Dcp2 is phosphorylated during stress
Several stresses, including glucose deprivation and growth to high cell density, lead to enhanced P-body formation and/or inhibition of mRNA degradation, suggesting that some components 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
). Because Dcp2 is the critical catalytic component of the decapping enzyme, we examined whether Dcp2 was phosphorylated 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 immunopurifying Flag-tagged Dcp2 from cells with or without stress conditions and then performing a Western blot on the immunopurified material with an anti–phospho-Ser–specific antibody.
We observed that hydrogen peroxide exposure, glucose deprivation, or growth to stationary phase all led to the appearance of phosphorylated Dcp2 (). In contrast, in mid-log cultures, phosphorylated Dcp2 was not detected, although Dcp2 immunopurified to similar levels as during stress conditions, as judged by a Western blot (). These results demonstrate that Dcp2 is phosphorylated in response to hydrogen peroxide treatment, glucose deprivation, or growth to high cell density.
Figure 1. Dcp2 is phosphorylated in response to stress in vivo. Wild-type (WT) and ste20Δ 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 (more ...)
Ste20 is required for Dcp2 phosphorylation
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 Moreover, a genetic screen identified Ste20 as being important in stress granule formation in yeast (unpublished data).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 ste20Δ strain.
We observed that after hydrogen peroxide treatment, glucose deprivation, or growth to high cell density, the ste20Δ strain showed reduced phosphorylation of Dcp2 as compared with a wild-type strain (). Thus, Ste20 either directly phosphorylates Dcp2 or is required for stress-induced phosphorylation 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 phosphorylate Dcp2.
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 experiments, a wild-type or kinase-dead mutant allele of Ste20 tagged with GFP at its genomic locus (Ahn et al., 2005
) was immunoprecipitated with an anti-GFP antibody. The resulting immunopellet 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 wild-type Ste20 immunopellet, but not Ste20 kinase-dead allele (K649R), led to the labeling of an ~130-kD band, which is likely to be autophosphorylation of Ste20-GFP, and a band running at ~35 kD (). Although this band is larger than the expected size of Dcp2 102–300 (27 kD with tags included), this band comigrates with the major Coomassie-stained band in Dcp2 102–300 preparations, which we have verified by excision of the band followed by mass spectroscopy to be Dcp2 102–300. Additional data that the phosphorylated ~35-kD 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 demonstrating that Ste20 purified from E. coli was also able to phosphorylate Dcp2 102–300 (Fig. S1 B).
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 (more ...)
Ser137 is a target for Dcp2 phosphorylation in yeast
To determine the significance of Dcp2 phosphorylation, we desired 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 phosphorylation 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 comparison 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 phosphorylation in vitro with immunopurified Ste20 from yeast or affinity-purified Ste20 from E. coli
. We observed that the extent of Dcp2 phosphorylation by Ste20 in vitro was reduced after either the S137A or S211A mutations ( and Fig. S1 B). Moreover, the double-mutant S137A, S211A, showed almost a complete loss to phosphorylation in vitro ( 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 phosphorylation on Ser137 when Dcp2 was incubated with Ste20 and ATP (). This provides direct evidence that Ste20 phosphorylates Dcp2 on Ser137.
In vivo, the specificity of phosphorylation might be influenced 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 mutant showed reduced phosphorylation (). 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 phosphorylated to levels similar to wild-type Dcp2 (unpublished data). These observations argue that S137 is required for phosphorylation in vivo by Ste20 and is likely to be the major site of phosphoryl 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 mimetic (S137E) on mRNA decapping, the formation of P-bodies, and stress granules. In addition, because Ste20 affects Dcp2 phosphorylation, we also examined the effects of a ste20Δ in these assays. Strikingly, we observed that dcp2Δ strains expressing the dcp2-S137A allele grew more slowly than strains expressing the wild-type 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 affect decapping
Because Dcp2 is the decapping enzyme, we first asked whether alteration of the phosphorylation site affected its catalytic activity. S137 is located adjacent to, but does not overlap, the active site of Dcp2 (She et al., 2006
; Deshmukh et al., 2008
). Given this, we purified the catalytic domain of Dcp2 from E. coli
(residues 102–300) either as wild-type or with the S137A or S137E mutations and assayed the catalytic ability of this protein in vitro with a cap-labeled 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 dcp2Δ strains transformed with plasmids expressing Dcp2 wild type, dcp2-S137A, or dcp2-S137E. We examined mRNA decay during both mid-log 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 mid-log cultures and during glucose deprivation ( 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 ste20Δ and wild-type strains both in mid-log and stress conditions ( 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).
Figure 3. Dcp2 phosphorylation is not required for mRNA decay. (A) dcp2Δ 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; (more ...)
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 P-bodies (Teixeira et al., 2005
). We transformed Dcp2-GFP expression plasmids either with or without the S137A, S137E, or S211A mutations in wild-type strains and examined the subcellular location of Dcp2 in mid-log cultures as well as those exposed to glucose deprivation and grown to stationary phase.
We observed that the Dcp2-S137A-GFP failed to accumulate in P-bodies during glucose deprivation and growth to high cell density (). In contrast, the Dcp2 wild-type, Dcp2-S211A, and Dcp2-S137E proteins all accumulated in P-bodies. The failure of Dcp2-S137A proteins to accumulate in P-bodies 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 P-bodies.
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 (more ...)
If Dcp2 phosphorylation is required for its accumulation in P-bodies, strains lacking Ste20, which affects Dcp2 phosphorylation, should also show a defect in the accumulation of Dcp2 in P-bodies. Moreover, if this defect is largely caused by the loss of Dcp2 phosphorylation, then a charge-mimetic allele of Dcp2 would be predicted to restore Dcp2 accumulation in P-bodies in a ste20Δ strain. To test these predictions, we transformed a ste20Δ strain with GFP-tagged versions of either wild-type Dcp2 or the different mutant alleles and examined their location during mid-log growth and after glucose deprivation.
This experiment revealed the following important observations. First, we observed that the accumulation of the wild-type Dcp2-GFP protein in P-bodies was reduced in ste20Δ strains (). The accumulation of Dcp2-GFP in P-bodies in the ste20Δ was not reduced as much as the accumulation of the Dcp2-S137A protein in a wild-type 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 ste20Δ strain during glucose deprivation (unpublished data). Nevertheless, the reduction in Dcp2-GFP accumulation in P-bodies in the ste20Δ strain provides additional evidence that phosphorylation of Dcp2 promotes its accumulation in P-bodies during stress.
A second important observation was that the charge-mimetic dcp2-S137E allele rescued the defect in Dcp2 accumulation seen in the ste20Δ strain (). This observation strongly argues that the defect in Dcp2 accumulation in P-bodies in the ste20Δ strain is caused by the failure of Dcp2 to get phosphorylated.
A third observation was that the dcp2-S137E mutant did not accumulate above normal levels in P-bodies during mid-log growth (). This argues that phosphorylation of Dcp2 is not sufficient by itself to induce large P-bodies, although Dcp2 phosphorylation is necessary during stress responses for the accumulation of Dcp2 in P-bodies.
Dcp2 phosphorylation affects stress granule formation but not P-body formation
The aforementioned observations indicated that phosphorylation of Dcp2 was required for its accumulation in P-bodies during stress. This could be because phosphorylation of Dcp2 is required for P-bodies to form or because Dcp2 phosphorylation specifically affects Dcp2 accumulation in P-bodies. Given this, we examined how P-bodies formed in the various Dcp2 alleles as well as in ste20Δ strains using Edc3-mCherry as a marker of P-bodies. Moreover, because recent results suggest that P-bodies promote the formation of stress granules in yeast (Buchan et al., 2008
), we also examined how the Dcp2-S137A and -S137E alleles and the ste20Δ affected stress granule formation in the same experiments using a Pab1-GFP fusion protein as a marker of yeast stress granules (Buchan et al., 2008
). Thus, either wild-type, ste20Δ, or various dcp2 mutant strains were transformed with a centromere plasmid expressing Edc3-mCherry (a P-body marker) and Pab1-GFP (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 dcp2Δ strains expressing the dcp2-S137A allele still produced P-bodies as judged by the accumulation of Edc3-mCherry (). Moreover, as seen previously, dcp2Δ strains also formed robust P-bodies during glucose deprivation and formed enhanced P-bodies during mid-log growth, presumably caused by a defect in mRNA decapping (Sheth and Parker, 2003
; Teixeira and Parker, 2007
). These results demonstrate that neither Dcp2 phosphorylation nor Dcp2 itself is required for P-body formation per se and, therefore, demonstrates that phosphorylation of Dcp2 is required for Dcp2 accumulation in P-bodies.
Figure 5. Dcp2 phosphorylation affects stress granule but not P-body formation. Wild-type (WT; BY4742), ste20Δ (yRP2547), or dcp2Δ (yRP1358) strains were transformed with Pab1-GFP/Edc3-mCherry plasmid (pRP1658; Buchan et al., 2008), grown to OD (more ...)
Second, we observed that Dcp2 wild-type strains efficiently formed stress granules, whereas the dcp2Δ and dcp2-S137A strains showed reduced accumulation of stress granules (, Pab1-GFP). These results suggest that Dcp2 and its phosphorylation are required for optimal stress granule formation. We also observed that ste20Δ strains showed reduced stress granule formation during glucose deprivation as compared with wild-type cells, although P-bodies formed normally in the ste20Δ strain (). This observation argues that Ste20 enhances stress granule formation either through phosphorylation of Dcp2 and/or by phosphorylation of additional proteins. The observed changes in Pab1 accumulation were not caused by reduction 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 phosphorylation of Dcp2. This predicts that the dcp2-S137E allele would restore stress granule formation in the ste20Δ strain. To test this possibility, we expressed the wild-type or dcp2-S137E alleles in a ste20Δ strain and examined P-body and stress granule formation in response to glucose deprivation. We observed that the ste20Δ strain expressing the Dcp2-S137E protein showed partially restored stress granule formation as compared with the wild-type strain, whereas ste20Δ strains expressing exogenous wild-type Dcp2 still showed a defect in stress granule formation (). The ability of the charge-mimetic form of Dcp2 to restore stress granule formation in the ste20Δ strain provides additional evidence that phosphorylated Dcp2 promotes stress granule formation and also provides strong evidence 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 glucose deprivation
One possible mechanism by which Dcp2 phosphorylation promoted stress granule formation is that Dcp2 phosphorylation alters protein–protein interactions within mRNPs accumulating in P-bodies, 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 P-bodies (Buchan et al., 2008
). Interestingly, the Dhh1 protein, which interacts with Dcp2 (Decker et al., 2007
) and localizes to P-bodies, is required for optimal stress granule formation (Buchan et al., 2008
). This suggested a possible 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 wild-type, S137A, and S137E Dcp2 variants with Dhh1 in wild-type or ste20Δ strains.
In wild-type strains, we observed that during mid-log, Dhh1 coimmunoprecipitated with Dcp2, and this coimmunoprecipitation was unaffected by the S137E or S137A alleles. This indicates that Dcp2 can interact with Dhh1 independent of the phosphorylation status of Dcp2 during mid-log 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 (). Similarly, in ste20Δ strains, Dhh1 and Dcp2 coimmunoprecipitated during mid-log cultures but showed reduced interaction during glucose deprivation (). Strikingly, the S137E allele of Dcp2 could restore the interaction with Dhh1 during glucose deprivation in the ste20Δ strain. These observations argue that the interaction between Dhh1 and Dcp2 is altered during stress either directly or indirectly and that phosphorylation of Dcp2 is required to maintain the interaction of Dhh1 and Dcp2 under stress conditions.
Figure 6. Dcp2 phosphorylation is required for its interaction with Dhh1p during glucose deprivation. (A) Wild-type (WT; BY4742) strains were transformed with expression plasmid of Dhh1-GFP (pRP 1151; Coller et al., 2001) controlled by its own promoter and centromeric (more ...)
The requirement for Dcp2 phosphorylation to maintain interactions with Dhh1 and to assemble into P-bodies 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 repression, forming an initial P-body containing Edc3 and Dhh1 (as well as other proteins), then phosphorylated Dcp2 would be recruited to this complex, which predicts that Dhh1 accumulation in P-bodies would be independent of Dcp2 phosphorylation. In an alternative model, the order of assembly would be formation of a P-body containing Edc3 (as well as other proteins), which then recruits phosphorylated Dcp2, leading to the recruitment of Dhh1, which predicts that Dhh1 accumulation in P-bodies would be dependent of Dcp2 phosphorylation. Thus, we examined the subcellular location of Dhh1-GFP in various mutants affecting Dcp2 phosphorylation with or without stress.
Consistent with earlier results (Teixeira and Parker, 2007
), we observed that Dhh1 accumulated in P-bodies during mid-log in a dcp2Δ strain. More importantly, we observed that Dhh1-GFP was also present in P-bodies in dcp2-S137A and ste20Δ strains (). This indicates that Dhh1 recruitment into P-bodies is independent of Dcp2 or its phosphorylation status and is therefore upstream of Dcp2 recruitment into P-bodies.
Dcp2 phosphorylation affects the expression and decay of certain mRNAs
The aforementioned results raised the possibility that phosphorylation of Dcp2 might affect the decay of some, but not all, transcripts. To identify mRNAs whose degradation might be affected by Dcp2 phosphorylation, we performed microarray analysis comparing the dcp2-S137E and dcp2-S137A alleles with wild-type Dcp2, which led to several important observations.
Most importantly, in dcp2-S137E cells, no mRNAs were down-regulated more than twofold, and 40 mRNAs out of ~6,200 were up-regulated more than twofold, which suggests that Dcp2 phosphorylation stabilizes a subset of mRNAs. Strikingly, the mRNAs up-regulated in the dcp2-S137E stain were overrepresented in ribosomal protein mRNAs (), suggesting that Dcp2 phosphorylation led to preferential stabilization of this class of mRNAs. Moreover, by direct measurement of mRNA decay rates, we validated that two ribosomal protein mRNAs (Rpl26a and Rpp1b) showed slower rates of mRNA degradation in the dcp2-S137E strain as compared with either wild-type or dcp2-S137A strains (). Collectively, these results argue that phosphorylation of Dcp2 at S137 stabilizes a subclass of mRNAs enriched in ribosomal mRNAs.
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 (more ...)
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 (), that were increased in both the dcp2-S137E and dcp2-S137A strains as compared with wild-type and, therefore, may be mRNAs whose degradation is normally enhanced by S137. We also observed a class of mRNAs, preferentially enriched in mRNAs involved in amino acid synthesis or heat response, which were unregulated in the dcp2-S137A strain and unaffected in the dcp2-S137E strain. Finally, we observed a class of mRNAs enriched in iron transporters, which were unaffected in the dcp2-S137E strain but down-regulated in the dcp2-S137A strain. Collectively, these results indicate that modification of Dcp2 on S137 impacts the levels of several mRNAs by both direct and indirect mechanisms.