Spatial patterning in multicellular eukaryotes involves the interaction of multiple signaling pathways that respond to morphogenetic signals in the organism to control cell-fate decisions and morphogenetic movements. These events lead to the proper positioning and differentiation of the cell types along an anterior-posterior axis. We have identified two pathways that regulate cell-type differentiation and spatially patterning in Dictyostelium. One pathway uses the MEK kinase MEKKalpha that contains an F-box and WD40 repeats. Activity of MEKKalpha is spatially and temporally regulated by protein degradation via a ubiquitination/deubiquitination regulatory network involving the ubiquitin conjugating enzyme UBC1 and the deubiquitinating enzyme UBP1. MEKKalpha is required for cell fate decisions and proper spatial patterning within the multicellular organism. The second pathway includes Spalten, a novel signaling component containing an N-terminal regulatory domain with homology to heterotrimeric G proteins a subunits and alpha C-terminal serine/threonine protein phosphatase PP2C. Spalten phosphatase activity is required to induce cell-type differentiation by dephosphorylating a substrate. Second site suppressor screens have identified a kinase, designated ARCK1, which has structure similarities to mammalian Raf-1. ARCK1 appears to function to phosphorylate the Spalten substrate and to maintain cells in an undifferentiated state. Spalten and ARCK1 thus function on a pathway that regulates cell fate decisions in Dictyostelium. Our goals include the identification of other components of these regulatory cascades and elucidation of the mechanisms by which signaling molecules control their activity and how these pathways regulate cell fate decisions in Dictyostelium. We will employ a combination of molecular, genetic, and biochemical approaches to achieve these goals. Because of the simplicity of the developmental system and the facility with which one can pursue multiple approaches simultaneously to analyze gene function, it is expected that significant progress will be made in achieving these goals. Moreover, as much of our analysis is directed at pathways that have counter parts in other systems, our work should shed light onto how regulatory cascades, such ubiquitin-mediated protein degradation, can be use to control complex developmental pathways, including spatial patterning and cell-type differentiation in eukaryotes in general.
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