Snf1 cooperates with the CWI MAPK pathway to mediate the degradation of Med13 following oxidative stress

Abstract

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCF^Grr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second SCF^Grr1 responsive degron in Med13. Deletion of Snf1 resulted in nuclear retention of cyclin C and failure to induce mitochondrial fragmentation. This degron was able to confer oxidative-stress-induced destruction when fused to a heterologous protein in a Snf1 dependent manner. Although snf1∆ mutants failed to destroy Med13, deleting the degron did not prevent destruction. These results indicate that the control of Med13 degradation following H₂O₂ stress is complex, being controlled simultaneously by CWI and MAPK pathways.

Keywords: AMPK; Cdk8; H2O2 stress; MAPK; Med13; SCFGrr1; Snf1; cyclin C; signal transduction; ubiquitin mediated destruction.

Figure 1. FIGURE 1:

(A) Cdk8 module regulation by the CWI MAPK pathway. H₂O₂ stimulates cell wall sensors Wsc1, Mtl1 and Mid2, leading to activation of Rho1 that in turn triggers the cell wall integrity (CWI) MAPK pathway by activating Protein Kinase C (Pkc1). Activation of this cascade triggers the MAPK, Slt2, to directly phosphorylate cyclin C, an event required for the 1^st step towards its release from the nucleus. The second step requires Slt2 to directly phosphorylate Med13-degron^742-844, which targets it for ubiquitin mediated degradation by SCF^Grr1. Cyclin C-Cdk8 activity is needed to prime the degron before it is recognized by SCF^Grr1 . (B) Outline of the AMPK pathway in yeast. It remains unknown how this pathway is activated in response to H₂O₂ stress. The gray box represents the Snf1 kinase complex (see text for details).

Figure 2. FIGURE 2: Med13 contains two H₂O₂ stress responsive degrons.

(A) Cartoon of the results from ProteinPredict^® analysis of yeast Med13. The amino and carboxyl terminal domains are structured and separated by a large intrinsic disordered region. The positions of the two degrons are indicated. (B) Yeast two hybrid analysis of degron^571-650 and Grr1 derivatives. Yeast PJ69-4a cells harboring Med13-activating domain plasmid (pDS15) and either pAS2, pAS-Grr1 or pAS2-Grr1∆F∆L binding domain plasmids were grown on -LEU, -TRP drop out medium to select for both plasmids (left panel) and -TRP, -LEU, -HIS -ADE (right panel) to test for Med13-Grr1 interaction. (C) Wild-type (RSY10) or grr1∆ cells (RSY1770) harboring degron^571-650 (pDS15) were treated with 0.4 mM H₂O₂ for the timepoints indicated and Med13^571-650-HA levels analyzed by Western blot. Tub1 levels were used as loading controls. (D) Degradation kinetics of the degron^571-650 constructs shown in C. Values represent averages ± SD from a total of at least two Western blots from two independent experiments.

Figure 3. FIGURE 3: Snf1, Sak1 and at least one β subunit are required for degradation of Med13 following H₂O₂ stress.

(A) Wild-type (RSY10), snf1∆ (RSY2080), sak1∆ (YPDahl17) and gal83∆ sip1∆ sip2∆ (MSY557) cells harboring full length Med13-HA (pKC801) were treated with 0.4 mM H₂O₂ for the timepoints indicated and Med13-HA levels analyzed by Western blot. Tub1 levels were used as a loading control. (B) snf1∆ cells harboring Med13-HA (pKC803) and either wild- type Snf1 (JG1193), a vector control (pRS316) or snf1^K84R (JG1338) were treated and analyzed as described in A. (C and D) Degradation kinetics of the Med13-HA shown in A and B. Values represent averages ± SD from a total of at least two Western blots from independent experiments.

Figure 4. FIGURE 4: Snf1 activation does not mediate Med13 degradation.

(A) Mid-log -type cells (RSY10) growing in 2% glucose (T=0) were switched to media containing 0.05% glucose. Snf1 phosphorylation and Snf1 itself were detected as described in materials and methods for the timepoint indicate. (B) Wild-type cells harboring Med13-HA (pKC801) were switched to media containing 0.05% glucose. Med13-HA levels were detected by Western blot analysis for the timepoints indicated. Tub1 was used as a loading control. (C) As in (A) except that Snf1 phosphorylation was monitored after 0.4 mM H₂O₂ treatment in the strains indicated.

Figure 5. FIGURE 5: Snf1 phosphorylates degron^571-650.

(A) Mid-log wild-type (RSY10) or snf1∆ cultures (RSY202) harboring degron^571-650 (pDS15) were subjected to an H₂O₂ timecourse experiment and protein extracts analyzed by Western blot. Tub1 levels were used as loading controls. (B) Degradation kinetics of degron^571-650 shown in (A) and (C). Values represent averages ± SD from a total of at three Western blots from independent experiments. (C) As in (A) except that degron^{571-650,S587A, S634A, S636A} (pDS56) was analyzed in wild-type cells. (D) Co-immunoprecipitation analysis of Snf1-myc and Cdk8-HA. Mid log wild-type cells harboring Snf1-myc and Cdk8-HA on single copy plasmids were treated with 0.4 mM H₂O₂ for the timepoints shown. Protein extracts were immunoprecipitated with anti-HA, separated by SDS-PAGE Western analysis and the membrane probed with the antibodies shown. [] represents no IP antibody and the asterisk represents the heavy chain. See Fig. S3C for vector control. (E) Potential phospho-sites in Med13^571-650. (F) Upper panels: Kinase assays using Snf1 and Snf1^K84R-myc (kinase dead) immunoprecipitated from yeast protein extracts prepared from either wild type (left panel) or cdk8∆ cells (right panel) and Med13-degron^571-650 (GST-Med13^{571-906,S608A} purified from E. coli) as the substrate. The reactions were separated by SDS PAGE and subject to autoradiography. Lower panels: Coomassie stained gels showing the input used in the kinase assays.

Figure 6. FIGURE 6: Other potential Snf1 sites in Med13 are not required for its degradation following H₂O₂ stress.

(A) Map of Med13 outlying the positions of the two Med13 degrons, the consensus Snf1 target site and potential Snf1 sites, identified by published proteomic screens. (B) and (C) Wild-type (RSY10) cultures harboring the NLS-Med13-HA constructs shown were grown to mid-log phase (0 h) then treated with 0.4 mM H₂O₂ for the indicated times. Med13-HA levels were determined by Western blot analysis. Tub1 levels were used as a loading control. (D) Mid-log wild type or snf1∆ cultures (RSY202) harboring HA tagged Med13 degron742-844 (pDS32) were subjected to an H₂O₂ timecourse experiment and protein extracts analyzed by Western blot. Tub1 levels were used as loading controls.

Figure 7. FIGURE 7: Cyclin C remains predominantly nuclear following H₂O₂ stress in snf1∆.

(A) Fluorescence microscopy of mid-log phase wild-type and snf1∆ cells harboring a cyclin C-YFP expression plasmid (pBK38). Cells were stained with Dapi to visualize the nucleus. (B) Quantification of the results obtained in (A). At least 200 cells were counted per timepoint from 3 individual isolates. The percent of cells (mean ± SEM) within the population displaying cytoplasmic cyclin C is given. * p < 0.05 difference from wild type. (C) Right panel: representative images of the two mitochondrial morphologies scored. Left panel: as in (B) except that percent of cells displaying fragmented mitochondria was scored. Representative images of the mitochondrial morphologies scored are shown in the left hand panel. The percent of cells (mean ± s.e.m.) within the population displaying fragmented mitochondria is given. * p < 0.05 difference from wild type. ** p < 0.01 difference from wild type. Bar = 13 µM.

Figure 8. FIGURE 8: Either Med13 degron is sufficient for Med13 degradation.

(A) med13∆ (RSY1701) cells harboring either Med13^{571-650deg∆-}HA (pKC805, upper panels) or Med13^{742-844deg∆-}HA plasmids (pKC814, lower panels) were treated with 0.4 mM H₂O₂ for the timepoints indicated and Med13^deg∆-HA levels analyzed by Western blot. Tub1 levels were used as a loading control. (B) Degradation kinetics of the results shown in (A). Values represent averages ± SD from a total of at least two Western blots from independent experiments. For clarity, the degradation kinetics of wild-type Med13-HA from previous experiments was included. (C) Model depicting how two SCF^Grr1 phospho-degrons mediate the destruction of Med13 following H₂O₂ stress. In unstressed cells cyclin C-Cdk8 phosphorylates degron^742-844 but both degrons are protected by an unknown mechanism from Snf1 and Slt2 kinase activity (depicted by the red circle). Following H₂O₂ stress Snf1 and Slt2 are activated and permitted access to the now exposed degrons. This results in SCF^Grr1 mediated degradation of Med13 and cyclin C nuclear release (not shown).