We plan to focus on a set of genes that play essential roles in terminal differentiation of Dictyostelium. Three genes were chosen from among those discovered in our on-going near- saturation mutagenesis screen. Conserved domains in the predicted products of these genes suggest that they may function in a network that coordinates morphogenesis and encapsulation. The cAMP dependent protein kinase, PKA, plays a critical role throughout development in Dictyostelium and triggers terminal differentiation of spores during culmination. Since the activity of PKA is determined by the internal levels of cAMP, the relative rates of synthesis and degradation of this cyclic nucleotide are central to its regulation. One of the genes we propose to study in depth, acrA, encodes a novel adenylyl cyclase that plays an essential role during late development. We will characterize strains in which the catalytic subunit of PKA is overexpressed and both of the genes encoding developmental adenylyl cyclases, acaA and acrA, are inactivated to establish the extent to which Dictyostelium can develop without cAMP. Expression of acrA in only one of the two major cell types, prespore or prestalk cells, will be analyzed to test whether there is intercellular communication through cAMP during terminal differentiation. The increase in cAMP that is observed during culmination not only depends on ACR but also on the inhibition of the cytoplasmic phosphodiesterase RegA. There are several sites in the carboxy- terminal domain of RegA that are phosphorylated by a MAP kinase. Phosphorylation of these sites by the MAP kinase ERK2 results in an increase in cAMP. The other two genes that we propose to study in depth, splA and mkcB, encode proteins that show significant similarity to Raf-like protein kinases that activate MAP kinase cascades. Null mutations in either of these genes reduce the extent of encapsulation dramatically. We will determine whether dominant activated forms of these protein kinases can rescue the block in spore formation in either splA- or mkcB- strain as well as make sporulation of wild type cells independent of prestalk derived signals. The results will test the hypothesis that a secreted signal peptide, SDF-2, that binds to the histidine kinase receptor, DhkA, activates the MAP kinase cascade, possibly by directly interacting with Sp1A. Together these studies will help to establish a signal transduction pathway that can serve as a model for studies in other systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060447-02
Application #
6387057
Study Section
Special Emphasis Panel (ZRG1-CDF-1 (01))
Program Officer
Anderson, Richard A
Project Start
2000-07-01
Project End
2004-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$270,621
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Loomis, William F (2008) cAMP oscillations during aggregation of Dictyostelium. Adv Exp Med Biol 641:39-48
Stepanovic, Vesna; Wessels, Deborah; Daniels, Karla et al. (2005) Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis. Eukaryot Cell 4:775-86
Wessels, Deborah; Brincks, Rebecca; Kuhl, Spencer et al. (2004) RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum. Eukaryot Cell 3:646-62
Winckler, Thomas; Iranfar, Negin; Beck, Peter et al. (2004) CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development. Eukaryot Cell 3:1349-58
Zhang, Hui; Heid, Paul J; Wessels, Deborah et al. (2003) Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum. Eukaryot Cell 2:62-75
Iranfar, Negin; Fuller, Danny; Loomis, William F (2003) Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum. Eukaryot Cell 2:664-70
Maeda, Mineko; Sakamoto, Haruyo; Iranfar, Negin et al. (2003) Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses. Eukaryot Cell 2:627-37
Anjard, Christophe; Loomis, William F; Dictyostelium Sequencing Consortium (2002) Evolutionary analyses of ABC transporters of Dictyostelium discoideum. Eukaryot Cell 1:643-52
Sasik, R; Iranfar, N; Hwa, T et al. (2002) Extracting transcriptional events from temporal gene expression patterns during Dictyostelium development. Bioinformatics 18:61-6
Chae, Soo-Cheon; Fuller, Danny; Loomis, William F (2002) Altered cell-type proportioning in Dictyostelium lacking adenosine monophosphate deaminase. Dev Biol 241:183-94

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