doi: 10.1128/EC.05305-11.
Epub 2012 Nov 16.
Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 signaling pathway in response to thermal stress
Affiliations
- PMID: 23159519
- PMCID: PMC3535842
- DOI: 10.1128/EC.05305-11
Item in Clipboard
Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 signaling pathway in response to thermal stress
Woei C Lam et al.
Eukaryot Cell.
2013 Jan.
Display options
Format
Display options
Format
Abstract
To initiate and establish infection in mammals, the opportunistic fungal pathogen Cryptococcus neoformans must survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase in Saccharomyces cerevisiae. We identified three C. neoformans Rho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion of RHO10 also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion of RHO11 yielded phosphorylation similar to that of wild type. Surprisingly, like a rho10Δ strain, a strain with a deletion of both RHO10 and RHO11 displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway in C. neoformans.
Figures
C. neoformans has three Rho GTPase homologues. Alignment of three identified C. neoformans (Cn) Rho1 homologues, Rho1, Rho10, and Rho11, with S. cerevisiae (Sc) Rho1p. Boxed in light gray are conserved domains for GTP hydrolysis, effector (putative site for interaction with target proteins), Rho insert (essential for Rho kinase activation), and putative membrane localization, including the prenylation site CAAX, where A represents an aliphatic amino acid (note that this site is conserved in CnRho1 and Rho11 but not in Rho10). Boxed in dark gray are the conserved glycine at amino acid position 15 and glutamine at position 64 that were mutated to valine and leucine, respectively, in the C. neoformans Rho1 protein to generate constitutively active point mutants.
C. neoformans
RHO1 is essential. KN99α and PCTR4-RHO1, a strain with the endogenous RHO1 promoter replaced by a copper-repressible promoter, were grown overnight in YPD medium containing 200 μM BCS at 30°C and then diluted to an OD650 of 1.0 in PBS. Five microliters of 10-fold serial dilutions were plated onto YPD plates containing either 200 μM BCS to allow expression of RHO1 from the copper promoter or 25 μM CuSO4 to repress expression of RHO1 from this promoter. The ability of cells to grow was analyzed after 2 and 5 days (shown) at 30°C.
The rho1G15V and rho1Q64L point mutants as well as both rho10Δ and rho10Δ rho11Δ strains are temperature sensitive, and this sensitivity is remediated by osmotic stabilization only for strains containing a deletion of RHO10. Cultures of strains were grown overnight in YPD medium at 30°C then diluted to an OD650 of 1.0. Five microliters of 10-fold serial dilutions were plated onto YPD plates with or without 1 M sorbitol (Sorb) and grown at indicated temperatures for 5 days. Strain names or genotypes are indicated to the left of each panel.
Mpk1 phosphorylation is induced in wild-type cells in response to thermal stress and the anti-Mpk1 phospho-antibody is specific for phosphorylation of Mpk1. (A) KN99α cells harboring the Mpk1 Flag-tagged construct were grown overnight in YPD medium at 24°C and then split the next morning into two aliquots. To one aliquot, an equal volume of YPD medium at room temperature was added, and cells were grown for an additional 15, 30, 45, and 60 min at 24°C. To the second aliquot, an equal volume of YPD medium prewarmed to 55°C was added, and the cells were heat shocked for the same number of minutes at 39°C. Western blots containing 50 μg of total protein from cell lysates were analyzed by probing sequentially with anti-Mpk1 phospho-antibody and then anti-Flag antibody after the membrane was stripped. (B) The Mpk1 Flag-tagged and mpk1Δ strains were treated as described for panel A, but heat shocked for 1 h only. Blots were probed sequentially with anti-Mpk1 phospho-antibody, anti-Flag antibody, and finally anti-β actin antibody for a loading control after the membrane was stripped.
Mutants that harbor either rho1G15V or rho1Q64L in place of endogenous Rho1 are constitutively active independent of heat shock, and Mpk1 phosphorylation is increased with thermal stress compared to that observed at permissive temperatures. KN99α, rho1G15V, rho1G15V::RHO1, rho1Q64L, and rho1Q64L::RHO1 strains were grown overnight in YPD medium at 24°C and then split the next morning into two aliquots. To one aliquot, an equal volume of YPD medium at room temperature was added, and cells were grown an additional hour at 24°C. To the second aliquot, an equal volume of YPD medium prewarmed to 55°C was added, and the cells were grown for one additional hour at 39°C. Western blots containing 50 μg of total protein from cell lysates were analyzed by probing sequentially with anti-Mpk1 phospho-antibody and then anti-Flag antibody after the membrane was stripped. All strains are in the Mpk1 Flag-tagged background.
A strain with a deletion of RHO10 demonstrates constitutive Mpk1 phosphorylation independent of heat shock, phosphorylation is increased at high temperature, and both rho11Δ and rho10Δ rho11Δ strains confer wild-type Mpk1 phosphorylation. Strains were grown overnight in YPD medium at 24°C and then split the next morning into two aliquots. To one aliquot, an equal volume of YPD medium at room temperature was added, and cells were grown an additional hour at 24°C. To the second aliquot, an equal volume of YPD medium prewarmed to 55°C was added, and the cells were grown for one additional hour at 39°C. Western blots containing 50 μg of total protein from cell lysates were analyzed by probing sequentially with anti-Mpk1 phospho-antibody and then anti-Flag antibody after the membrane was stripped. All strains are in the Mpk1 Flag-tagged background. Strain names and/or genotypes are given above the blots.
RHO1 and RHO10 are necessary for protection against cell wall stress but RHO11 is dispensable for this response. Cultures of strains were grown overnight in YPD medium at 30°C then diluted to an OD650 of 1.0. Five microliters of 10-fold serial dilutions were plated onto YPD plates plus the indicated inhibitor (SDS, sodium dodecyl sulfate; CFW, calcofluor white) and grown at 30°C for 5 days. Strain names or genotypes are indicated to the left of the panels.
A strain with a deletion of RHO10 but not both RHO10 and RHO11 is sensitive to nitrosative stress. Cultures of strains were grown overnight in YPD medium at 30°C and then diluted to an OD650 of 1.0. Five microliters of 10-fold serial dilutions were plated onto freshly made YNB, pH 4.0, plates containing 1 mM NaNO2 and grown for 5 days at 30°C.
The constitutively active rho1G15V and rho1Q64L mutant strains display decreased capsule size compared to wild-type cells. Capsule was induced by growing cells on DMEM plates in the presence of 5% CO2 for 5 days at 30°C. Cells were stained with a 1:4 India ink-H2O solution, and capsule diameter was measured for a minimum of 100 cells per strain. (A) Differences in capsule diameters were tested for statistical significance using a one-way ANOVA with a Dunnett's t posthoc test to compare each mutant or deletion strain to the WT (see Materials and Methods). Open circles represent measurements that were >1.5 to 3.0 times the interquartile range. Asterisks represent measurements that were greater than three times the interquartile range. (B) Microscopic representation of India ink-stained cells for each strain at a magnification of ×ばつ400.
The PKC1 pathway components Bck1 and Mkk2 are necessary but Pkc1 is dispensable for phosphorylation of Mpk1 in response to thermal stress in C. neoformans. (A) KN99α, bck1Δ, and mkk2Δ strains were grown overnight in YPD medium at 24°C and then split the next morning into two aliquots. To one cell aliquot, an equal volume of YPD medium at room temperature was added, and cells were grown an additional hour at 24°C. To the other cell aliquot, an equal volume of YPD medium prewarmed to 55°C was added, and the cells were grown for one additional hour at 39°C. Western blots containing 50 μg of total protein from cell lysates were analyzed by probing sequentially with anti-Mpk1 phospho-antibody and then anti-Flag antibody after the membrane was stripped. All strains are in the Mpk1 Flag-tagged background. Identical results were obtained when experiments with KN99α, bck1Δ, and mkk2Δ strains were done using YPD medium containing 1 M sorbitol. (B) KN99α and pkc1Δ strains in the Mpk1 Flag-tagged background were treated identically to samples described in panel A, but all YPD medium contained 1 M sorbitol due to the conditional lethality of the pkc1 deletion strain.
Model for C. neoformans Rho GTPases in protection against thermal stress and their relationship to the PKC1 cell integrity pathway. (A) Under growth conditions at 24°C, Rho1 is not active, and Rho11 is negatively inhibited by Rho10. This leads to inactivation of the cell integrity pathway as measured by Mpk1 phosphorylation. (B) Under thermal stress conditions (39°C for 1 h), Rho1 signals the activation of the cell integrity pathway, and Mpk1 is phosphorylated. Rho10 is inactivated, thereby releasing inhibition of Rho11, resulting in activation of the cell integrity pathway. Rho10 may also positively regulate an unknown protein or process that leads to the activation of the thermal stress response independent of Mpk1 phosphorylation (dashed arrows). For thermal stress response, Pkc1 is dispensable; therefore, Rho proteins likely interact with downstream components of the PKC1 pathway (brackets and solid arrows). Rho1, Rho10, and Rho11 act in concert and require balance to regulate the precise and critical amount of Mpk1 phosphorylation needed for growth and combating thermal stress.
References
-
- Etienne-Manneville S, Hall A. 2002. Rho GTPases in cell biology. Nature 420:629–635 - PubMed
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Miscellaneous