK1 promoted cell exit from
G2 /M-phase into G1 -phase. Thus phospho-regulation of KIBRA
by CDK1 and CDC14 is required for proper cell-cycle progression
under spindle damage stress. It will be interesting to explore how
these cells could enter G1 -phase in the presence of Taxol and what
the fate of these cells may be.
We also counted the mitotic index and found indeed that
cells expressing KIBRA-4SA could not arrest efficiently in
mitosis, as revealed by a significant decrease in the number
of cells stained by anti-(phospho-Ser10 histone H3) antibody
c The Authors Journal compilation c 2012 Biochemical Society
upon treatment with Taxol (Supplementary Table S1 at
http://www.BiochemJ.org/bj/447/bj4470093add.htm). However,
we found that all of these cell lines (Tet-control, Tet-KIBRA
and Tet-KIBRA-4SA) responded to nocodazole (an agent that
arrests cells in mitosis by depolymerizing microtubules) similarly
with regard to cell-cycle distribution (Supplementary Table S2 at
http://www.BiochemJ.org/bj/447/bj4470093add.htm) or mitotic
index (Supplementary Table S3 at http://www.BiochemJ.org/
bj/447/bj4470093add.htm).
Anti-mitotic agents, such as Taxol, have been widely used
for treatment of several types of cancers, including ovarian
and breast cancer [24,25]. Although these drugs are known
to induce apoptotic cell death, the biochemical mechanisms
and signalling pathways underlying the toxicity are not clearly
CDK1 and CDC14 regulate KIBRA phosphorylation
Figure 7
101
Phospho-regulation of KIBRA by CDK1 and CDC14 controls cell-cycle progression
(A) Characterization of Tet-On-inducible MCF-7 cells expressing WT KIBRA or KIBRA-4SA mutant. The cell lines were treated with doxycycline (Dox, 0.2 μg/ml) as indicated. Total protein lysates
were subjected to Western blot analysis with the indicated antibodies. Molecular masses (M) are indicated in kDa. (B) The cell lines in (A) were treated with or without Dox for 48 h. The fraction of
cells in various cell-cycle phases was analysed by flow cytometry. Results from one representative experiment (performed in triplicate) are shown. Three independent experiments showed similar
results. (C) The cell lines in (A) were induced by Dox for 24 h and treated further with Taxol (10 nM) for 24 or 36 h. Flow cytometry was carried out as in (B).
understood [59]. Interestingly, Lats2 knockdown sensitized cells
to the spindle poison nocodazole [60]. Furthermore, the Hippo
pathway effectors YAP and TAZ confer resistance to Taxol in
ovarian and breast cancer cells respectively [61,62]. Thus the
Hippo signalling pathway may also function as a sensor linking
the signal elicited by spindle poisons to apoptotic cell death.
Future studies are needed to determine the role of KIBRA and its
phosphorylation in anti-mitotic drug-induced apoptosis and the
underlying mechanisms/signalling pathways. Such studies may
have implications in cancer treatment with anti-mitotic drugs since
Taxol-resistance is a major clinical challenge in Taxol-treated
patients [26].
AUTHOR CONTRIBUTION
Jixin Dong and Ming Ji designed and wrote the paper. Ming Ji, Shuping Yang and Ling
Xiao performed the experiments, analysed the data and interpreted the results. Yuanhong
Chen and Lin Zhang provided technical support and purified GST-fusion proteins, and
also interpreted the data. All authors reviewed and approved the paper before submission.
ACKNOWLEDGEMENTS
We thank J. Lukas (Danish Cancer Society, Denmark) for Myc–CDC14A/B constructs.
The HA–CDC25A and its DN mutant constructs were from Kay-Uwe Wagner (University
of Nebraska Medical Center, who had received them originally from Jacob Falck Hansen,
Novo Nordisk Biotechnology Fund, Denmark). We also thank Dr Joyce Solheim, Dr Robert
Lewis and Dr Keith Johnson for a critical reading and comments on the paper.
FUNDING
This work was supported in part by the National Center for Research Resources (NCRR), a
component of the National Institutes of Health (NIH) [grant number 5P20GM103489] and
a grant from the Nebraska Cancer and Smoking Disease Research Program (to J.D.).
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doi:10.1042/BJ20120751
SUPPLEMENTARY ONLINE DATA
Phospho-regulation of KIBRA by CDK1 and CDC14 phosphatase controls
cell-cycle progression
Ming JI, Shuping YANG, Yuanhong CHEN, Ling XIAO, Lin ZHANG and Jixin DONG1
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A.
Figure S2 MAPKs are not responsible for KIBRA phosphorylation during
Taxol treatment
GST–KIBRA-M and -C proteins were used as substrates for in vitro kinase assays with
asynchronized ( − ) or Taxol-treated ( + ) HeLa cell lysates in the presence of kinase inhibitors
as indicated. The MEK/ERK inhibitor U0126 was used at 20 μM, SP600125 (a JNK inhibitor)
was used at 20 μM, and SB203580 (10 μM) was used for inhibiting p38. 32 P incorporation was
revealed by autoradiography. The Western blot (WB) shows the substrate loading. Molecular
masses (M) are indicated in kDa.
Figure S1 GST–KIBRA-N, -M and -C proteins were used as substrates for
in vitro kinase assays with purified CDK5
MBP (myelin basic protein) was used as a positive control. The products were separated by
SDS/PAGE and transferred on to PVDF membranes, followed by autoradiography and Western
blotting (WB). Molecular masses (M) are indicated in kDa.
Figure S3 GST–KIBRA-M and GST–KIBRA-M-4SA were used as substrates
for in vitro kinase assays with purified Aurora-A kinase
The products were separated by SDS/PAGE and transferred on to PVDF membranes, followed
by Western blotting. Molecular masses (M) are indicated in kDa.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
M. Ji and others
Figure S4
CDK1-mediated phosphorylation of KIBRA does not affect Hippo–YAP signalling activity
(A) HEK-293T cells were transfected with FLAG–KIBRA or FLAG–KIBRA-4SA. At 48 h after transfection, KIBRA was immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates were
probed with an anti-NF2/Merlin antibody. Total protein lysates before immunoprecipitation were also included for Western blot analysis. (B) HEK-293T cells were transfected with various DNAs as
indicated. Cells were treated with vehicle or Taxol as indicated. Total protein lysates were subjected to Western blot analysis with the indicated antibodies. Molecular masses (M) are indicated in kDa.
IP, immunoprecipitation.
Table S1
Mitotic index of cells expressing KIBRA or KIBRA mutant
Cells were treated with Taxol at 1 μM for 16 h. The mitotic index is revealed by
anti-(phospho-Ser10 histone H3) antibody staining and flow cytometry. Numbers in parentheses
show the values for three repeats.
Cell line
Mitotic index
Tet-control
Tet-KIBRA
Tet-KIBRA-4SA
40.0 (38;42;40)
39.3 (41;37;40)
21.0 (20;19;24)
Table S2
Cell-cycle distribution of cells expressing KIBRA or KIBRA mutant
MCF-7 cells were first treated with doxycycline (200 ng/ml for 2 days) and then treated with
nocodazole (100 ng/ml) for 24 h. Cells were then analysed by flow cytometry. Values are the
percentage of cells in each phase.
Cell line
G1 -phase
G2 /M-phase
S-phase
Tet-control
Tet-KIBRA
Tet-KIBRA-4SA
7.68
9.43
8.62
77.8
75.4
72.8
14.5
15.1
18.6
Table S3
Mitotic index of cells expressing KIBRA or KIBRA mutant
Cells were treated with nocodazole at 100 ng/ml for 16 h. The mitotic index is revealed by
anti-(phospho-Ser10 histone H3) antibody staining and flow cytometry. Numbers in parentheses
represent three repeats.
Cell line
Mitotic index
Tet-control
Tet-KIBRA
Tet-KIBRA-4SA
28.0 (24;29;31)
25.7 (23;28;26)
25.0 (24;22;29)
Received 4 May 2012/9 July 2012; accepted 11 July 2012
Published as BJ Immediate Publication 11 July 2012, doi:10.1042/BJ20120751
c The Authors Journal compilation c 2012 Biochemical Society