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 826
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George Tsaprailis for MS analysis, and the Parker laboratory for support
and discussions. Mass spectrometric data were acquired by the Arizona
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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