Targets downstream of Cdk8 in Dictyostelium development.
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.
Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU UK. catherine.pears@bioch.ox.ac.uk
David M
DM
Greene
Gareth
G
Bloomfield
Jason
J
Skelton
Alasdair
A
Ivens
Catherine J
CJ
Pears
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000235:genetics; D015152:Blotting, Northern; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D056850:Cyclin-Dependent Kinase 8 / Q000235:genetics* / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000235:genetics; D020440:Gene Duplication; D046228:Microarray Analysis; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D010641:Phenotype; D040901:Proteomics; D033761:Spores, Protozoan / Q000235:genetics / Q000378:metabolism
D015111:4-Hydroxyphenylpyruvate Dioxygenase; C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D064429:Fatty Acid Synthases; D017868:Cyclic AMP-Dependent Protein Kinases; D056850:Cyclin-Dependent Kinase 8
Mol Cell Proteomics. 2006 Dec;5(12):2228-43
16926386
Science. 1997 Jul 11;277(5323):251-4
9211856
Nature. 2002 Jul 4;418(6893):79-85
12097910
Mol Cell Biol. 1999 May;19(5):3338-48
10207058
Development. 2005 Apr;132(8):1885-93
15790964
Dev Biol. 2005 Aug 1;284(1):25-36
15964562
J Biol Chem. 1997 Feb 14;272(7):3883-6
9020088
EMBO J. 2007 Feb 21;26(4):1045-54
17290221
Genome Biol. 2008 Apr 22;9(4):R75
18430225
PLoS One. 2010 May 07;5(5):e10543
20479885
Genome Biol. 2004;5(10):R80
15461798
Development. 2004 Jul;131(13):3147-56
15175247
Gene. 1995 Aug 30;162(1):129-34
7557400
Biochim Biophys Acta. 1972 Sep 19;284(1):90-100
4627454
Genes Dev. 2001 May 1;15(9):1078-92
11331604
J Proteome Res. 2006 Mar;5(3):589-98
16512674
Cell Microbiol. 2006 Mar;8(3):438-56
16469056
Nature. 2005 May 5;435(7038):43-57
15875012
Mol Cell. 2004 Nov 19;16(4):509-20
15546612
Biochim Biophys Acta. 1972 May 12;268(2):601-4
5026314
Nucleic Acids Res. 2004 Oct 26;32(19):e143
15507682
Dev Biol. 2004 Jul 1;271(1):49-58
15196949
Trends Biochem Sci. 2005 May;30(5):250-5
15896743
Genes Cells. 2000 Jan;5(1):43-55
10651904
Bioinformatics. 2005 May 1;21(9):2067-75
15657102
Proc Natl Acad Sci U S A. 1990 Nov;87(21):8247-51
2236037
J Comput Biol. 2002;9(1):55-66
11911795
FEBS Lett. 1995 Jul 17;368(2):381-4
7628643
Dev Growth Differ. 2002 Jun;44(3):213-23
12060071
Microbiol Mol Biol Rev. 1998 Sep;62(3):684-94
9729606
Cell. 1998 Nov 25;95(5):717-28
9845373
Nature. 2008 Sep 25;455(7212):547-51
18794900
Nature. 2003 Jan 9;421(6919):187-90
12520306
Development. 1992 Jul;115(3):785-90
1330484
Dev Biol. 2006 Apr 15;292(2):519-32
16469305
Microbiology (Reading). 2007 Feb;153(Pt 2):619-630
17259634
J Biol Chem. 1963 Dec;238:4091-7
14086750
Eukaryot Cell. 2007 Feb;6(2):245-52
17085634
Stat Appl Genet Mol Biol. 2004;3:Article3
16646809