Fission yeast mitogen-activated protein kinase Sty1 interacts with translation factors

Abstract

Signaling by stress-activated mitogen-activated protein kinase (MAPK) pathways influences translation efficiency in mammalian cells and budding yeast. We have investigated the stress-activated MAPK from fission yeast, Sty1, and its downstream protein kinase, Mkp1/Srk1, for physically associated proteins using tandem affinity purification tagging. We find Sty1, but not Mkp1, to bind to the translation elongation factor eukaryotic elongation factor 2 (eEF2) and the translation initiation factor eukaryotic initiation factor 3a (eIF3a). The Sty1-eIF3a interaction is weakened under oxidative or hyperosmotic stress, whereas the Sty1-eEF2 interaction is stable. Nitrogen deprivation causes a transient strengthening of both the Sty1-eEF2 and the Sty1-Mkp1 interactions, overlapping with the time of maximal Sty1 activation. Analysis of polysome profiles from cells under oxidative stress, or after hyperosmotic shock or nitrogen deprivation, shows that translation in sty1 mutant cells recovers considerably less efficiently than that in the wild type. Cells lacking the Sty1-regulated transcription factor Atf1 are deficient in maintaining and recovering translational activity after hyperosmotic shock but not during oxidative stress or nitrogen starvation. In cells lacking Sty1, eIF3a levels are decreased, and phosphorylation of eIF3a is reduced. Taken together, our data point to a central role in translational adaptation for the stress-activated MAPK pathway in fission yeast similar to that in other investigated eukaryotes, with the exception that fission yeast MAPK-activated protein kinases seem not to be directly involved in this process.

FIG. 1.

Sty1, but not Mkp1, is coimmunoprecipitated with eEF2 in undisturbed log-phase cells and after oxidative and hyperosmotic stress. Strains expressing HA-tagged eEF2, myc-tagged Sty1, or myc-tagged Mkp1, alone or in combinations as indicated, were grown to an OD₅₉₅ of ∼0.5 in YES medium. Protein preparations were made from cells without stress or after exposure to 1 mM H₂O₂ for 20 min or 1 M KCl for 15 min. Following bicinchoninic acid protein quantification, equal amounts from each sample were used for IP of eEF2-HA with HA probe. (A) Interacting Sty1-myc and Mkp1-myc were detected with anti-myc antibodies. A strain expressing only Sty1-myc (JM1698) was used as a negative control for co-IP. Western blots of total protein (smaller amounts loaded than for the co-IP lanes) show the positions of myc-tagged Sty1 and Mkp1. (B) eEF2 interacting with Sty1 was detected with anti-HA antibodies. A strain expressing only eEF2-HA (EA129) was used as a negative control.

FIG. 2.

Sty1, but not Mkp1, is coimmunoprecipitated with eIF3a in undisturbed log-phase cells, and the interaction weakens after oxidative and hyperosmotic stress. (A) Cells were grown and exposed to stress, and protein samples were prepared and loaded as for Fig. 1 except that strains expressing eIF3a-HA were used in place of eEF2-HA. (B) eIF3a interacting with Sty1 was detected with anti-HA antibodies. A strain expressing only eIF3a-HA (EA128) was used as a negative control. (C) Western blots of cells expressing eIF3a-HA in either a wild-type (EA128) or sty1Δ (DN7) background. (D) Western blot of cells expressing eIF3a-HA under stress conditions, to show that the level is unchanged.

FIG. 3.

The intensities of the Sty1-eEF2, Sty1-eIF3a, and Sty1-Mkp1 interactions change after nitrogen deprivation. Strains expressing eEF2-HA and Sty1-myc (A), eIF3a-HA and Sty1-myc (B), or Mkp1-HA and Sty1-myc (C) were grown to an OD₅₉₅ of ∼0.25 in YES medium. After a brief centrifugation, the cells were resuspended in EMM minimal medium without NH₄Cl. Protein samples were prepared and loaded as for Fig. 1 at the times indicated after transfer to nitrogen-free medium. (A and B) Top rows, Sty1-myc coprecipitated with eEF2-HA (A) or eIF3a-HA (B). Second rows, Western blots from the same protein preparations as above, to show the total amount of eEF2-HA (A) or eIF3a-HA (B). Third and fourth rows, Western blots from the same protein preparations probed for α-tubulin and Cdc2 as loading controls. (C) Sty1-myc coprecipitated with Mkp1-HA.

FIG. 4.

Polysomal contents of wild-type and sty1 cells after hyperosmotic stress. 972h⁻ and sty1 cells were grown to an OD₅₉₅ of ∼0.5 and subjected to 0.6 M KCl. Extracts were prepared at various times after shock, separated on sucrose gradients, and analyzed online as described in Materials and Methods. (A) Polysome profiles obtained at three different times after hyperosmotic shock. The positions of the 40S, 60S, 80S, and polysomal peaks are indicated in one of the panels. (B) Relative polysome ratios calculated as polysome/(monosome + polysome) areas at five different times after hyperosmotic shock for wild-type and sty1 cells (averages from two to four independent experiments). The ratio before stress (0 min) for each strain was set to 1, and values for subsequent time points are indicated relative to that value.

FIG. 5.

Polysomal contents of wild-type and sty1 cells after oxidative stress. 972h⁻ and sty1 cells were grown to an OD₅₉₅ of ∼0.5 and exposed to 1 mM H₂O₂. Polysomal extracts were prepared and analyzed as for Fig. 4. (A) Polysome profiles obtained at three different times after oxidative stress as for Fig. 4A. (B) Relative polysome ratios at five times after oxidative stress as for Fig. 4B.

FIG. 6.

Polysomal contents of wild-type and sty1 cells after nitrogen deprivation. 972h⁻ and sty1 cells were grown to an OD₅₉₅ of ∼0.25 and after a brief centrifugation were resuspended in nitrogen-free EMM medium. (A) Polysome profiles obtained at three different times after nitrogen deprivation as for Fig. 4A. (B) Relative polysome ratios at five times after nitrogen deprivation as for Fig. 4B.

FIG. 7.

Polysomal contents of atf1 mutant cells in different stress conditions. Cells were grown and lysates were prepared as for Fig. 4 to 6. Values for wild-type or sty1 cells are taken from Fig. 4 to 6 for comparison. Cells were exposed to nitrogen deprivation, 0.6 M KCl, or 1 mM H₂O₂ for the indicated times.

FIG. 8.

Phosphorylation of eIF3a in wild-type and sty1 mutant cells. Wild-type (EA128) or sty1 mutant (DN7) cells expressing eIF3a-HA were harvested before treatment or after treatment with stress agents as indicated. Nondenaturing protein lysates were prepared as for Fig. 1 to 3, and HA-tagged eIF3a was purified with HA probe. Approximately equal amounts of total eIF3a were loaded in each lane. The position of a protein size marker is shown to the right. The eIF3a band is indicated; the band visible just above it in the SYPRO staining corresponds to eIF3c copurifying under native conditions, which migrates slightly slower than eIF3a (46). Upper panel, phosphorylated eIF3a detected with ProQ Diamond phosphoprotein stain. Lower panel, total eIF3a detected with SYPRO Ruby protein stain.

FIG. 9.

2D gel electrophoresis analysis of eEF2 isoforms after stress treatments. Wild-type (upper row) or sty1 mutant (lower row) cells expressing HA-tagged eEF2 were treated as indicated. Lysates were prepared and proteins separated on 2D gels, blotted, and probed with anti-HA antibodies. The arrows (A, C, I, and J) point to the predominant isoform.