PMC

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 ste20Δ 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.

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; ; 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 ste20Δ was transformed 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.

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; ), 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 examined with a microscope. In addition, the dcp2Δ strain was transformed with wild-type or S137A Flag-Dcp2 plasmid (pRP983 or pRP1676) before glucose deprivation. The ste20Δ strain containing Pab1-GFP/Edc3-mCherry plasmids was transformed with wild-type or S137E Flag-Dcp2 (pRP983 or pRP1679). Images were quantified as in and in Materials and methods. Error bars indicate SD. Bar, 5 µm.

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 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. IB, immunoblot.

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; ) 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 ste20Δ (yRP2547) strains was cotransformed with plasmids expressing Dhh1-GFP and plasmids expressing Flag-tagged wild-type or S137E Dcp2 as indicated. (C) Wild-type, ste20Δ, or dcp2Δ (yRP1358) strains were transformed with a plasmid expressing Dhh1-GFP, or in some cases, the dcp2Δ 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.

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.

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 digested 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 ste20Δ strains. Cells were grown in mid-log phase and exposed to glucose deprivation stress. Cell lysates were examined to detect phosphorylated Dcp2 by SDS-PAGE and immunoblot (IB) assay.