The P Body Protein Dcp1a Is Hyper-phosphorylated
Adva Aizer, Pinhas Kafri, Alon Kalo, Yaron Shav-Tal*
The Mina and Everard Goodman Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel
Processing bodies (PBs) are non-membranous cytoplasmic structures found in all eukaryotes. Many of their components
such as the Dcp1 and Dcp2 proteins are highly conserved. Using live-cell imaging we found that PB structures disassembled
as cells prepared for cell division, and then began to reassemble during the late stages of cytokinesis. During the cell cycle
and as cells passed through S phase, PB numbers increased. However, there was no memory of PB numbers between
mother and daughter cells. Examination of hDcp1a and hDcp1b proteins by electrophoresis in mitotic cell extracts showed a
pronounced slower migrating band, which was caused by hyper-phosphorylation of the protein. We found that hDcp1a is a
phospho-protein during interphase that becomes hyper-phosphorylated in mitotic cells. Using truncations of hDcp1a we
localized the region important for hyper-phosphorylation to the center of the protein. Mutational analysis demonstrated the
importance of serine 315 in the hyper-phosphorylation process, while other serine residues tested had a minor affect. Live-
cell imaging demonstrated that serine mutations in other regions of the protein affected the dynamics of hDcp1a
association with the PB structure. Our work demonstrates the control of PB dynamics during the cell cycle via
Citation: Aizer A, Kafri P, Kalo A, Shav-Tal Y (2013) The P Body Protein Dcp1a Is Hyper-phosphorylated during Mitosis. PLoS ONE 8(1): e49783. doi:10.1371/
Editor: Claude Prigent, Institut de Ge ´ne ´tique et De ´veloppement de Rennes, France
Received June 13, 2012; Accepted October 15, 2012; Published January 2, 2013
Copyright: ? 2013 Aizer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants to YST by the Israel Science Foundation (grant 250/06) and the Alon Fellowship. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Yaron.Shav-Tal@biu.ac.il
Processing bodies (P bodies, PBs) are cytoplasmic structures
involved in mRNA decay and mRNA storage. PB structures are
detected in all eukaryotes and many of their components are
conserved from yeast cells to mammals. PB numbers and size are
quite dynamic. Mammalian cells harbor between 3–9 distinct PBs,
although these numbers can vary. PBs are 100–300 nms in
diameter and are composed of aggregates of electron dense fibrils
as observed by electron microscopy. They are readily detectable
when cytoplasmic mRNA levels are elevated, and tend to
disassemble when mRNA levels drop [1,2].
PB detection is based mainly on their protein components, such
as the decapping enzyme Dcp2 and the exonuclease Xrn1, hinting
to their possible role in 59R39 mRNA degradation pathways [3–
5]. Still, even when dispersed in the cytoplasm, PB enzymes do not
lose their ability to function in mRNA decay . The 59 cap
structure of mRNA is removed by Dcp2, an enzyme that requires
interaction with other proteins for full functionality. In yeast, the
Dcp1p protein is a requisite for Dcp2 function [6–8], while in
human cells additional proteins are required for the Dcp1-Dcp2
interaction [9,10]. The C-terminus of Dcp1 is a trimerization
domain and is required for the decapping activity of the decapping
complex . Human cells carry two hDcp1 homologues, hDcp1a
and hDcp1b, encoded by two separate genes. The functional
difference between the two is unknown and most studies have used
the hDcp1a variant as a PB marker.
PB structures are mRNA-protein complexes that are not
membrane surrounded. Photobleaching experiments used for
measuring protein dynamics in living cells have demonstrated
that most PB components exhibit a continuous flux between the
cytoplasm and the PB. Uniquely, hDcp2 in PBs shows very low
recovery rates after photobleaching indicating that it is a core PB
protein , in comparison to proteins like hDcp1a that are
continuously exchanging with the cytoplasmic pool. While a
variety of conditions affect PB formation and disassembly, for
instance cell cycle stage , cell proliferation rates, nutrient
availability and translational stress, the signals that control PB
assembly and disassembly are not well understood.
In a previous study we quantified PB mobility in living human
cells and demonstrated PB interactions with the microtubule
network . This association has been observed in yeast  and
neuronal cells . We showed that PBs disassembled when
transcription and translation were inhibited. Additionally, we
found that the disruption of the microtubule network caused an
opposite effect of PB assembly . In this study we focused our
attention on the time-frame of cell division during which the
transcription and translation processes are inhibited, together with
microtubule network disassembly. Using live-cell microscopy we
demonstrate an increase in PB numbers during S phase, the
disappearance of PB structures before mitosis, and their reassem-
bly during cytokinesis. We further analyze the phosphorylation
modifications occurring on hDcp1a at the time of cell division.
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PB disassembly and assembly during cell division
We examined the fate of PBs during cell division. Previous
studies, in which PBs/GWBs were marked with an antibody to
GW182, found that PBs disassembled upon entry to mitosis .
Using antibodies to endogenous hDcp1b (Figure 1) and other PB
markers (hDcp1a, hDcp2, Hedls; data not shown) we found that
all antigens showed the same behavior and dispersed throughout
the cytoplasm of human U2OS cells during mitosis, indicating that
the entire PB structure disassembles. The same phenomenon was
observed in long-term live cell imaging of a GFP-Dcp1a U2OS
stable cell line that allowed the visualization of PB dynamics
throughout the cell cycle. Most PBs disappeared during or several
minutes before nuclear envelope breakdown (Figure 2 and Movie
S1). Occasionally, PBs disappeared some minutes after. The
reassembly of PBs after mitosis occurred several minutes after
nuclear envelope assembly (Figure 2 and Movie S1). Interestingly,
PBs were also observed in the retraction regions formed during cell
mobility (Top daughter cell, Figure 2 and Movie S1).
We did not observe a correlation between the number of PBs
present within the cell before and after mitosis. For instance, in
Figure 2 there were two detectable PBs in the pre-mitotic cell,
while after mitosis the two daughter cells contained 4 or 7 PBs,
respectively. To carefully examine PB numbers during the cell
cycle we made use of the Fucci system that allows the identification
of cells in various phases of the cell cycle using two fluorescent cell
cycle markers. The two Fucci vectors encode for Cdt1 or Geminin,
proteins whose levels fluctuate differentially throughout the cell
cycle. Cdt1 levels peak in G1 phase, and then as cells transition
into S phase, Cdt1 levels fall and Geminin levels rise, remaining
high until the cells are back in G1. Cells control Cdt1 and
Geminin levels post-translationally using ubiquitination to target
the unwanted proteins for proteasomal degradation. We expressed
a combination of mCherry-Cdt1 and AmCyan1-Geminin in
U2OS cells and labeled PBs using an anti-Hedls antibody
(Figure 3A). Counting PBs at different steps of the cell cycle
showed that PB numbers increased from an average of 462 per
cell during G1 and G1/S to an average of 763 in S/G2, and were
not detectable during mitosis (Figure 3B). The gradual change in
PB numbers through the cell cycle could also be followed in living
cells expressing GFP-Dcp1a and the Fucci markers (Figure 3C,D
and Movie S2). An increase in PB numbers was observed during
late S, which then remained steady until cell division. Finally, the
daughter cells contained less PBs than the mother cell prior to
mitosis (Figure 3C,D and Movie S2). Altogether, we find that
visible PB structures increase in number as the cell approaches S
phase, and then finally disassemble during mitosis during which
PB components are dispersed in the dividing cell. PB structures
reform immediately after daughter cell formation.
Dcp1a protein is hyper-phosphorylated during mitosis
It is known that phosphorylation regulates the assembly/
disassembly of structures during mitosis . We next examined
whether post-translational modifications are occurring on PB
components during PB disassembly, focusing on the hDcp1a
protein. Western blots to endogenous hDcp1a showed that
changes in PB integrity during the cell cycle did not involve a
reduction in protein levels. Instead, slower band migration was
observed for the endogenous hDcp1a protein coming from protein
extracts of synchronized mitotic cells (nocodazole block), indicat-
ing the occurrence of post-translational modifications on the
protein during mitosis (Figure 4A). Several slow migrating hDcp1a
bands were observed in mitotic cells, while cells synchronized to
G1/S (thymidine block) showed a faster migrating doublet similar
to untreated cells (Figure 4A). The same mobility patterns were
observed in HeLa cells (Figure S1A). Slower migrating bands were
not observed in cells treated with nocodazole for only 30 minutes
(not shown), a treatment that disassembles microtubules , nor
with cycloheximide for up to 4 hours (Figure 4B and S1A), a
treatment that inhibits translation.
Treatment of protein extracts from mitotic cells with a
phosphatase led to higher mobility of hDcp1a and to the
disappearance of the slower migrating bands (Figure 4B and
S1A). Moreover, the band from the phosphatase-treated extract
Figure 1. PB assembly and disassembly during the cell cycle.
Immunofluoresence staining of endogenous hDcp1b (green), a-tubulin
(red), DNA (Hoechst, blue) and DIC images show that PB structures
disassemble during cell division. (Bar 20 mm).
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did not present as a doublet as seen in the extracts from untreated
cells, and had even higher mobility than the hDcp1a protein bands
from the control and G1/S blocked cells. The hyper-phosphor-
ylation of Dcp1a was also observed when synchronizing cells by
the means of noscapine, which interferes with microtubule
function and thus with cell division (Figure 4C). Altogether, this
means that during interphase hDcp1a is a phospho-protein, that
later undergoes hyper-phosphorylation prior to or during mitosis.
Hyper-phosphorylation was detected also for GFP-Dcp1a and
GFP-Dcp1b (Figure S1B).
Amino acids situated within the 200–380 aa region of
the Dcp1a protein are phosphorylated during mitosis
To identify the region in hDcp1a undergoing hyper-phosphor-
ylation during mitosis we first generated a series of GFP-Dcp1a
truncated constructs containing different regions of the protein.
We first determined which is the smallest domain of Dcp1a that
still assembles in PBs. Figure 5A shows that hDcp1a truncated of
its mid- and C-terminal regions (protein containing only aa’s 1–
200) could assemble in PBs while the shorter N-terminal 1–150 aa
protein did not assemble in PBs. All the truncated proteins were
tested and did not have a dominant negative effect on the
formation of endogenous PBs in the transfected cells.
The N-terminal 1–133 region in hDcp1a is similar to the N-
terminal regions of hDcp1b, and to two regions of the S. cerevisiae
Dcp1p protein homologue . This region is important for
decapping activity, since site-specific mutations in two residues of
hDcp1a (D20A and R59A) reduced the decapping activity of
immunoprecipitated complexes . We therefore generated
additional constructs based on the smallest assembling truncated
protein (1–200) in which the regions with the critical amino acids
(aa’s 20 and 59) were removed. Truncated versions containing the
region 75–200 of hDcp1a assembled in PBs indicating that these
specific N-terminal residues (in region 1–75) were not necessary for
assembly into PBs. However, further dissection of this region
showed that truncated proteins containing amino acids 150–200
or 100–200 were not able to assemble into PBs. To identify the
region of hDcp1a that undergoes hyper-phosphorylation we
expressed these truncated forms of GFP-Dcp1a that assemble in
PBs, performed a nocodazole block and collected mitotic cells.
The N-terminal portion of Dcp1a (75–200 or the 1–200 regions)
did not show a mobility shift in Western blots (Figure 5C),
meaning that this probably was not the region responsible for the
Next we generated N-terminally truncated forms of hDcp1a
(200–582 or 380–582) lacking the 75–200 region. These were still
able to assemble into PBs (Figure 5B), implying that it is not
necessarily a contiguous region in the protein that is responsible
for hDcp1a targeting to PBs. Finally, hyper-phosphorylation in
mitotic cells was detected for the truncated 1–380 form lacking the
Figure 2. PB assembly and disassembly during cell division in living cells. Cells stably expressing GFP-Dcp1a were simultaneously imaged in
GFP and DIC showing the assembly and disassembly of PBs from a movie acquired for 14 hours. Cells were imaged every 6 min. Red arrows: PBs in
the cell before mitosis. White arrows: PBs in daughter cells after mitosis. Yellow arrow head: PB in a retraction fiber.
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Figure 3. PB numbers increase as cells reach S/G2 phase of the cell cycle. (A) The Fucci markers mCherry-Cdt1 (red) and AmCyan1-Geminin
(cyan) were expressed in U2OS cells and then cells were stained with an anti-Hedls (green) antibody to mark PBs. (Bar 20 mm). It was possible to
detect the cell cycle phase using the intensity combination of the red and cyan markers in the cell, as explained in scheme below. (B) The number of
PBs in each cell was counted and assigned a cell cycle phase according to the Fucci colors. The plot designates the average PB number in each phase
(G1 n=40, G1/S n=15, S/G2 n=40, M n=10). Error bars represent STDEV and a T-Test was performed. (C) U2OS cells stably expressing GFP-Dcp1a
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C-terminus (Figure 5C). We therefore concluded that the region
200–380 contains amino acid residues that are hyper-phosphor-
ylated during mitosis.
Serine 315 is involved in Dcp1a hyper-phosphorylation
The above mentioned 200–380 region of hDcp1a contains a
high number of putative phosphorylations sites (21 serines, 14
threonines and 1 tyrosine) (Figure S2). A previous study provided a
scored list of serine and threonine residues that are prone to
phosphorylation during mitosis . We chose to focus on serines
in positions 315 and 319 (found within the 200–380 region of
hDcp1a), which received high phosphorylation scoring in this
analysis. As a control we chose serines 522 and 523 from the C-
terminus, which were also highly scored but according to the
truncations are probably not phosphorylated during mitosis
Using site specific mutagenesis we mutated all 4 serines to
alanines in the GFP-Dcp1a construct, and generated the following
S522+523A. All mutated plasmids were then transiently transfect-
ed into U2OS cells. In all cells the expressed mutated hDcp1a
proteins were found in PBs (Figure 6A), and disassembled during
mitosis (Figure 6B). We stably integrated the wild type and
mutated plasmids into U2OS cells. Counting of PBs in the cell
lines showed a statistically significant reduction in PB numbers in
the cells expressingGFP-Dcp1a
(Figure 7A). This could indicate that these mutated forms were
not able to correctly assemble in PBs. Therefore, we next
examined whether there was a change in the dynamics of the
hDcp1a protein during association and dissociation with PB
structures, due to these mutations. The association/dissociation
rates of PB components are measured using the fluorescence
recovery after photobleaching (FRAP) method . FRAP
recovery rates demonstrated that indeed the S522,523A mutations
caused a significantly faster interchange of the hDcp1a protein
within the PBs (t1/2of recovery=3.3 sec for wild type hDcp1a,
and 2.28 sec for S522,523A) indicating a problem in association
with the PB structure (Figure 7B). Moreover, there was a change in
the immobile Dcp1a fraction within the PB, which was typically
32% in cells expressing wild type hDcp1a, and was reduced to
20% with the S522,523A mutant, once again indicating an
assembly defect for this mutant. However, the S315A and S319A
mutations did not significantly change the dynamics (Figure 7B).
We then examined whether the hyper-phosphorylation occur-
ring on GFP-Dcp1a during mitosis as seen by a mobility shift in
Western blots, was affected by the serine mutations in hDcp1a.
Figure 8A shows that hDcp1a hyper-phosphorylation in mitotic
cells was highly reduced in the S315A mutant (Figure 8A and
S3A,B), although the endogenous hDcp1a protein was hyper-
phosphorylated as usual (Figure S3A). In contrast, the S319A and
the S522,523A mutants continued to show hyper-phosphorylated
bands in the mitotic cell extracts (Figure 8A and S3A,B), although
the mobility shift was less pronounced in S319A. Altogether, these
results demonstrate that serine residue S315 is a key player in the
hyper-phosphorylation that Dcp1a undergoes during mitosis.
The Dcp1-Dcp2 interaction is required for 59 mRNA decap-
ping from yeast to metazoa. In humans this interaction occurs in
multimeric decapping complexes that require enhancers of
decapping 3 and 4 (EDC3, EDC4), and the DEAD-box RNA
helicase DDX6. A structural study showed that Dcp1a proteins
can form trimers via a C-terminal trimerization domain and can
also heterodimerize with Dcp1b . Dcp1a can interact with
Dcp2 and EDC4 independently of its interaction with EDC3 and
DDX6. PB size can change in response to a variety of cell cycle
and metabolic signals, and this may depend on the oligomerization
capabilities of some of its components such as hDcp1a
[5,12,13,20]. Indeed, the oligomerization traits of hDcp1a can
lead to the formation of very large PBs observed when
fluorescently tagged hDcp1a is highly overexpressed in human
cells . hDcp1a dynamic properties in PBs as measured by
FRAP were dependent on PB size, and the comparison of FRAP
recoveries of different PB proteins has suggested that hDcp1a
shuttles in and out of PBs independently of the RNA substrates
were co-transfected with AmCyan1-Geminin and mCherry-Cdt1 and imaged for 15 hours. Frames show the cytoplasmic GFP-Dcp1a signal together
with nuclear AmCyan1-Geminin staining that looks green due to the filter used. The plot represents the relative intensity analysis of all markers as
quantified throughout the movie. Red – mean Cdt1 intensity, cyan – mean Geminin intensity, green – number of PBs.
Figure 4. Dcp1a is hyper-phosphorylated during cell division. Western blot analysis of (A) endogenous hDcp1a protein in U2OS cell extracts
during interphase (untreated), metaphase (nocodazole block, Noc) and at G1/S (thymidine block, Thy), showed the appearance of slower migrating
Dcp1a bands in metaphase cells. (B) Treatment of U2OS protein extracts from metaphase cells with a phosphatase (Noc+PPase) caused a reduction in
the molecular weight of hDcp1a, compared to untreated, G1/S blocked (Thy), and metaphase blocked cells (Noc). This demonstrated that Dcp1a is
hyper-phosphorylated during mitosis. Treatment with cycloheximide (Cyclo) for 1 or 4 hrs did not change the mobility of hDcp1a indicating that
hyper-phosporylation is cell cycle dependent. (C) Shift in mobility due to hyper-phosphorylation in mitotic cells is seen using two different cell cycle
blockers, nocodazole (Noc) and noscapine. Similarly, phosphatase treatment (Nos+PPase) caused a reduction in the molecular weight of Dcp1a from
noscapine treated cells. Tubulin was used as a loading control.
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Figure 5. Phosphorylation of Dcp1a occurs in 200–380 region of the protein. GFP constructs containing different fragments of the hDcp1a
protein were transfected into U2OS cells and their assembly into PBs was monitored. The symbol ! indicates accumulation in PBs and the
symbol6indicates no accumulation. (A) C-terminal truncations showing that region 1–200 is important for assembly into PBs. (B) N-terminal
truncations. (Bar 20 mm). (C) Change in SDS-PAGE mobility in extracts from mitotic cells was detected for the 1–380 aa GFP-Dcp1a truncated protein,
but not in the 1–200 and 75–200 aa forms. The blots were reacted with anti-GFP. Tubulin was used as a loading control.
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While studying PB dynamics in living cells we found that we
could follow PB assembly and disassembly during the cell cycle.
The numbers of detectable PBs increases as cells proceed through
S phase, and remain high until cell division. PBs disassemble
during mitosis and reassemble immediately when interphase
resumes. There are less PBs in daughter cells compared to the
number of PBs in the mother cell before division. Since
phosphorylation is a major regulator of structural integrity in cells
during mitosis we decided to examine the phosphorylation profile
of hDcp1a during mitosis. We found that hDcp1a and hDcp1b
proteins undergo hyper-phosphorylation during mitosis. Several
findings have indicated that hDcp1a is a phospho-protein under
normal conditions. The yeast Dcp1 protein was found to migrate
as two bands in SDS-PAGE experiments and was later shown to
be a phospho-protein [21,22]. Similar results were observed in a
variety of mammalian cell lines . Interestingly, the relative
intensity of these differentially migrating bands changed during
mouse brain development or following differentiation of P19
neuronal cells, culminating with only one hDcp1a band. These
data demonstrated that hDcp1a phosphorylation levels can be
reduced during differentiation. This study also examined a
number of serine and threonine residues and found that mutation
of S142, S144, S319, T321 or S353, had no effect on the mobility
of YFP-Dcp1a. However, when mutated at S315 the hDcp1a
Figure 6. Serine mutated Dcp1a proteins assembled into PBs. (A) Serine to alanine mutated GFP-Dcp1a proteins assembled in PBs in U2OS
cells, and (B) disassembled during mitosis. Enlarged insets are boxed. DNA was counterstained with Hoechst. (Bar 20 mm).
Figure 7. Serine 522 and 523 mutations affect Dcp1a association/dissociation dynamics in PBs. (A) Plot showing the average number of
PBs in cells expressing the different serine mutated forms of Dcp1a. A statistically significant reduction in PB numbers was seen in cells expressing the
S522,523A and S319A mutations (n=40). Error bars represent STDEV and a T-Test was performed. (B) PBs containing the different serine mutated
proteins were photobleached and the kinetics of recovery were analyzed. The curves represent an average of 20 PBs in 10 cells. The recovery curves
were statistically different as seen by Mann-Whitney (non parametric test). The recovery curves were fit by Matlab and the t1/2recovery times and
fixed fractions were calculated and showed a reduction in Dcp1a association with the PB structure in cells expressing the S522,423A mutant.
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protein migrated as a single band, and when a triple mutation
S315/S319/T321 was expressed, the migration was even slower.
Following arsenate treatment hDcp1a became hyper-phosphory-
lated but the change in mobility was not affected by separately
mutating S315, S319 or T321, whereas the triple S315/S319/
T321 mutant did not show a mobility shift . Altogether, these
data demonstrate that hDcp1a is a phospho-protein under normal
conditions, and can be hyper-phosphorylated following arsenate
Phosphatase treatment of the hyper-phosphorylated form of
hDcp1a from mitotic cells showed that the protein in interphase is
already a phospho-protein and accumulates additional phospho-
modifications during mitosis. The region important for hDcp1a
hyper-phosphorylation during mitosis was identified within the
center of the protein (aa’s 200–380). We chose to mutate two
serine amino acids within this region (315 and 319) and two serine
residues from the C-terminus (522 and 523). These amino acid
positions were shown to be highly prone to phosphorylation by a
proteomic screen . Serine 315 mutation had a dramatic effect,
while the others did not. During mitosis, very little hyper-
phosphorylation was detected on the S315A mutated protein.
Since the hyper-phosphorylation occurs probably on several
hDcp1a residues as deduced from the significant mobility shifts,
it would suggest that phosphorylation on serine 315 is critical for
initiation of this process during mitosis. On the other hand, even
though stably overexpressed, this mutated protein did not have a
negative effect on PB integrity and could in fact assemble in PBs.
The mutations in the C-terminus of hDcp1a (residues 522 and
523) were not important for the hyper-phosphorylation of the
protein during mitosis. However, these amino acid substitutions
changed the association rates of hDcp1a with the PBs as measured
by FRAP, indicating that this region plays an important role in
association with the PB structure. Indeed, the C-terminal portion
of hDcp1a has been shown to be an essential region for the
trimerization of hDcp1a, in which it folds into three kinked a-
helices, and is also important for localization to PBs .
Under endogenous conditions of cell division, when cells divide
and cease to transcribe in conjunction with the loss of the MT
network, PBs disassemble. We found that Dcp proteins are hyper-
phosphorylated during cell division, in particular hDcp1a. On the
other hand, drug treatments such as nocodazole or cycloheximide
that increase PB abundance, did not affect the phosphorylation
status of the protein. Serine 315 of hDcp1a is important for
hDcp1a phosphorylation during translational stress such as
arsenate treatment  or sorbitol or anisomycin . The latter
study showed that S315 is phosphorylated by the JNK kinase in
response to IL-1 treatment, and that hDcp1a phosphorylation
during stress is associated with PB dispersal. Interestingly,
decapping activity of S315 mutant hDcp1a was not abolished.
Other PB proteins are phospho-proteins, such as GW182 ,
Pat1 in yeast , and Dcp2 in yeast . S. cerevisiae Dcp2 is
phosphorylated by the Ste20 kinase during a variety of stresses
such as glucose deprivation, oxidative stress, and high cell density,
and in this case the phosphorylation is required for the localization
in PBs . Pat1 in yeast is phosphorylated by PKA, and this
disrupts interaction with other PB components such as Dhh1 .
Overall, it is possible that PB assembly, disassembly and even
cytoskeletal association and transport are controlled by phosphor-
Why then is Dcp1a hyper-phosphorylated during mitosis? This
might be a mechanism for protecting mRNAs from degradation
during cell division. mRNAs must not be degraded during mitosis
otherwise the cell would enter a new cycle with no available
mRNAs or proteins. Indeed, most translation shuts down during
mitosis  probably via mechanisms that regulate initiation .
It was suggested that much of the mRNAs are retained in
polysomes during mitosis . Also, mRNAs are stabilized when
translation inhibitors are added to cells [31–33]. It has been
hypothesized  that mRNA stabilization is a requirement for
the cell during cell division (and similarly when using translation
inhibitors). This hypothesis postulated that mRNA stabilization
during mitosis cannot possibly depend on the binding of thousands
of stabilizing molecules to mRNAs, but most likely relies on the
inactivation of mRNA degradation factors during mitosis. Our
data suggest that PB disassembly during mitosis is regulated by
specific phosphorylation events that disrupt the interactions
between key PB components, which may lead to inactivation of
the decapping machinery. PB dispersal alone does not protect
mRNAs from degradation since even under such conditions PB
proteins maintain their competence for mRNA decay [2,35].
However, phosphorylation of yeast Dcp2 had a positive effect on
mRNA stability of several mRNAs  as did the phosphorylation
of hDcp1a on several IL-1 responsive mRNAs . It therefore
seems plausible to suggest that disassembly of PBs in conjunction
with hyper-phosphorylation of key components such as hDcp1a
would render PB proteins inactive during cell division, thus
providing a time-frame during which mRNA decay is significantly
reduced and to allow for fast recovery of post-mitotic cellular
Materials and Methods
Plasmids and site directed mutagenesis
The GFP-Dcp1a was previously described . The truncated
forms of GFP-Dcp1a were obtained by the following restriction
reactions on GFP-Dcp1a: 1–200: digested with XbaI; 1–150:
digested with PstI. For 150–200: GFP-Dcp1a was digested with
HindIII and PstI, and the fragment was subcloned into the
peGFP-C1 vector. For 75–200: This region was amplified by RT-
PCR from GFP-Dcp1a using primers – ATAGAATTCGCA-
Figure 8. Serine 315 is important for the hyper-phosphoryla-
tion of Dcp1a during cell division. Top - The S319A and S522,523A
mutated GFP-Dcp1a proteins showed prominent hyper-phosphoryla-
tion patterns compared to the S315A protein. Bottom - blot comparing
the mobility shifts of the mutated proteins from mitotic cell extracts
showing that S315A is the least affected.
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TAACTCTTCTACCGT, digested with EcoRI and XbaI and
subcloned into peGFP-C1. For 100–200: The amplified 75–224
region was digested with HindIII and XbaI and then subcloned
The previously constructed GFP-Dcp1a plasmid was mutated
using the QuikChange II Site-Directed Mutagenesis kit (Strata-
gene). Serine 315 was mutated to alanine using primers – CTACA
AACAGGGGAACGGGATTGTGTAG. Serine 319 was mutat-
ed to alanine using primers - CGTTGAGCCCTGTTCTCGCC
Both serines 315 and 319 were mutated to alanine using primers
Both serines at 522 and 523 were mutated to alanine using
primers - CCTTGAGAGGAAAGCCGCGCCCCTTCTCCTC-
Cell culture and transfections
Human U2OS cells were maintained in low glucose DMEM
(Biological Industries, Israel) and HeLa cells in high glucose
DMEM (Gibco) containing 10% FBS (HyClone). The following
U2OS stable lines were generated: GFP-Dcp1a, GFP-Dcp1a
S315A, GFP-Dcp1 S319A, GFP-Dcp1a S522,523A. For Western
blotting of GFP-Dcp1a truncations, cells were transiently trans-
fected with Lipofectamine (Invitrogen). The Fucci system
(Clontech) was used for cell cycle phase detection. For G1 phase
detection, pRetroX-G1-Red vector (mCherry-hCdt1) was used,
and for S/G2 the Phase pRetroX-SG2M-Cyan vector (AmCyan-
hGeminin). Fucci vectors were transiently transfected into U2OS
cells with the PolyJet transfection reagent (SignaGem Laborato-
Immunofluorescence was performed as previously described
. Primary antibodies: rabbit anti-hDcp1a, rabbit anti-hDcp1b,
anti-Dcp2 (J. Lykke-Andersen, University of Colorado, Boulder,
CO), mouse anti-hDcp1a (Abnova), mouse anti-Hedles (Santa
Cruz), rabbit anti-a-tubulin (Abcam). Secondary Abs: anti-rabbit
and anti-mouse Cy3 (Jackson ImmouResearch). Nuclei were
counterstained with Hoechst 33342 and coverslips were mounted
in mounting medium.
For the various treatments cells were treated with: 600 nM
nocodazole, 5 mg/ml cycloheximide (Sigma). For cell cycle
synchronization, cells were arrested at G1/S using a thymidine
block. Briefly, cells were cultured for 1 day and then incubated in
medium containing 5 mM thymidine (Sigma) for U2OS cells and
2 mM thymidine for HeLa cells for 24 hours. For a mitotic block,
cells were incubated with medium containing 600 nM nocodazole
or 25 mM noscapine for 16 hrs. Mitotic cells were washed off the
plates. Cells were then washed briefly and cell extracts were
prepared for Western blotting.
SDS-PAGE and Western blotting were performed as previously
described . When appropriate, extracts were further treated
with 400 u of lambda protein phosphatase (New England Biolabs)
for 1 hr at 30uC. Primary antibodies used were mouse anti-
hDcp1a (Abnova), rabbit anti-hDcp1a, (J. Lykke-Andersen),
mouse anti-a-tubulin (Abcam), mouse anti-GFP (Covance), mouse
anti-GFP (Roche). The secondary antibody was a HRP-conjugat-
ed goat anti-rabbit or anti-mouse IgG (Sigma). Immunoreactive
bands were detected by the Enhanced Chemiluminescence kit
Fluorescence microscopy, live-cell imaging and data
Wide-field fluorescence images were obtained using the Cell‘R
system based on an Olympus IX81 fully motorized inverted
microscope (606PlanApo objective, 1.42 NA) fitted with an Orca-
AG CCD camera (Hamamatsu), rapid wavelength switching, and
driven by the Cell‘R software. For time-lapse imaging, cells were
plated on glass-bottomed tissue culture plates (MatTek, Ashland,
MA) in medium containing 10% FCS at 37uC. The microscope is
equipped with an enclosure incubator which includes temperature
and CO2 control (Life Imaging Services, Reinach, Switzerland).
For long-term imaging of the cell cycle, several cell positions were
chosen and recorded by a motorized stage (Scan IM, Ma ¨rzha ¨user,
Wetzlar-Steindorf, Germany). In these experiments, cells were
typically imaged in 3D (4 Z planes per time point) every 6 minutes,
at 3.33 mm steps (Figure 2) or every 15 minutes at 2 mm steps
(Figure 3). For presentation of the movies, the 4D image sequences
were transformed into a time sequence using the maximum
projection option in the Cell‘R software.
FRAP experiments were performed using a 3D-FRAP system
(Photometrics) built on an Olympus IX81 microscope (636 Plan-
Apo, 1.4 NA) equipped with an EM-CCD (Quant-EM, Roper),
491 nm laser, Lambda DG-4 light source (Sutter), XY&Z stages
(Prior), and driven by MetaMorph (Molecular Devices). Experi-
ments were performed at 37uC with 5% CO2 using a live cell
chamber system (Tokai). For each acquisition, PBs were bleached
using the 491 nm laser. Six pre-bleach images were acquired.
Post-bleach images were acquired with a sequence of 2 time
frequencies: 57 images every 350 msec and 12 images every 3 sec.
The experiments were analyzed using ImageJ macros previously
described . Data from at least 10 experiments for each cell line
were collected and the averaged FRAP measurements were fitted
division in living cells. Cells stably expressing GFP-Dcp1a
were simultaneously imaged in GFP and DIC showing the
assembly and disassembly of PBs during a movie of 14 hours. Cells
were imaged every 6 min.
PB assembly and disassembly during cell
stable for GFP-Dcp1a were co-transfected with AmCyan1-
Geminin and mCherry-Cdt1 and imaged for 15 hours. The
movie shows the cytoplasmic GFP-Dcp1a signal together with
nuclear AmCyan1-Geminin staining (looks green due to the filter
used). The increase in PBs during S can be seen for the marked cell
and also for the unlabeled cell above, which also undergoes mitosis
at a similar time. Cells were imaged every 15 min.
PB numbers during the cell cycle. U2OS cells
mitosis. (A) Treatment of U2OS or HeLa protein extracts
before SDS-PAGE with a phosphatase (Noc+PPase) caused a
Hyper-phosphorylation of Dcp1a during
Dcp1a Hyper-Phosphorylation in Mitosis
PLOS ONE | www.plosone.org9 January 2013 | Volume 8 | Issue 1 | e49783
reduction in the molecular weight of hDcp1a, compared to Download full-text
untreated, G1/S blocked (Thy), and metaphase blocked cells
(Noc), and the appearance of slower migrating Dcp1a bands. This
demonstrated that Dcp1a is hyper-phosphorylated during mitosis.
Treatment with cycloheximide (Cyclo) for 1 or 4 hrs did not
change the mobility of hDcp1a indicating that hyper-phosporyla-
tion is cell cycle dependent. (B) Shift in mobility due to hyper-
phosphorylation in mitotic cells is seen also for GFP-Dcp1a and
GFP-Dcp1b using an anti-GFP antibody. Phosphatase (Noc+P-
Pase) treatment caused a reduction in the molecular weight of
GFP-Dcp1a and Dcp1b, compared to control. Tubulin was used
as a loading control.
protein. The central region of Dcp1a is marked in red (200–380,
as used in figure 5). Serine, threonine, and tyrosine residues are
marked in green. Mutated amino acids are marked in yellow.
Putative phosphorylation sites in the hDcp1a
tion of GFP-Dcp1a. (A) No hyper-phosphorylation of hDcp1a
S315A mutated protein was observed in mitotic cells (Noc)
expressing GFP- hDcp1a S315A (100 kD) compared to the
endogenous Dcp1a protein (70 kD) which did show hyper-
phosphorylated Dcp1a bands. The blot was reacted with anti-
Dcp1a. (B) The S319A and S522,523A mutated GFP-Dcp1a
proteins showed prominent hyper-phosphorylation patterns com-
pared to the S315A protein. Tubulin was used as a loading
Mutation S315A reduces hyper-phosphoryla-
We are grateful to J. Lykke-Andersen (University of Colorado, Boulder,
CO) for providing antibodies. We thank A. Shraga for his assistance.
Conceived and designed the experiments: AA YST. Performed the
experiments: AA PK AK. Analyzed the data: AA. Wrote the paper: YST.
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PLOS ONE | www.plosone.org10 January 2013 | Volume 8 | Issue 1 | e49783