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Targets downstream of Cdk8 in Dictyostelium development.

Greene DM, Bloomfield G, Skelton J, Ivens A, Pears CJ - BMC Dev. Biol. (2011)

Bottom Line: Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background.Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU UK. catherine.pears@bioch.ox.ac.uk

ABSTRACT

Background: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.

Results: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).

Conclusions: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

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Expression of constitutively active PKA in cdk8-2 cells. (A) Western analysis was used to confirm expression of the FLAG-PKAC protein in the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] cell lines. Samples were collected from vegetatively growing cells of each strain and resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with the α-FLAG antibody. The blot was reprobed with an α-actin antibody to control for loading. (B) Developmental phenotype of the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] strain. Cells were spotted onto a lawn of K. aerogenes and incubated at 22°C. Photographs were taken after 5 days. Each colony is approximately 1 cm in diameter.
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Figure 4: Expression of constitutively active PKA in cdk8-2 cells. (A) Western analysis was used to confirm expression of the FLAG-PKAC protein in the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] cell lines. Samples were collected from vegetatively growing cells of each strain and resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with the α-FLAG antibody. The blot was reprobed with an α-actin antibody to control for loading. (B) Developmental phenotype of the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] strain. Cells were spotted onto a lawn of K. aerogenes and incubated at 22°C. Photographs were taken after 5 days. Each colony is approximately 1 cm in diameter.

Mentions: The early developmental phenotype of the cdk8-2 cells suggested a potential gene target for regulation by Cdk8 activity. The cAMP dependent protein kinase (PKA) enzyme is vital for both aggregation and formation of spore cells (reviewed in [16]). Many aggregation-deficient strains which can be rescued by exogenous pulses of cAMP can also be rescued by restoring expression of the catalytic subunit of PKA, pkaC. As no expression of pkaC could be detected in cdk8- cells ([11] and data not shown), it was hypothesised that this may be responsible for the defect in aggregation. In order to address this question, the pDXA[act15::FLAG-pkaC] plasmid which expresses the PKA-C protein with an N-terminal FLAG-tag was transformed into the cdk8-2 and Ax2bsR to create the cdk8-2[FLAG-pkaC] and Ax2bsR[FLAG-pkaC] strains. Cell extracts from these strains were analysed by western blot to confirm expression of the 75kDa FLAG-PKA-C protein in both strains (Figure 4A).


Targets downstream of Cdk8 in Dictyostelium development.

Greene DM, Bloomfield G, Skelton J, Ivens A, Pears CJ - BMC Dev. Biol. (2011)

Expression of constitutively active PKA in cdk8-2 cells. (A) Western analysis was used to confirm expression of the FLAG-PKAC protein in the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] cell lines. Samples were collected from vegetatively growing cells of each strain and resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with the α-FLAG antibody. The blot was reprobed with an α-actin antibody to control for loading. (B) Developmental phenotype of the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] strain. Cells were spotted onto a lawn of K. aerogenes and incubated at 22°C. Photographs were taken after 5 days. Each colony is approximately 1 cm in diameter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037916&req=5

Figure 4: Expression of constitutively active PKA in cdk8-2 cells. (A) Western analysis was used to confirm expression of the FLAG-PKAC protein in the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] cell lines. Samples were collected from vegetatively growing cells of each strain and resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with the α-FLAG antibody. The blot was reprobed with an α-actin antibody to control for loading. (B) Developmental phenotype of the Ax2bsR[FLAG-pkaC] and cdk8-2[FLAG-pkaC] strain. Cells were spotted onto a lawn of K. aerogenes and incubated at 22°C. Photographs were taken after 5 days. Each colony is approximately 1 cm in diameter.
Mentions: The early developmental phenotype of the cdk8-2 cells suggested a potential gene target for regulation by Cdk8 activity. The cAMP dependent protein kinase (PKA) enzyme is vital for both aggregation and formation of spore cells (reviewed in [16]). Many aggregation-deficient strains which can be rescued by exogenous pulses of cAMP can also be rescued by restoring expression of the catalytic subunit of PKA, pkaC. As no expression of pkaC could be detected in cdk8- cells ([11] and data not shown), it was hypothesised that this may be responsible for the defect in aggregation. In order to address this question, the pDXA[act15::FLAG-pkaC] plasmid which expresses the PKA-C protein with an N-terminal FLAG-tag was transformed into the cdk8-2 and Ax2bsR to create the cdk8-2[FLAG-pkaC] and Ax2bsR[FLAG-pkaC] strains. Cell extracts from these strains were analysed by western blot to confirm expression of the 75kDa FLAG-PKA-C protein in both strains (Figure 4A).

Bottom Line: Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background.Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU UK. catherine.pears@bioch.ox.ac.uk

ABSTRACT

Background: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.

Results: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)).

Conclusions: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

Show MeSH
Related in: MedlinePlus