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. 2007 Mar;6(3):521-32.
doi: 10.1128/EC.00343-06. Epub 2007 Jan 12.

Cdc37p is required for stress-induced high-osmolarity glycerol and protein kinase C mitogen-activated protein kinase pathway functionality by interaction with Hog1p and Slt2p (Mpk1p)

Patricija Hawle  1 , Danielle Horst, Jan Paul Bebelman, Xiao Xian Yang, Marco Siderius, Saskia M van der Vies
Affiliations

Cdc37p is required for stress-induced high-osmolarity glycerol and protein kinase C mitogen-activated protein kinase pathway functionality by interaction with Hog1p and Slt2p (Mpk1p)

Patricija Hawle et al. Eukaryot Cell. 2007 Mar.

Abstract

The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.

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Figures

FIG. 1.
FIG. 1.
Western blot of Cdc37pWt and Cdc37pS14 mutant proteins. Cells expressing HA-tagged versions of wild-type Cdc37p or one of the three mutant forms carrying a replacement of S14 by Leu, Ala, or Glu were grown in YPD at 24°C to mid-log phase. Total-cell extracts were analyzed by Western blotting using an anti-HA antibody. After being stripped, the blots were reprobed with anti-Hsp90, stripped again, and finally probed with anti-G6PDH to assess loading.
FIG. 2.
FIG. 2.
Phenotypic analysis of the cdc37 and cka2 mutants. (A) Sensitivity of cdc37 mutants to osmotic and cell wall stress. Cells expressing wild-type Cdc37p or its S14A, S14E, S17A, or S17E mutant version (strain DH231, DH232, DH233, DH234, or DH235, respectively) were grown on YPD plates containing no additive, 2 M sorbitol, or 20 μg/ml CFW. (B) Sensitivity of the cka2 mutant to osmotic and cell wall stresses. Strains YDH6 and YDH8, containing the wild-type CKA2 gene and the cka2-8 mutant allele, respectively, were grown on YPD plates containing either no additive, 2 M sorbitol, or 20 μg/ml CFW.
FIG. 3.
FIG. 3.
Analysis of Hsp90 coimmunoprecipitation with Cdc37pS14 mutant proteins. Yeast strains expressing plasmid-encoded, HA-tagged versions of wild-type Cdc37p (DH211), Cdc37pS14A (DH212), or Cdc37S14E (DH213) were grown in YPD at 24°C to mid-log phase. Cell extracts were prepared, and an aliquot of cell-free proteins was loaded onto an anti-HA agarose column. Bound protein was eluted, and 15% of the eluate was subjected to Western blot analysis using an anti-HA or anti-Hsp90 antibody. The wild-type strain TM141, expressing untagged Cdc37p, was used as a control.
FIG. 4.
FIG. 4.
Analysis of HOG pathway activation in the cdc37 S14 mutants during osmotic shock. (A) Analysis of Hog1p activation in the cdc37-S14 mutant strains. Strains expressing wild-type Cdc37p (DH231) or its S14A (DH232) or S14E (DH233) mutant version were grown to mid-log phase in YPD medium and then given an osmotic shock by the addition of 2 M sorbitol. Aliquots were taken at the indicated time points, and cell extracts were prepared and subsequently analyzed by Western blotting using anti-phospho-p38 MAPK and an anti-Hog1p antibody. Loading was assessed by probing with an anti-G6PDH antibody. The same blot was used for all antibody analyses. Bar graphs represent quantification (using Quantity One software from Bio-Rad) of the level of phosphorylated Hog1p relative to the total amount of Hog1p. To avoid interference with quantification analysis due to different blot backgrounds, a Quantity One tool was selected that subtracts the image background from each selected signal. (B) Analysis of STL1-LacZ reporter gene expression in cdc37-S14 mutant strains. Strains PH311, PH312, and PH313, which contain the STL1-LacZ reporter construct and express wild-type Cdc37 and the S14A and S14E mutant versions, respectively, were grown to mid-log phase in YPD medium. Osmotic stress was induced by the addition of 1 M sorbitol, and cells were allowed to grow for an additional 2 h. β-Galactosidase activity in cell extracts was determined (filled bars). Extracts from cells grown in the absence of sorbitol were used as a control (open bars). Error bars, standard deviations from the means of four experiments.
FIG. 5.
FIG. 5.
Effects of high osmotic stress on the interaction of Cdc37p with Hsp90 and Hog1p. (A) A C-terminal 13cmyc tag stabilizes Hog1p in the cdc37-S14A mutant. Strains PH401, PH402, and PH403, expressing HA-tagged wild-type Cdc37p and the S14A and S14E mutant versions, respectively, and 13cmyc epitope-tagged Hog1p were grown to mid-log phase in YPD medium and treated with 2 M sorbitol for 30 min. Cell extracts were prepared, and each extract was analyzed by Western blotting using anti-HA and an anti-cmyc antibody. An anti-G6PDH antibody was used as a loading control. (B) Hog1p-cmyc does not suppress the osmosensitive phenotype of the cdc37-S14A strain. Cells expressing wild-type Cdc37p or its S14A or S14E mutant version and containing 13cmyc epitope-tagged Hog1p (strains PH401, PH402, and PH403, respectively) were grown on YPD plates containing no additive or 2 M sorbitol. (C) Coimmunoprecipitation of Hsp90 and Hog1p with wild-type and mutant Cdc37p before and during osmotic stress. An aliquot of the same cell-free protein extracts analyzed in panel A was loaded onto an anti-HA agarose column (lanes 5 to 10). Cell extracts of strain PH404, containing 13cmyc-tagged Hog1 but no HA-tagged Cdc37 (lanes 1 and 2), and strain DH211, containing HA-tagged Cdc37Wt but no 13cmyc-tagged Hog1 (lanes 3 and 4), were used as controls. Bound protein was eluted by 2× SDS sample buffer. To avoid coelution of the heavy chain of the anti-HA antibody, which results in overlapping protein bands of endogenously phosphorylated Hog1 with the anti-HA heavy chain on a Western blot, bound protein of the control sample, containing endogenous Hog1, was eluted with 100 μg/ml HA peptide (see Materials and Methods). Fifteen percent of the total eluate was subjected to Western blot analysis using an anti-HA, anti-Hsp90, anti-phospho-Hog1p, or anti-cmyc antibody. The same blot was used for all analyses.
FIG. 6.
FIG. 6.
Analysis of PKC pathway activation in cdc37-S14 mutants during CFW stress. (A) Slt2p activation in cdc37-S14 mutant strains. Strains expressing wild-type Cdc37p (DH231) or the S14A (DH232) or S14E (DH233) mutant version were grown to mid-log phase in YPD medium and exposed to cell wall stress by the addition of CFW (20 μg/ml). Aliquots were taken at the indicated time points, and cell extracts were prepared and analyzed by Western blotting, using an anti-phospho-p42/44 MAPK antibody to detect phosphorylated Slt2p. The total amount of Slt2p was determined by using an anti-Slt2p antibody. Loading was assessed by means of an anti-G6PDH antibody. The same blot was used for all analyses. Bar graphs represent quantification of the level of phosphorylated Slt2p relative to total Slt2p. (B) Analysis of YIL117c-LacZ reporter gene expression in cdc37-S14 mutant strains. Strains PH301, PH302, and PH303, containing the Rlm1p-responsive YIL117c-LacZ reporter gene constructs and expressing wild-type Cdc37p and the S14A and S14E mutant versions, respectively, were grown to mid-log phase in YPD at 24°C and subsequently exposed to 20 μg/ml CFW in YPD for 2 h. Cell extracts were prepared, and β-galactosidase activity was measured (filled bars). Extracts from cells grown in the absence of CFW were used as a control (open bars). Error bars, standard deviations from the means of four experiments.
FIG. 7.
FIG. 7.
Effects of CFW stress on the interaction of Cdc37p with Hsp90 and Slt2p. (A) Analysis of protein levels of Hsp90, Slt2p, and Cdc37p during CFW stress in cdc37-S14 mutant strains. Strains DH211, DH212, and DH213, expressing HA-tagged wild-type Cdc37p and its S14A and S14E mutant versions, respectively, were grown to mid-log phase in YPD at 24°C and treated with 20 μg/ml CFW for 1 h. Cell extracts were prepared and analyzed by Western blotting using an anti-HA antibody, an anti-Hsp90 antibody, an anti-phospho-p42/44 MAPK antibody to detect phosphorylated Slt2p, and an anti-Slt2p antibody. Loading was assessed by means of an anti-G6PDH antibody. Untreated cells from each strain, as well as CFW-treated and untreated cells from strain TM141 expressing untagged Cdc37p, were used as controls. (B) Coimmunoprecipitation of Hsp90 and Slt2p with wild-type and mutant Cdc37p before and during CFW stress. An aliquot of the same cell-free protein extracts analyzed in panel A was loaded onto an anti-HA agarose column. Bound protein was eluted, and 15% of the total eluate was subjected to Western blot analysis using an anti-HA, anti-phospho-p42/44 MAPK, or anti-Slt2p antibody. (C) Coimmunoprecipitation of phosphorylated Slt2pHis-Wt with wild-type Cdc37p. Cell cultures of strain PH501 or PH502, expressing wild-type Slt2pHis or a nonphosphorylatable Slt2pHis mutant (T190A Y192F), respectively, were treated with 20 μg/ml CFW for 1 h. Total-cell extracts (lanes labeled “Input”) and anti-HA-agarose-retained protein (lanes labeled “IP” [immunoprecipitation]) were analyzed by Western blotting using an anti-HA antibody to detect Cdc37-HA, an anti-His antibody to detect Slt2His, and anti-phospho-p42/44 MAPK to detect phosphorylated Slt2p.
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