The Rockefeller University Press $30.00
J. Cell Biol. Vol. 189 No. 5 813–827
Correspondence to R. Parker: email@example.com
Abbreviations used in this paper: MS, mass spectrometry; P-body, process-
The proper control of translation, mRNA degradation, and the
subcellular localization of mRNAs are important aspects of the
regulation of gene expression in eukaryotic cells. Eukaryotic
mRNAs are typically degraded in a process initiated by de
adenylation, which can lead to 3 to 5 degradation but often
allows mRNA decapping and 5 to 3 degradation (Parker and
Song, 2004; Garneau et al., 2007). Decapping is in competi
tion with translation initiation (Coller and Parker, 2004) and
occurs by a process involving a set of decapping activator and
enhancer proteins that can inhibit translation and/or assemble a
translationally repressed mRNP, which contains the decapping
enzyme and is capable of decapping and 5 to 3 degradation
(for review see Parker and Sheth, 2007).
mRNAs that are not engaged in translation can accumu
late in a variety of RNA–protein granules in the cytosol. For
example, untranslating mRNAs complexed with the decapping
machinery accumulate in foci referred to as processing bod
ies (Pbodies; for reviews see Anderson and Kedersha, 2006;
Eulalio et al., 2007; Parker and Sheth, 2007). Pbodies, and/or
the mRNPs within them, have been implicated in mRNA de
capping, nonsensemediated decay, mRNA storage, general
translation repression, µRNAmediated repression, and viral
life cycles (for reviews see Anderson and Kedersha, 2006;
Eulalio et al., 2007; Parker and Sheth, 2007). Transcripts within
Pbodies are in dynamic exchange with the translating pool of
mRNAs and can either be degraded or can return to translation
(Brengues et al., 2005; Bhattacharyya et al., 2006).
Pbodies can partially overlap in yeast (Brengues and
Parker, 2007; Hoyle et al., 2007; Buchan et al., 2008) or dock in
mammalian cells (Kedersha et al., 2005; Wilczynska et al., 2005),
with a second cytoplasmic RNA–protein structure referred to as
a stress granule. Stress granules are dynamic aggregates of un
translating mRNAs in conjunction with some translation initia
tion factors (e.g., eIF4E and eIF4G) and several RNAbinding
proteins with the precise composition of stress granules depen
dent on the stress or organism examined (for reviews see
degradation are reprogrammed to stabilize bulk mRNAs
and to preferentially translate mRNAs required for the
stress response. During stress, untranslating mRNAs ac-
cumulate both in processing bodies (P-bodies), which
contain some translation repressors and the mRNA deg-
radation machinery, and in stress granules, which con-
tain mRNAs stalled in translation initiation. How signal
transduction pathways impinge on proteins modulating
ranslation and messenger RNA (mRNA) degrada-
tion are important sites of gene regulation, particu-
larly during stress where translation and mRNA
P-body and stress granule formation and function is un-
known. We show that during stress in Saccharomyces
cerevisiae, Dcp2 is phosphorylated on serine 137 by the
Ste20 kinase. Phosphorylation of Dcp2 affects the decay
of some mRNAs and is required for Dcp2 accumulation
in P-bodies and specific protein interactions of Dcp2 and
for efficient formation of stress granules. These results
demonstrate that Ste20 has an unexpected role in the
modulation of mRNA decay and translation and that
phosphorylation of Dcp2 is an important control point for
Dcp2 phosphorylation by Ste20 modulates
stress granule assembly and mRNA decay in
Je-Hyun Yoon,1,2 Eui-Ju Choi,3 and Roy Parker1,2
1Department of Molecular and Cellular Biology and 2Howard Hughes Medical Institute, University of Arizona, Tucson, AZ 85721
3Laboratory of Cell Death and Human Diseases, School of Life Science, Korea University, Seoul 136-701, South Korea
© 2010 Yoon et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 189 • NUMBER 5 • 2010 814
Because Dcp2 is the critical catalytic component of the de
capping enzyme, we examined whether Dcp2 was phosphory
lated during glucose deprivation (10 min), oxidative stress
induced by hydrogen peroxide (1 mM for 30 min), or at high
cell density. We examined Dcp2 phosphorylation by immuno
purifying Flagtagged Dcp2 from cells with or without stress
conditions and then performing a Western blot on the immuno
purified material with an anti–phosphoSer–specific antibody.
We observed that hydrogen peroxide exposure, glucose
deprivation, or growth to stationary phase all led to the appear
ance of phosphorylated Dcp2 (Fig. 1). In contrast, in midlog
cultures, phosphorylated Dcp2 was not detected, although Dcp2
immunopurified to similar levels as during stress conditions, as
judged by a Western blot (Fig. 1). These results demonstrate
that Dcp2 is phosphorylated in response to hydrogen peroxide
treatment, glucose deprivation, or growth to high cell density.
Ste20 is required for Dcp2
To identify protein kinases that potentially phosphorylate Dcp2,
we focused on protein kinases activated during stress. Previous
work has shown that oxidative stress activates the yeast MAPK
pathway, including Ste20, Ste11, Ste7, Fus3, and Kss1. (Staleva
et al., 2004). Moreover, a genetic screen identified Ste20 as
being important in stress granule formation in yeast (unpublished
data). Thus, we hypothesized that Ste20 might be responsible
for the phosphorylation of Dcp2 during stress, which we tested
by examining whether Dcp2 was phosphorylated during stress
in a ste20 strain.
We observed that after hydrogen peroxide treatment, glu
cose deprivation, or growth to high cell density, the ste20
strain showed reduced phosphorylation of Dcp2 as compared
with a wildtype strain (Fig. 1). Thus, Ste20 either directly
phosphorylates Dcp2 or is required for stressinduced phos
phorylation of Dcp2 in Saccharomyces cerevisiae by activating
a downstream kinase. However, strains lacking the Ste11 and
Ste7 proteins, which are downstream of Ste20 in its canonical
MAPK pathway, still show phosphorylation of Dcp2 during
glucose deprivation (Fig. S1), arguing that Ste20 might directly
Ste20 can directly phosphorylate Dcp2
To determine whether Ste20 could directly phosphorylate Dcp2,
we immunopurified Ste20 from yeast and determined whether it
could phosphorylate recombinant Dcp2 in vitro. In these exper
iments, a wildtype or kinasedead mutant allele of Ste20 tagged
with GFP at its genomic locus (Ahn et al., 2005) was immuno
precipitated with an antiGFP antibody. The resulting immuno
pellet was mixed with the Dcp2 catalytic domain (amino acids
102–300) purified from Escherichia coli in the presence of
radioactive ATP. If Ste20 is capable of directly phosphorylating
Dcp2, Dcp2 phosphorylation should be observed and should be
dependent on the kinase activity of Ste20.
Incubation of Dcp2 102–300 with the wildtype Ste20
immunopellet, but not Ste20 kinasedead allele (K649R), led
to the labeling of an 130kD band, which is likely to be auto
phosphorylation of Ste20GFP, and a band running at 35 kD
Anderson and Kedersha, 2006; Buchan and Parker, 2009).
Stress granules are generally not present in normal cells and
form in response to defects in translation initiation, including
decreased function of eIF2 or eIF4A (Kedersha et al., 2002;
Dang et al., 2006; Mazroui et al., 2006), or heat shock or glu
cose deprivation in yeast (Brengues and Parker, 2007; Hoyle
et al., 2007; Buchan et al., 2008; Grousl et al., 2009). Pbodies
also increase during stress in both yeast and mammals presum
ably because of increases in the pool of nontranslating mRNPs
(Kedersha et al., 2005; Teixeira et al., 2005; Wilczynska et al.,
2005). At a minimum, the presence of Pbodies and stress gran
ules serves as microscopic markers for pools of biochemically
distinct mRNPs, although the additional properties of the larger
aggregates remain to be identified.
The interaction of Pbodies and stress granules suggests
that mRNA might be exchanged between these two compart
ments in a process involving remodeling of the composite
mRNPs. Consistent with that possibility, stress granules in yeast
during glucose deprivation are dependent on Pbodies for their
formation and commonly form in association with preexisting
Pbodies (Buchan et al., 2008). These observations led to the
suggestion that, at least during glucose deprivation in yeast,
mRNAs might primarily move from Pbodies to stress granules
in a process that would require exchange of Pbody components
on the mRNA for the proteins seen associated with the mRNA
in stress granules such as translation initiation factors (Buchan
et al., 2008). Such a remodeling of the mRNP would be ex
pected to impact on the fate of the transcript because transition
ing from a Pbody mRNP to a stress granule state would reduce
the possibility of mRNA degradation and promote reentry into
translation. Thus, important and unresolved issues are how
individual mRNPs within Pbodies and stress granules are
remodeled and how external stimuli activate signaling path
ways to alter these interactions, thereby modulating translation
and mRNA degradation.
Signaling pathways that activate different MAPKs control
many cellular responses to external cues. In Saccharomyces
cerevisae, three of the MAPK cascades are activated by the
Ste20 protein kinase, which serves as a MAPKKKK. We now
show that during stress, Ste20 also directly phosphorylates the
decapping enzyme on Ser137. Phosphorylation of Dcp2 affects
the decay of some mRNAs, is required for Dcp2 accumulation
in Pbodies and specific protein interactions of Dcp2, and for
efficient formation of stress granules. These results demonstrate
that Ste20 has an unexpected role in the modulation of mRNA
decay and translation and that phosphorylation of Dcp2 is an
important control point for mRNA decapping.
Dcp2 is phosphorylated during stress
Several stresses, including glucose deprivation and growth to
high cell density, lead to enhanced Pbody formation and/or
inhibition of mRNA degradation, suggesting that some compo
nents of the mRNA degradation machinery might be modified
under these conditions (Jona et al., 2000; Benard, 2004; Teixeira
et al., 2005; Greatrix and van Vuuren, 2006; Hilgers et al., 2006).
815 Phosphorylation of Dcp2 by Ste20 • Yoon et al.
phosphorylation in vitro with immunopurified Ste20 from yeast
or affinitypurified Ste20 from E. coli. We observed that the ex
tent of Dcp2 phosphorylation by Ste20 in vitro was reduced after
either the S137A or S211A mutations (Fig. 2 B and Fig. S1 B).
Moreover, the doublemutant S137A, S211A, showed almost a
complete loss to phosphorylation in vitro (Fig. 2 B and Fig. S1 B).
We interpret these results to indicate that both S137 and S211
can serve as sites of Ste20 phosphorylation in vitro.
To verify that Dcp2 was phosphorylated by Ste20 on
Ser137, we phosphorylated Dcp2 in vitro with recombinant
Ste20 purified from E. coli and analyzed the products by mass
spectrometry (MS; see Materials and methods). We observed
phosphorylated peptide fragments corresponding to phosphory
lation on Ser137 when Dcp2 was incubated with Ste20 and ATP
(Fig. 2 C). This provides direct evidence that Ste20 phosphory
lates Dcp2 on Ser137.
In vivo, the specificity of phosphorylation might be influ
enced by additional factors. Thus, we examined how the S137A
and S211A mutations affected the phosphorylation of Dcp2 in
yeast during glucose deprivation. We observed that the S137A
mutant was no longer phosphorylated, whereas the S211A mu
tant showed reduced phosphorylation (Fig. 2 D). All proteins
were equivalently immunopurified based on Western analysis
for the Flag epitope fused to Dcp2. We also observed that the
S137A mutant showed reduced phosphorylation after hydrogen
peroxide treatment, whereas the S211A mutant was phosphory
lated to levels similar to wildtype Dcp2 (unpublished data).
These observations argue that S137 is required for phosphory
lation in vivo by Ste20 and is likely to be the major site of phos
phoryl group addition by Ste20 in cells. However, there may
be additional sites, including S211A, that are phosphorylated
in some stresses. Additional evidence that S137 is the key site
for phosphorylation during stress is the phenotypes of a charge
mimetic allele at this position (see below).
Consequences of Dcp2 phosphorylation
The aforementioned results argued that Dcp2 is phosphorylated
on S137A during stress by Ste20. To determine the role of Dcp2
phosphorylation, we examined the consequences of mutations
that either prevent phosphorylation (S137A) or are charge mi
metic (S137E) on mRNA decapping, the formation of Pbodies,
and stress granules. In addition, because Ste20 affects Dcp2
phosphorylation, we also examined the effects of a ste20 in
these assays. Strikingly, we observed that dcp2 strains ex
pressing the dcp2S137A allele grew more slowly than strains
expressing the wildtype or S137E allele (see Discussion). This
indicates that phosphorylation of Dcp2 is required for optimal
growth rate. More detailed experiments to understand the role of
Dcp2 phosphorylation are described in Materials and methods.
Dcp2 phosphorylation does not generally
Because Dcp2 is the decapping enzyme, we first asked whether
alteration of the phosphorylation site affected its catalytic activ
ity. S137 is located adjacent to, but does not overlap, the active
site of Dcp2 (She et al., 2006, 2008; Deshmukh et al., 2008).
Given this, we purified the catalytic domain of Dcp2 from E. coli
(Fig. 2 A). Although this band is larger than the expected size of
Dcp2 102–300 (27 kD with tags included), this band comigrates
with the major Coomassiestained band in Dcp2 102–300 prep
arations, which we have verified by excision of the band fol
lowed by mass spectroscopy to be Dcp2 102–300. Additional
data that the phosphorylated 35kD migrating band is Dcp2
102–300 is the detection of phosphopeptides from Dcp2 after
kinasing with Ste20 (see below). These results argue that Ste20
can directly phosphorylate Dcp2, which is confirmed by dem
onstrating that Ste20 purified from E. coli was also able to phos
phorylate Dcp2 102–300 (Fig. S1 B).
Ser137 is a target for Dcp2
phosphorylation in yeast
To determine the significance of Dcp2 phosphorylation, we de
sired to identify the Ser and/or threonine residues where yeast
Dcp2 was phosphorylated and then use genetic approaches to
address the function of phosphorylation. Because the phosphory
lation sites in vitro were localized in the catalytic domain of
yeast Dcp2 (residues 102–300), we examined this portion of
Dcp2 for possible Ste20 phosphorylation sites based on com
parison with the sites mapped in histone H2B and Ste11 (Wu
et al., 1995; Ahn et al., 2005). This analysis identified Ser137
(S137) and 211 (S211) as possible Ste20 phosphorylation sites.
We tested this possibility by mutating these sites to alanine
either individually or in combination and examining their
Figure 1. Dcp2 is phosphorylated in response to stress in vivo. Wild-type
(WT) and ste20 strains in the BY4741 background were transformed
with Flag-Dcp2 expression plasmid (pRP983) and grown to reach OD
600 of 0.50–0.6. Cells were treated with 1 mM hydrogen peroxide for
30 min or deprived of glucose for 10 min. For examination at high cell
density, cells were grown in synthetic media for 48 h. Cell lysates were
immunoprecipitated (IP) with anti-Flag antibody–conjugated agarose and
probed with anti—phospho-Ser antibody (pSer; see Materials and methods).
To detect Flag-Dcp2, the membrane was reprobed with anti-Flag antibody.
JCB • VOLUME 189 • NUMBER 5 • 2010 816
Figure 2. Ste20 phosphorylates Dcp2 in vitro and in vivo. (A) GFP-tagged wild-type (WT) or kinase-dead Ste20 (K649R) integrated at the genomic locus
was immunoprecipitated with anti-GFP antibody. The resulting immunopellets were incubated with Dcp2 catalytic domain (residues 102–300; pRP1211)
purified from E. coli in the presence of radioactive ATP for 30 min at 30°C. The reaction mixture was subjected to SDS-PAGE and autoradiograph. (B) Dcp2
S137 and/or S211 residue was mutated to an alanine residue with site-directed mutagenesis (pRP1678, pRP1684, or pRP1685). The recombinant protein
was purified from E. coli and mixed with Ste20 purified from yeast for in vitro phosphorylation analysis. (C) His-tagged Dcp2 (102–300) was incubated
with (top) or without (bottom) GST-Ste20 purified from E. coli (pRP2135) in the presence of nonradioactive ATP. The resulting reaction mixtures were di-
gested with trypsin for MS analysis. Product b and y ions were indicated in the peptide sequences and mass spectrum, respectively. Key m/z values were
indicated to compare mass shift after phosphorylation. The b4, b5, and b6 peptides, which show a shift corresponding to phosphorylation, are marked
with colored arrows. (D) Plasmids expressing Dcp2 wild type, S137A, or S211A (pRP983, pRP1676, or pRP1682, respectively) were transformed into
wild-type or ste20 strains. Cells were grown in mid-log phase and exposed to glucose deprivation stress. Cell lysates were examined to detect phosphory-
lated Dcp2 by SDS-PAGE and immunoblot (IB) assay.
817 Phosphorylation of Dcp2 by Ste20 • Yoon et al.
different mutant alleles and examined their location during
midlog growth and after glucose deprivation.
This experiment revealed the following important obser
vations. First, we observed that the accumulation of the wild
type Dcp2GFP protein in Pbodies was reduced in ste20
strains (Fig. 4). The accumulation of Dcp2GFP in Pbodies in
the ste20 was not reduced as much as the accumulation of the
Dcp2S137A protein in a wildtype strain. This suggests that
additional kinases might be able to phosphorylate Dcp2 at low
levels. Consistent with this possibility, we observed that after long
exposures, low levels of phosphorylated Dcp2 could be detected
in the ste20 strain during glucose deprivation (unpublished
data). Nevertheless, the reduction in Dcp2GFP accumulation in
Pbodies in the ste20 strain provides additional evidence that
phosphorylation of Dcp2 promotes its accumulation in Pbodies
A second important observation was that the chargemimetic
dcp2S137E allele rescued the defect in Dcp2 accumulation seen
in the ste20 strain (Fig. 4). This observation strongly argues
that the defect in Dcp2 accumulation in Pbodies in the ste20
strain is caused by the failure of Dcp2 to get phosphorylated.
A third observation was that the dcp2S137E mutant did
not accumulate above normal levels in Pbodies during midlog
growth (Fig. 4). This argues that phosphorylation of Dcp2 is not
sufficient by itself to induce large Pbodies, although Dcp2
phosphorylation is necessary during stress responses for the ac
cumulation of Dcp2 in Pbodies.
Dcp2 phosphorylation affects stress
granule formation but not P-body formation
The aforementioned observations indicated that phosphoryla
tion of Dcp2 was required for its accumulation in Pbodies dur
ing stress. This could be because phosphorylation of Dcp2 is
required for Pbodies to form or because Dcp2 phosphorylation
specifically affects Dcp2 accumulation in Pbodies. Given this,
we examined how Pbodies formed in the various Dcp2 alleles
as well as in ste20 strains using Edc3mCherry as a marker of
Pbodies. Moreover, because recent results suggest that Pbodies
promote the formation of stress granules in yeast (Buchan et al.,
2008), we also examined how the Dcp2S137A and S137E
alleles and the ste20 affected stress granule formation in the
same experiments using a Pab1GFP fusion protein as a marker
of yeast stress granules (Buchan et al., 2008). Thus, either wild
type, ste20, or various dcp2 mutant strains were transformed
with a centromere plasmid expressing Edc3mCherry (a Pbody
marker) and Pab1GFP (a stress granule marker) protein fusions
and their subcellular location examined with and without glucose
deprivation. These experiments revealed the following points.
First, we observed that dcp2 strains expressing the dcp2
S137A allele still produced Pbodies as judged by the accumu
lation of Edc3mCherry (Fig. 5). Moreover, as seen previously,
dcp2 strains also formed robust Pbodies during glucose dep
rivation and formed enhanced Pbodies during midlog growth,
presumably caused by a defect in mRNA decapping (Sheth and
Parker, 2003; Teixeira and Parker, 2007). These results demon
strate that neither Dcp2 phosphorylation nor Dcp2 itself is re
quired for Pbody formation per se and, therefore, demonstrates
(residues 102–300) either as wildtype or with the S137A or
S137E mutations and assayed the catalytic ability of this pro
tein in vitro with a caplabeled substrate based on the MFA2
mRNA. We observed that the S137A or S137E mutation did
not substantially alter the decapping activity of Dcp2 in vitro
(Fig. S2). Based on this, we suggest that phosphorylation does
not directly inhibit or stimulate Dcp2 enzymatic activity.
To examine the effects of these lesions on decapping in
vivo, we examined the decay of the MFA2pG reporter mRNA,
which is under control of the GAL promoter, in dcp2 strains
transformed with plasmids expressing Dcp2 wild type, dcp2
S137A, or dcp2S137E. We examined mRNA decay during both
midlog growth, in which Dcp2 is generally not phosphorylated
and mRNA decay is normal, and during glucose deprivation, in
which Dcp2 is phosphorylated and mRNA decay is inhibited,
primarily by a block to deadenylation (Hilgers et al., 2006).
We observed that the decay rate of the MFA2pG mRNA
was largely unaffected by the S137A or S137E alleles of Dcp2
in both midlog cultures and during glucose deprivation (Fig. 3 A
and Fig. S3). However, we did observe that the total levels of
mRNA were consistently reduced in the S137E strain for unknown
reasons (≤40% mRNA compared with wild type). Nevertheless,
the main implication is that Dcp2 phosphorylation on S137 does
not globally alter mRNA decay in vivo. Similarly, we observed
that the decay of the MFA2pG reporter was the same in ste20
and wildtype strains both in midlog and stress conditions
(Fig. 3 B and Fig. S3). We interpret these results to indicate that
phosphorylation of Dcp2 does not globally alter mRNA decay,
although it remains possible that Dcp2 phosphorylation affects
the decapping of a subset of mRNAs (see below).
Dcp2 phosphorylation affects its
localization in P-bodies
We also investigated the effect of Dcp2 phosphorylation on the
subcellular location of Dcp2. During stresses such as glucose
deprivation and high cell density, Dcp2 accumulates in Pbodies
(Teixeira et al., 2005). We transformed Dcp2GFP expression
plasmids either with or without the S137A, S137E, or S211A
mutations in wildtype strains and examined the subcellular
location of Dcp2 in midlog cultures as well as those exposed to
glucose deprivation and grown to stationary phase.
We observed that the Dcp2S137AGFP failed to accumu
late in Pbodies during glucose deprivation and growth to high
cell density (Fig. 4). In contrast, the Dcp2 wildtype, Dcp2
S211A, and Dcp2S137E proteins all accumulated in Pbodies.
The failure of Dcp2S137A proteins to accumulate in Pbodies
is not because of changes in its expression levels (Fig. S4).
These observations argue that phosphorylation of Dcp2 is
required for its efficient accumulation in Pbodies.
If Dcp2 phosphorylation is required for its accumulation in
Pbodies, strains lacking Ste20, which affects Dcp2 phosphoryla
tion, should also show a defect in the accumulation of Dcp2 in
Pbodies. Moreover, if this defect is largely caused by the loss of
Dcp2 phosphorylation, then a chargemimetic allele of Dcp2
would be predicted to restore Dcp2 accumulation in Pbodies in a
ste20 strain. To test these predictions, we transformed a ste20
strain with GFPtagged versions of either wildtype Dcp2 or the
JCB • VOLUME 189 • NUMBER 5 • 2010 818
phosphorylation are required for optimal stress granule for
mation. We also observed that ste20 strains showed reduced
stress granule formation during glucose deprivation as com
pared with wildtype cells, although Pbodies formed normally
in the ste20 strain (Fig. 5). This observation argues that Ste20
enhances stress granule formation either through phosphorylation
that phosphorylation of Dcp2 is required for Dcp2 accumula
tion in Pbodies.
Second, we observed that Dcp2 wildtype strains effi
ciently formed stress granules, whereas the dcp2 and dcp2
S137A strains showed reduced accumulation of stress granules
(Fig. 5, Pab1GFP). These results suggest that Dcp2 and its
Figure 3. Dcp2 phosphorylation is not re-
quired for mRNA decay. (A) dcp2 strain
(yRP1358) expressing Gal-MFA2pG mRNA
was transformed with centromere plasmids
expressing either wild-type, S137A, or S137E
Dcp2-GFP (pRP1275, pRP1677, or pRP1680,
respectively; Coller and Parker, 2005; this
study). Cells were grown in synthetic media
containing galactose, and transcription was
repressed by changing to glucose media.
At each time point, cells were harvested, and
total RNA was analyzed by Northern blotting.
(B) BY4741 wild-type or ste20 was trans-
formed with GAL-MFA2pG plasmid and grown
in minimal media with galactose. MFA2pG
transcription was blocked by glucose addition,
and MFA2pG mRNA levels were examined
over time by Northern blotting.
819 Phosphorylation of Dcp2 by Ste20 • Yoon et al.
restore stress granule formation in the ste20 strain. To test
this possibility, we expressed the wildtype or dcp2S137E al
leles in a ste20 strain and examined Pbody and stress gran
ule formation in response to glucose deprivation. We observed
that the ste20 strain expressing the Dcp2S137E protein
showed partially restored stress granule formation as compared
with the wildtype strain, whereas ste20 strains expressing
of Dcp2 and/or by phosphorylation of additional proteins. The
observed changes in Pab1 accumulation were not caused by re
duction of protein expression (Fig. S4).
The requirement for Dcp2 phosphorylation for stress
granule formation suggests that the requirement for Ste20 in
stress granule formation is at least in part caused by phosphory
lation of Dcp2. This predicts that the dcp2S137E allele would
Figure 4. Dcp2 phosphorylation is necessary
for its accumulation in P-bodies. A wild-type
(WT) strain (BY4741) was transformed with
wild-type, S137A, S137E, or S211A Dcp2-
GFP (pRP1683) plasmids. Cells were grown
in synthetic media to mid-log and deprived
of glucose for 10 min or grown to high cell
density before microscopic examination on a
microscope. A ste20 strain in the BY4741
background was transformed with wild-type
or S137E Dcp2-GFP plasmid and exposed to
glucose deprivation or high OD stress. Cells
having at least one Dcp2 foci were counted by
ImageJ for quantification (see Materials and
methods). Error bars indicate SD. Bar, 5 µm.
JCB • VOLUME 189 • NUMBER 5 • 2010 820
alters protein–protein interactions within mRNPs accumulating
in Pbodies, and thereby leads to mRNP transitions that transform
an mRNA into a stress granule mRNP. This possibility is also raised
by the observation that stress granule formation in S. cerevisiae is
promoted by preexisting Pbodies (Buchan et al., 2008). Interest
ingly, the Dhh1 protein, which interacts with Dcp2 (Decker et al.,
2007) and localizes to Pbodies, is required for optimal stress
granule formation (Buchan et al., 2008). This suggested a pos
sible mechanism whereby Dcp2 phosphorylation might alter the
Dhh1–Dcp2 interaction and thereby affect the ability of Dhh1 to
promote stress granule formation. Given this, we examined the
coimmunoprecipitation of wildtype, S137A, and S137E Dcp2
variants with Dhh1 in wildtype or ste20 strains.
exogenous wildtype Dcp2 still showed a defect in stress gran
ule formation (Fig. 5). The ability of the chargemimetic form
of Dcp2 to restore stress granule formation in the ste20 strain
provides additional evidence that phosphorylated Dcp2 pro
motes stress granule formation and also provides strong evi
dence that at least part of the role of Ste20 in stress granule
formation is to phosphorylate Dcp2.
Dcp2 phosphorylation is required to
maintain Dhh1–Dcp2 interactions during
One possible mechanism by which Dcp2 phosphorylation pro
moted stress granule formation is that Dcp2 phosphorylation
Figure 5. Dcp2 phosphorylation affects stress
granule but not P-body formation. Wild-type
(WT; BY4742), ste20 (yRP2547), or dcp2
(yRP1358) strains were transformed with
Pab1-GFP/Edc3-mCherry plasmid (pRP1658;
Buchan et al., 2008), grown to OD 600 of
0.5–0.6 in synthetic media, and deprived
of glucose for 10 min before the localization of
Pab1p-GFP and Edc3p-mCherry were exam-
ined with a microscope. In addition, the dcp2
strain was transformed with wild-type or S137A
Flag-Dcp2 plasmid (pRP983 or pRP1676) be-
fore glucose deprivation. The ste20 strain
containing Pab1-GFP/Edc3-mCherry plasmids
was transformed with wild-type or S137E Flag-
Dcp2 (pRP983 or pRP1679). Images were
quantified as in Fig. 4 and in Materials and
methods. Error bars indicate SD. Bar, 5 µm.
Phosphorylation of Dcp2 by Ste20 • Yoon et al.
of mRNA decay rates, we validated that two ribosomal protein
mRNAs (Rpl26a and Rpp1b) showed slower rates of mRNA
degradation in the dcp2S137E strain as compared with either
wildtype or dcp2S137A strains (Fig. 7, B and C). Collectively,
these results argue that phosphorylation of Dcp2 at S137 stabi
lizes a subclass of mRNAs enriched in ribosomal mRNAs.
Our microarray results also revealed other alterations in
mRNA levels in response to alterations at S137. We observed
that there was a class of mRNAs, preferentially enriched in
mitochondrial function (Fig. 7 A), that were increased in both
the dcp2S137E and dcp2S137A strains as compared with wild
type and, therefore, may be mRNAs whose degradation is nor
mally enhanced by S137. We also observed a class of mRNAs,
preferentially enriched in mRNAs involved in amino acid syn
thesis or heat response, which were unregulated in the dcp2
S137A strain and unaffected in the dcp2S137E strain. Finally,
we observed a class of mRNAs enriched in iron transporters,
which were unaffected in the dcp2S137E strain but down
regulated in the dcp2S137A strain. Collectively, these results
indicate that modification of Dcp2 on S137 impacts the levels of
several mRNAs by both direct and indirect mechanisms.
Ste20 modulates mRNA decay by
phosphorylation of Dcp2
Our observations indicate that Dcp2 is phosphorylated by Ste20
during certain stresses. Specifically, during glucose deprivation,
hydrogen peroxide exposure or growth to high cell density,
immunopurified Dcp2 reacts with antibody against phosphoSer
in a manner dependent on Ste20 (Fig. 1). Moreover, immuno
purified or recombinant Ste20 can phosphorylate the catalytic
region of Dcp2 in vitro on Ser137 as confirmed by MS analysis
(Fig. 2). Phosphorylation of Dcp2 by Ste20 is primarily on
Ser137 because mutation of Ser137 to alanine reduces phos
phorylation of Dcp2 by Ste20 in vitro and in vivo (Fig. 2 B,
Fig. S1 B, and Fig. S2 D). In addition, substitution of Ser137
with a chargemimetic allele, S137E, suppresses some of the
defects seen in a ste20 strain (Figs. 4 and 5). These results
reveal an unexpected function of Ste20, modifying the decapping
enzyme, and demonstrate that activation of Ste20 leads to a bi
furcated response, whereby phosphorylation of a downstream
MAPK cascade leads to transcriptional regulation (Herskowitz,
1995), and Ste20 directly impinges on molecules involved in
posttranscriptional control, thereby affecting the rates of trans
lation and/or decay of certain mRNAs.
An unresolved issue is the cellular compartment wherein
Ste20 phosphorylates Dcp2. Ste20 shuttles between the nucleus
and cytoplasm, and during oxidative stress accumulates in the
nucleus (Ahn et al., 2005). Because Dcp2 also shuttles between
the nucleus and cytosol (Grousl et al., 2009), it could be that
Ste20 phosphorylates Dcp2 in the nucleus. However, because
some Ste20 remains in the cytosol during oxidative stress (Ahn
et al., 2005) and we have not observed accumulation of Ste20 in
the nucleus during glucose deprivation or growth to high cell
concentrations (unpublished data), it could be that Dcp2 is phos
phorylated by cytoplasmic Ste20.
In wildtype strains, we observed that during midlog,
Dhh1 coimmunoprecipitated with Dcp2, and this coimmuno
precipitation was unaffected by the S137E or S137A alleles.
This indicates that Dcp2 can interact with Dhh1 independent of
the phosphorylation status of Dcp2 during midlog growth. In
contrast, during glucose deprivation, we observed that the
S137A allele showed reduced ability to coimmunoprecipitate
Dhh1, although it was expressed at normal levels (Fig. 6 A).
Similarly, in ste20 strains, Dhh1 and Dcp2 coimmunoprecipi
tated during midlog cultures but showed reduced interaction
during glucose deprivation (Fig. 6 B). Strikingly, the S137E
allele of Dcp2 could restore the interaction with Dhh1 during
glucose deprivation in the ste20 strain. These observations
argue that the interaction between Dhh1 and Dcp2 is altered
during stress either directly or indirectly and that phosphoryla
tion of Dcp2 is required to maintain the interaction of Dhh1 and
Dcp2 under stress conditions.
The requirement for Dcp2 phosphorylation to maintain
interactions with Dhh1 and to assemble into Pbodies suggested
two possible models by which these events could be occurring,
which can be distinguished by the subcellular location of Dhh1
during stress. In one model, stress induces translation repres
sion, forming an initial Pbody containing Edc3 and Dhh1 (as
well as other proteins), then phosphorylated Dcp2 would be re
cruited to this complex, which predicts that Dhh1 accumulation
in Pbodies would be independent of Dcp2 phosphorylation. In
an alternative model, the order of assembly would be formation
of a Pbody containing Edc3 (as well as other proteins), which
then recruits phosphorylated Dcp2, leading to the recruitment
of Dhh1, which predicts that Dhh1 accumulation in Pbodies
would be dependent of Dcp2 phosphorylation. Thus, we exam
ined the subcellular location of Dhh1GFP in various mutants
affecting Dcp2 phosphorylation with or without stress.
Consistent with earlier results (Teixeira and Parker, 2007),
we observed that Dhh1 accumulated in Pbodies during midlog
in a dcp2 strain. More importantly, we observed that Dhh1
GFP was also present in Pbodies in dcp2S137A and ste20
strains (Fig. 6 C). This indicates that Dhh1 recruitment into
Pbodies is independent of Dcp2 or its phosphorylation status
and is therefore upstream of Dcp2 recruitment into Pbodies.
Dcp2 phosphorylation affects the
expression and decay of certain mRNAs
The aforementioned results raised the possibility that phosphory
lation of Dcp2 might affect the decay of some, but not all, tran
scripts. To identify mRNAs whose degradation might be affected
by Dcp2 phosphorylation, we performed microarray analysis
comparing the dcp2S137E and dcp2S137A alleles with wild
type Dcp2, which led to several important observations.
Most importantly, in dcp2S137E cells, no mRNAs were
downregulated more than twofold, and 40 mRNAs out of
6,200 were upregulated more than twofold, which suggests
that Dcp2 phosphorylation stabilizes a subset of mRNAs. Strik
ingly, the mRNAs upregulated in the dcp2S137E stain were
overrepresented in ribosomal protein mRNAs (Fig. 7 A), sug
gesting that Dcp2 phosphorylation led to preferential stabiliza
tion of this class of mRNAs. Moreover, by direct measurement
JCB • VOLUME 189 • NUMBER 5 • 2010 822
interactions. First, the S137A allele of Dcp2 fails to accumulate
in Pbodies during stress, although Pbodies form as judged by
Edc3p accumulation (Figs. 4 and 5). Second, ste20 strains
still form Pbodies during glucose deprivation, as judged by
Dcp2 phosphorylation affects its
accumulation in P-bodies
Several observations indicate that phosphorylation of Dcp2 by
Ste20 affects its accumulation in Pbodies and its protein–protein
Figure 6. Dcp2 phosphorylation is required for its interaction with Dhh1p during glucose deprivation. (A) Wild-type (WT; BY4742) strains were trans-
formed with expression plasmid of Dhh1-GFP (pRP 1151; Coller et al., 2001) controlled by its own promoter and centromeric replication origin. Again,
these strains were cotransformed with Flag-tagged wild-type (pRP983), S137A (pRP1676), or S137E (pRP1679) Dcp2 plasmids. Cells were grown in
synthetic media to reach OD 600 of 0.5–0.6 with or without glucose deprivation for 10 min. Cell lysates were immunoprecipitated (IP) with anti-Flag
antibody–conjugated beads, and copurified Dhh1p-GFP was detected with anti-GFP antibody after SDS-PAGE. (B) Wild-type or ste20 (yRP2547) strains
was cotransformed with plasmids expressing Dhh1-GFP and plasmids expressing Flag-tagged wild-type or S137E Dcp2 as indicated. (C) Wild-type,
ste20, or dcp2 (yRP1358) strains were transformed with a plasmid expressing Dhh1-GFP, or in some cases, the dcp2 strain was cotransformed with
plasmids expressing Dcp2-WT, S137A, or S137E as indicated. Dhh1 localization was examined with a microscope with or without stress. IB, immunoblot.
Bar, 5 µm.
823Phosphorylation of Dcp2 by Ste20 • Yoon et al.
Figure 7. Dcp2 phosphorylation is required for the expression and degradation of certain mRNAs. (A) The schematic of expression microarray analysis
and gene clustering of 109 genes, which are up-regulated or down-regulated more than twofold in dcp2-S137E or 137A allele. Number of genes found
in the microarray, total genes involved in specific gene ontology, and p-values were described. (B and C) Dcp2-WT, dcp2-S137A, or dcp2-S137E strains
(yRP2680, yRP2681, or yRP2682, respectively) were grown in synthetic media containing to reach mid-log. And the cells were incubated with 5 µg/ml
thiolutin for the indicated times, and total RNA was prepared at each time point for Northern blot analysis of Rpl26a or Rpp1b mRNA. The relative intensity
of each band was quantified for a half-life measurement with two independent experiments.
JCB • VOLUME 189 • NUMBER 5 • 2010 824
An unresolved issue is how phosphorylated Dcp2 promotes
stress granule formation. One possibility is that when mRNAs are
exiting translation, they can either assemble an mRNP capable
of accumulation into stress granules or an mRNP capable of as
sembling into Pbodies, and that phosphorylated Dcp2 limits the
accumulation of the Pbody type of mRNP, thereby promoting
stress granule formation. However, three observations make this
possibility unlikely. First, recent results indicate that Pbodies
promote the assembly of stress granules in yeast and are not
in competition with stress granule formation (Buchan et al.,
2008). Second, we observe that dcp2 strains are also defective
in stress granule formation (Fig. 5), which is inconsistent with
Dcp2 functioning in competition with stress granule formation.
Third, we observe that dcp2S137A and dcp2 strains not only
show reduced stress granules, they show increased size and in
tensity of Pbodies in midlog as judged by Edc3. Given this,
the simplest interpretation is that phosphorylation of Dcp2 in
creases the formation of stress granules by affecting remodeling
of mRNPs within Pbodies and thereby promoting the transition
of some mRNPs from Pbodies into stress granules, perhaps by
affecting Dhh1 function, which is required for optimal stress
granule assembly (Buchan et al., 2008). This model provides a
possible explanation for why dcp2S137A strains grow poorly
(unpublished data). In the absence of Dcp2 phosphorylation,
some mRNAs will be unable to exit Pbodies and reenter trans
lation, which is consistent with the small Pbody formation in
S137A strains even during midlog growth (Fig. 5).
Dcp2 phosphorylation stabilizes some
mRNAs encoding ribosomal proteins
Several observations argue that phosphorylation of Dcp2 modu
lates the levels and/or decay of some, but not all, yeast mRNAs.
Most importantly, microarray analysis of the dcp2S137E allele
revealed an increase in a set of mRNAs enriched in ribosomal
proteins (Fig. 7 A). Moreover, direct measurement of the mRNA
decay rates of some of these mRNAs revealed that they were
more stable in the dcp2S137E strain as compared with either
wildtype or dcp2S137A strains (Fig. 7, B and C). However,
this effect is limited to a subset of mRNA conditions because
the levels of many mRNAs were not changed in the different
Dcp2 alleles, and phosphorylation of Dcp2 did not affect the
decay of the MFA2pG reporter mRNA (Fig. 3). To date, we
have only been able to examine the effects of Dcp2 phosphory
lation during midlog growth because during the stresses we
have used to date, mRNA deadenylation is also inhibited, and
bulk mRNA is stabilized (Hilgers et al., 2006), thus obscuring
our ability to measure the effect of Dcp2 phosphorylation on
decapping of the total population of mRNAs. However, it is
likely that the role of Dcp2 phosphorylation under stress would
be to stabilize the subpopulation of mRNAs that are already
deadenylated. Because we only analyzed mRNAs that changed
greater than twofold in our microarray experiments, it is possible
that additional mRNAs are affected by Dcp2 phosphorylation to
a smaller degree. Thus, phosphorylation of Dcp2 stabilizes a
subset of mRNAs, particularly those involved in ribosome bio
genesis. Because the mRNAs involved in ribosome biogenesis
represent a large fraction of the total mRNA in the cell, this
accumulation of Edc3, but fail to recruit Dcp2 to these complexes
(Fig. 5). Third, the ability of Dcp2 to accumulate in Pbodies
in the ste20 strain can be restored by the chargemimetic
S137E allele of Dcp2 (Fig. 4). Fourth, the dcp2S137A allele in
wildtype cells and the wildtype Dcp2 in ste20 strains show
reduced coimmunoprecipitation with Dhh1 during glucose dep
rivation (Fig. 6). Moreover, the reduced interaction of Dcp2 and
Dhh1 in the ste20 strain can be restored by the S137E allele
(Fig. 6). Thus, phosphorylation of Dcp2 increases its coimmuno
precipitation with Dhh1 at least during glucose deprivation.
These results indicate that the phosphorylation of Dcp2 is
likely to change some aspect of its protein–protein interactions,
thereby affecting the assembly of an mRNP containing the
One surprising observation was that the Dhh1–Dcp2 inter
action is normal during midlog growth when Dcp2 is not
phosphorylated, yet requires Dcp2 to be phosphorylated during
glucose deprivation to maintain the interaction. This could be
because phosphorylation of Dcp2 is required to counterbalance
additional modifications to Dhh1 or Dcp2 during stress that di
rectly reduces their interaction. Alternatively, during stress, Dcp2
might interact with other factors that compete for Dhh1 binding
to Dcp2 and that phosphorylation decreases the binding to
allow Dhh1 to bind more tightly.
Dcp2 phosphorylation promotes stress
Several observations indicate that phosphorylation of Dcp2
by Ste20 affects the formation of stress granules. First, strains
lacking Dcp2 or Ste20 show reduced numbers and fainter
stress granules during glucose deprivation (Fig. 5). This is
not a result of defects in decapping per se because dcp1
strains, which are as defective as dcp2 strains in decapping,
show increased numbers of stress granules during glucose
deprivation (Buchan et al., 2008). Second, strains with the
dcp2S137A allele show a defect in forming stress granules
(Fig. 5). Third, the defect in stress granule formation in a
ste20 strain can be partially suppressed by expression of
the Dcp2S137E protein. This latter observation indicates
that at least part of the defect in stress granule formation of
the ste20 strain is the result of a failure to phosphorylate
Dcp2. These observations demonstrate that Dcp2 is not just
a decapping enzyme but plays a second role in stress gran
ule formation in a manner dependent on its phosphorylation.
This also provides additional evidence that stress granule
formation during glucose deprivation in yeast is dependent
on Pbodies (Buchan et al., 2008).
Our results suggest that Ste20 might also impact stress
granule formation through additional targets. This possibility is
based on the observations that although the ste20 strain shows
dramatically decreased stress granule formation during glucose
deprivation, a dcp2 strain only has partially reduced stress
granules. Moreover, the dcp2S137E allele only partially res
cued the ste20 defect in stress granule formation. Another
possible target for Ste20 affecting stress granule formation is
eIF4E, which was identified as a substrate for Ste20 in a genome
wide screen (Ptacek et al., 2005).
825 Phosphorylation of Dcp2 by Ste20 • Yoon et al.
To analyze phosphorylation in vivo, cells were grown in synthetic
media to mid-log (or stationary phases) and harvested with or without de-
scribed stresses. Cells were lysed, and Flag-Dcp2 (Dunckley and Parker,
1999) was purified as previously described (Beckham et al., 2007).
Immunopellets were run on SDS-PAGE and subjected to Western analysis
with antibody against phospho-Ser. Images are representative of three in-
Expression microarray analysis was performed as described previously
(Capaldi et al., 2008). dcp2 strain was transformed with expression plas-
mid of Dcp2-GFP wild-type, S137A, or S137E mutant. Cells were grown
in synthetic complete media to reach OD 600 of 0.5–0.6 and frozen
in dry ice. Total RNA was purified by phenol/chloroform and chloroform
extraction. 40 µg total RNA was converted into cDNA by SuperScript III
(Invitrogen) with 40 µg random primer (N9) and amino allyl UTP (Sigma-
Aldrich) and purified with the use of gel extraction kit (QIAGEN). 5 µg
purified DNA was labeled with NHS ester Cy3 or Cy5 (GE Healthcare) by
incubating in sodium bicarbonate for 5 h, and the free dye was removed
by gel extraction kit. 500 ng Cy3- or Cy5-labeled cDNA was hybridized to
a microarray with 6,200 60-base probes (G4140A arrays; Agilent Tech-
nologies) in hybridization buffer (Agilent Technologies) for 16 h at 65°C
in rotation chamber. The arrays were washed and scanned with the use
of a scanner (4000B; Axon). The resulting images were analyzed with
GenePix, and the all of the files were uploaded to Stanford Microarray
Database. The further clustering work was performed with Cluster 3.0
(Eisen Laboratory) and Java Tree view. Two independent microarray analy-
ses were performed (Table S5).
Liquid chromatography tandem MS
Mapping of phosphorylation on Dcp2 in vitro was performed as follows.
First, His-Dcp2 (102–300) was incubated with or without GST-Ste20 puri-
fied from E. coli for 2 h at 37°C. The reaction mixtures were digested in
100 mM ammonium bicarbonate by trypsin (10 µg/ml) at 37°C overnight
as previously described (Flannery et al., 1989). Liquid chromatography
MS/MS analyses of trypsin-digested proteins were performed using a lin-
ear quadrupole-ion trap mass spectrometer (LTQ; Thermo Fisher Scientific)
equipped with an HPLC system (Paradigm MS4; Michrom), an autosam-
pler (AS3000; SpectraSystems), and a nanoelectrospray source as de-
scribed previously (Andon et al., 2002; Lantz et al., 2007). Tandem MS
spectra of peptides were analyzed with a program that allows the correla-
tion of experimental tandem MS data with theoretical spectra generated
from known protein sequences (TurboSEQUEST version 3.1; Thermo Fisher
Scientific; Eng et al., 1994). The peak list (DTA files) for the search was
generated by Bioworks (version 3.1; Thermo Fisher Scientific). Parent pep-
tide mass error tolerance, fragment ion mass tolerance, and criteria used
for preliminary positive peptide identification are the same as previously
described (Cooper et al., 2003; Qian et al., 2005). All matched peptides
were confirmed by visual examination of the spectra. All spectra were
searched against an S. cerevisiae database (downloaded October 3,
2009; National Center for Biotechnology Information) and the primary se-
quence of DCP2. At the time of the search, the S. cerevisiae protein data-
base from contained 64,422 entries. The results were also validated using
XTandem, another search engine (Craig and Beavis, 2004), and with Scaf-
fold, a program that relies on various search engine results (i.e., Sequest,
XTandem, and MASCOT) and that uses Bayesian statistics to reliably iden-
tify more spectra (Keller et al., 2002; Nesvizhskii et al., 2003).
Online supplemental material
Fig. S1 shows Dcp2 phosphorylation during glucose deprivation in wild
type or various deletion strains of MAPK pathway in yeast and in vitro
phosphorylation assay results with Ste20 purified from E. coli. Fig. S2
shows in vitro decapping activity of wild-type or charge-mimetic Dcp2.
Fig. S3 shows the decay rate of MFA2pG during glucose deprivation in
wild type, Dcp2 mutant alleles, or the Ste20 deletion strain. Fig. S4 shows
expression level of Dcp2-GFP or Pab1-GFP in various alleles with Western
blot analysis. Tables S1–S5 detail properties of yeast strains, plasmids,
and oligonucleotides as well as quantitation of microscopic results and raw
data of microarray analysis. Online supplemental material is available at
We thank C. David Allis for yeast strains and expression plasmid, J. Ross Buchan
and Carolyn Decker for critical review of the manuscript, Anne Webb for as-
sistance with blind scoring of microscopic images, Carl Boswell for assistance
with microscopy, Andrew Capaldi for the support of microarray analysis,
suggests that during stress, Dcp2 phosphorylation contributes to
the formation of an mRNP that can selectively promote the re
modeling of this class of mRNPs from a Pbody state to a stress
granule complex. Such a remodeling would be expected to sta
bilize the mRNA by reducing its interactions with the decap
ping complex and possibly also promote its subsequent reentry
into translation. Thus, posttranslational modification of the de
capping enzyme by Ste20 plays an important role in modulating
the fate of cytoplasmic mRNAs.
Materials and methods
Yeast strains, growth conditions, plasmids, and oligonucleotides
The list of strains, plasmids, and oligonucleotides used in this work are
shown in Tables S1, S2, and S3, respectively. Strains were grown at 30°C
in a shaking water incubator in yeast/extract/peptone medium or synthetic
medium supplemented with the appropriate amino acid drop out solutions
and 2% glucose. All site-directed mutagenesis was performed using stan-
dard protocols, and resulting plasmids were verified by sequencing (Wang
and Malcolm, 1999).
Cells were grown to OD 600 of 0.5–0.6 in synthetic media with 2% glu-
cose. For glucose deprivation, cells were centrifuged and quickly washed
with synthetic medium lacking glucose. Pellets were resuspended in syn-
thetic medium lacking glucose and incubated for 10 min in a shaking
water bath at 30°C. Images were collected by a deconvolution microscope
(Deltavision RT; Applied Precision) with a UPlan S Apo 100× 1.4 NA
objective (Olympus). 512 × 512–pixel files were acquired with a camera
(CoolSNAP HQ; Photometrics) by 1 × 1 binning. Z-series images were
compiled with maximum intensity projections using ImageJ (National In-
stitutes of Health). Each image was blindly randomized and quantified as
described previously (Buchan et al., 2008). The counting and measure-
ment of foci size were performed using ImageJ with smoothing, threshold-
ing, and analyze particle functions. Quantitation datasets represent the
analysis of at least two independent experiments with at least 50 cells. The
details for quantitation are shown in Table S4.
Western blot analysis
Western blots were performed following standard protocols. The following
antibodies and their sources were used: anti–phospho-Ser antibody (BD),
anti-Flag antibody (Sigma-Aldrich), anti-GFP antibody (Covance), or anti-
actin antibody (Abcam). Goat anti–mouse HRP (Thermo Fisher Scientific)
was used as a secondary antibody.
mRNA decay assay
For in vivo mRNA decay assay, cells were grown to reach OD 600 of
0.5–0.6 in synthetic medium supplemented with either 2% glucose or
2% galactose at 30°C. Transcription was blocked by transferring cells to
4% glucose media (when grown in galactose) by adding tetracycline
(when using a tet-off reporter) or by adding thiolutin (when probing Rpl26a
or Rpp1b; Sigma-Aldrich). Total RNA was extracted and probed with
oRP140 (Muhlrad et al., 1994; Caponigro and Parker, 1995), 100 (Caponigro
et al., 1993), 1479, or 1480. The resulting images were acquired with a
phosphoimager (Molecular Dynamics) and quantified using 7S RNA as a
loading control (Caponigro et al., 1993). Images are representative of
two independent experiments.
To analyze phosphorylation of Dcp2 in vitro, the catalytic core of Dcp2
(amino acids 102–300) and Ste20 were purified from E. coli as previ-
ously described (Ahn et al., 2005; She et al., 2006). Ste20 was puri-
fied from yeast using either Ste20-TAP or Ste20-GFP fusion proteins
(Ahn et al., 2005). The TAP purification method was used as described
previously (Rigaut et al., 1999). To immunopurify the GFP-tagged pro-
tein, cell lysates were incubated with anti-GFP antibody (AnaSpec) for
2 h, and IgG-conjugated Sepharose bead (GE Healthcare) was used to
pull down antibody (Ryoo et al., 2004). The resulting immunopellet was
washed three times with lysis buffer and mixed with purified recombi-
nant proteins and radioactive ATP in the kinase reaction buffer as de-
scribed previously (Ryoo et. al., 2004).
JCB • VOLUME 189 • NUMBER 5 • 2010 826
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George Tsaprailis for MS analysis, and the Parker laboratory for support
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Proteomics Consortium supported by NIEHS (grant ES06694 to the SWEHSC),
National Institutes of Health/NCI (grant CA023074 to the AZCC), and the
BIO5 Institute of the University of Arizona. This work was supported by
the Howard Hughes Medical Institute and the National Institutes of Health
Submitted: 3 December 2009
Accepted: 28 April 2010
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