SIGNALING PATHWAYS THAT REGULATE DEVELOPMENT

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Our (and other) studies of the cell surface receptors for cAMP were the first to link 7-TMRs with morphogen signaling. In the multicellular aggregate, cAMP receptor (CAR) signaling regulates pathways that activate/de-activate protein kinase GSK3, which, in turn, establishes developmental patterning and cell fate specification. In certain respects, extracellular cAMP in Dictyostelium serves as a functional analog of Wnt, a secreted ligand that regulates multiple, but discrete pathways, during metazoan development. The Wnts organize planar cell polarity and coordinate cell migration, but also function as effective inhibitors of GSK3, a developmental switch for cell fate determination. Mutations of many genes in this pathway result in embryonic lethality and tumorigenesis in mice and humans. We had shown that ZAK1 is an activating tyrosine kinase of GSK3 and have now identified a related tyrosine kinase, ZAK2, that also regulates GSK3 function during Dictyostelium development; no additional family members exist. We have shown that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 are differentially required to regulate GSK3 within distinct differentiated cell populations. ZAK2 can also act in a non-autonomous manner to regulate cell-type differentiation. Finally, we show that efficient polarization of Dictyostelium toward cAMP depends on ZAK-mediated tyrosine phosphorylation of GSK3. Our results extend the complexity of GSK3 signaling during development and suggest that combinatorial regulation of GSK3 can differentially guide cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. beta-catenin stabilization in mammalian cells is required for Wnt stimulated promoter (e.g. Topflash) activity in mammalian cells. We have shown that Galphao and Galphaq mediate beta-catenin stabilization by Wnt. Inactivation of Galphao by RNAi depletion or by PTX treatment only partially inhibits beta-catenin stabilization, but PTX completely suppresses Wnt/Topflash activation. Since levels of cytosolic beta-catenin do not appear to simply predict transactivating function for gene expression, we investigated beta-catenin protein complexes in mammalian cells treated with Wnt and/or with PTX. Wnt promoted the accumulation of monomeric beta-catenin in the cytosol and interaction with Lef/Tcf transcription factors in the nucleus. Treatment with PTX induced the release of b-catenin from association with E-cadherin at the membrane to the cytosol, where it can dimerize with and potentially sequester beta-catenin. Although Wnt stimulation of PTX-treated cells also induced an increase in cytosolic and nuclear beta-catenin, most beta-catenin was in complex with alpha-catenin and was unable to effect induction of Wnt/Topflash. These data are consistent with other studies that infer that alpha-catenin functions as a beta-catenin signaling inhibitor, blocking beta-catenin/TCF complexes from binding to DNA. We are now extending our studies on developmental cellular signaling in Dictyostelium to the gamma-secretase/Presenilin (PS) pathway. Our studies are intended to further dissect these mechanisms and to identify novel targets of Presenilin signaling. Our ability to analyze these pathways in Dictyostelium can contribute to understanding essential signaling events that are conserved in humans. The gamma-secretase/PS complex, comprised of Presenilin (PS), Nicastrin (Nct), Aph1 and Pen2, is responsible for intramembrane proteolysis of type 1 single-pass transmembrane proteins and is essential for a variety of cellular and developmental functions. In some instances proteolysis induces the release of a transcription factor, while in others, mechanisms of PS signal transduction remain unknown. Principle substrates of gamma-secretase include beta-Amyloid Precursor Protein and Notch, and mutations in the gamma-secretase complex have been clearly linked to Alzheimer's disease, defects in Notch signaling, and embryonic lethality. Since Dictyostelium does not have orthologs of these traditional substrates, it presents a unique system to reveal novel PS functions. Analyses of a series of single and double mutations of the PS, Aph1, Pen2, and Nct genes show that the PS complex plays two important, yet distinct, roles in control of cell fate specification during Dictyostelium development. Putative novel substrates have been identified. Dictyostelium is exceptionally powerful for studying numerous aspects of cellular and developmental function. While the high efficiency of targeted gene disruption has enabled researchers to characterize many specific genes, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or redundancies between various pathways. We developed a robust system for production of multiple gene (knock-out) mutations, where cells remain sensitive to transformation for additional targeted or random mutagenesis and for functional expression studies of mutated or tagged proteins.