Role for YakA, cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells - PubMed (original) (raw)

Role for YakA, cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells

Alexandre Taminato et al. Mol Biol Cell. 2002 Jul.

Abstract

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyostelium cells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found that yakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation in pkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.

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Figures

Figure 1

Growth is inhibited by SNP, spermine NONOate, H2O2, or heat treatment. Wild-type cells were diluted to 1 × 106 cells/ml, and cells were incubated at 22°C in the presence of 500 μM SNP, 500 μM SNP, and 40 μM oxyhemoglobin, 500 μM spermine NONOate, or 500 μM H2O2. Alternatively, cells were incubated at 30°C with no additions. Cells were counted at the indicated times. Data corresponding to WT and WT treated with SNP and oxyhemoglobin overlap.

Figure 2

SNP induces death of yakA null cells. (A) Wild-type cells and yakA null cells were diluted to 5 × 105 cells/ml and SNP was added to 500 μM. Cells were counted at the indicated times. (B) Wild-type cells reestablish growth when SNP is removed. Wild-type cells were diluted to 1 × 106 cells/ml and SNP was added to 500 μM. SNP-containing media were kept continuously or washed off after 12, 24, or 48 h of incubation and cells were resuspended in SNP-free media. Cells were counted at the indicated times. (C) yakA null cells do not recover from the SNP-induced growth arrest when SNP is removed. yakA null cells were diluted to 5 × 105 cells/ml and SNP was added to 500 μM. SNP-containing media were kept continuously or washed off after 12, 24, or 48 h of incubation, and cells were resuspended in SNP free media. Cells were counted at the indicated times.

Figure 3

H2O2 induces death of yakA null cells. Wild-type cells and yakA null cells were diluted to 5 × 105 cells/ml and H2O2 was added to 500 μM. Cells were counted at the indicated times.

Figure 4

PKA is involved in the growth inhibition response induced by SNP. (A) Wild-type cells, yakA null cells, pkaC null cells, yakA null overexpressing pkaC (yakA − [pkaC/pkaC]), yakA/pkaC double, and yakA/pufA double mutants were diluted to 5 × 105 cells/ml, and SNP was added to 500 μM. Cells were counted at the indicated times. (B) Same as in A, with Y-axis expanded for better visualization.

Figure 5

pkaC and acaA null cells have a less pronounced growth inhibition. (A) Wild-type cells, yakA null cells, and pkaC null cells were diluted to 5 × 105 cells/ml and H2O2 was added to 500 μM. Cells were counted at the indicated times. (B) yakA null and pkaC null cells have similar initial responses to SNP treatment. Same as in A, with X- and Y-axes expanded for better visualization of the early response. (C) yakA null cells do not recover from the H2O2-induced growth arrest. Wild-type, yakA null, and pkaC null cells were diluted to 5 × 105 cells/ml and H2O2 was added to 500 μM. H2O2-containing media were washed off, and cells were resuspended in H2O2-free media after 12 or 24 h of incubation. Cells were counted at the indicated times.

Figure 6

SNP and H2O2 induce increased PKA activity and higher cAMP levels. (A) PKA activity measurements in the absence of added cAMP. Wild-type, yakA null, and pkaC null cells were diluted to 1 × 106 cells/ml and incubated with 500 μM SNP or H2O2 for 4, 12, or 24 h. Cells were counted, aliquots of 106 cells were collected, and the cell pellets were frozen for PKA activity measurements. The PKI-inhibited phosphorylation of kemptide in the absence of cAMP is shown. The values represent the mean ± SEM for six independent experiments. (B) PKA activity measurements in the presence of added cAMP. Wild-type, yakA null, and pkaC null cells were diluted to 1 × 106 cells/ml and incubated with 500 μM SNP for 24 h or H2O2 for 12 h. Cells were counted, aliquots of 106 cells were collected, and the cell pellets were frozen for PKA activity measurements. The PKI-inhibited phosphorylation of kemptide in the presence of 10 μM cAMP is shown. The values represent the mean ± SEM for six independent experiments. (C) cAMP measurements. Exponentially growing cells were diluted to 1 × 106 cells/ml and incubated with 500 μM SNP for 24 h or H2O2 for 12 h. Cells were counted, aliquots of 5 × 106 cells were collected, and the cell pellets were frozen for cAMP measurements. The values represent the mean ± SEM for six independent experiments.

Figure 7

yakA mRNA, pkaC mRNA, or PKA-C protein content is not altered by SNP or H2O2 treatment. (A) RNase protection assays were performed on RNA samples extracted from wild-type cells grown in the presence of bacteria and collected after 44 h (low cell density) or 50 h (high cell density) or axenically grown wild-type cells treated or not with SNP and H2O2 for 24 h. Total RNA was extracted from the cells and assayed using a [32P]CTP-labeled antisense yakA mRNA. (B) RNase protection assays were performed on RNA samples extracted from wild-type cells starved for 8 h on filters and collected at the aggregation stage or axenically grown wild-type cells treated or not with SNP for 24 h and H2O2 for 12 h. Total RNA was extracted from the cells and assayed using a [32P]CTP-labeled antisense pkaC mRNA. (C) Western blot analysis using PKA-C antibodies were performed on protein extracts of axenically grown wild-type cells treated or not with SNP and H2O2 for 12 or 24 h.

Figure 8

yakA null, pkaC null, and acaA null cells are more resistant to heat stress. Wild-type cells, yakA null cells, pkaC null cells, and acaA null cells were diluted to 5 × 105 cells/ml and grown in HL-5 at 27 or 30°C. Cells were counted at the indicated times.

Figure 9

Pathways proposed to mediate stress responses of Dictyostelium cells. The regulatory relationship between genes and events is described, with arrows representing a positive requirement for a gene or event and bars representing an inhibitory role. See DISCUSSION for details.

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Role for YakA, cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells - PubMed (original) (raw)