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Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress.

Muir A, Roelants FM, Timmons G, Leskoske KL, Thorner J - Elife (2015)

Bottom Line: Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress.These events are all Hog1-independent.Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

No MeSH data available.


Related in: MedlinePlus

Modification at T662 and isoforms of Ypk17A both accurately report authentic in vivo phosphorylation.(A) A ypk1∆ strain (JTY6142) expressing Ypk1-HA (pPL215) was grown to mid-exponential phase and diluted into fresh medium in the absence (−) or presence (+) of 1 M sorbitol (final concentration). After 1 min, the cells were collected by centrifugation for 5 min and lysed. The resulting extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-pYpk1(T662) antibody and anti-HA antibody, as described in ‘Materials and methods’. (B) Cells (BY4741) expressing Ypk17A-myc (pFR252) were grown, extracts prepared, treated with phosphatase, and analyzed as Figure 1D.DOI:http://dx.doi.org/10.7554/eLife.09336.006
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fig1s4: Modification at T662 and isoforms of Ypk17A both accurately report authentic in vivo phosphorylation.(A) A ypk1∆ strain (JTY6142) expressing Ypk1-HA (pPL215) was grown to mid-exponential phase and diluted into fresh medium in the absence (−) or presence (+) of 1 M sorbitol (final concentration). After 1 min, the cells were collected by centrifugation for 5 min and lysed. The resulting extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-pYpk1(T662) antibody and anti-HA antibody, as described in ‘Materials and methods’. (B) Cells (BY4741) expressing Ypk17A-myc (pFR252) were grown, extracts prepared, treated with phosphatase, and analyzed as Figure 1D.DOI:http://dx.doi.org/10.7554/eLife.09336.006

Mentions: We documented elsewhere using Phos-tag gel mobility shift that Ypk1 phosphorylation at T662, one of its well-characterized TORC2 sites, is eliminated when cells are subjected to hyperosmotic shock for 10 min (Lee et al., 2012), and the same effect is observed using a specific antibody (Niles et al., 2012) that monitors phosphorylation of Ypk1 at the same site (Figure 1—figure supplement 4A). Using Ypk17A, which also permits facile detection of mobility shifts arising from TORC2-specific phosphorylation (K Leskoske and FM Roelants, unpublished results) (Figure 1—figure supplement 4B), we followed the kinetics of this change. Loss of TORC2-mediated Ypk1 phosphorylation upon hyperosmotic shock occurs very rapidly (within 1 min) and persists for about 15 min (Figure 1D), but is transient. By 20 min after hyperosmotic shock, TORC2-mediated Ypk1 phosphorylation is again detectable and is nearly back to the pre-stress level by 75 min (Figure 1—figure supplement 5A). Rapid reduction in TORC2-mediated Ypk1 phosphorylation under hypertonic stress was still observed in mutants lacking the Sho1- or Sln1-dependent pathways that converge on Hog1 or Hog1 itself (Figure 1E) or CN (Figure 1F). Thus, loss of TORC2-mediated Ypk1 phosphorylation upon hyperosmotic shock occurs independently of other known response pathways.


Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress.

Muir A, Roelants FM, Timmons G, Leskoske KL, Thorner J - Elife (2015)

Modification at T662 and isoforms of Ypk17A both accurately report authentic in vivo phosphorylation.(A) A ypk1∆ strain (JTY6142) expressing Ypk1-HA (pPL215) was grown to mid-exponential phase and diluted into fresh medium in the absence (−) or presence (+) of 1 M sorbitol (final concentration). After 1 min, the cells were collected by centrifugation for 5 min and lysed. The resulting extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-pYpk1(T662) antibody and anti-HA antibody, as described in ‘Materials and methods’. (B) Cells (BY4741) expressing Ypk17A-myc (pFR252) were grown, extracts prepared, treated with phosphatase, and analyzed as Figure 1D.DOI:http://dx.doi.org/10.7554/eLife.09336.006
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4552222&req=5

fig1s4: Modification at T662 and isoforms of Ypk17A both accurately report authentic in vivo phosphorylation.(A) A ypk1∆ strain (JTY6142) expressing Ypk1-HA (pPL215) was grown to mid-exponential phase and diluted into fresh medium in the absence (−) or presence (+) of 1 M sorbitol (final concentration). After 1 min, the cells were collected by centrifugation for 5 min and lysed. The resulting extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-pYpk1(T662) antibody and anti-HA antibody, as described in ‘Materials and methods’. (B) Cells (BY4741) expressing Ypk17A-myc (pFR252) were grown, extracts prepared, treated with phosphatase, and analyzed as Figure 1D.DOI:http://dx.doi.org/10.7554/eLife.09336.006
Mentions: We documented elsewhere using Phos-tag gel mobility shift that Ypk1 phosphorylation at T662, one of its well-characterized TORC2 sites, is eliminated when cells are subjected to hyperosmotic shock for 10 min (Lee et al., 2012), and the same effect is observed using a specific antibody (Niles et al., 2012) that monitors phosphorylation of Ypk1 at the same site (Figure 1—figure supplement 4A). Using Ypk17A, which also permits facile detection of mobility shifts arising from TORC2-specific phosphorylation (K Leskoske and FM Roelants, unpublished results) (Figure 1—figure supplement 4B), we followed the kinetics of this change. Loss of TORC2-mediated Ypk1 phosphorylation upon hyperosmotic shock occurs very rapidly (within 1 min) and persists for about 15 min (Figure 1D), but is transient. By 20 min after hyperosmotic shock, TORC2-mediated Ypk1 phosphorylation is again detectable and is nearly back to the pre-stress level by 75 min (Figure 1—figure supplement 5A). Rapid reduction in TORC2-mediated Ypk1 phosphorylation under hypertonic stress was still observed in mutants lacking the Sho1- or Sln1-dependent pathways that converge on Hog1 or Hog1 itself (Figure 1E) or CN (Figure 1F). Thus, loss of TORC2-mediated Ypk1 phosphorylation upon hyperosmotic shock occurs independently of other known response pathways.

Bottom Line: Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress.These events are all Hog1-independent.Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

No MeSH data available.


Related in: MedlinePlus