Regulation of Signaling Pathways that Organize Developme

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Our laboratory is investigating molecular processes critical for developing a terminally differentiated organism from a homogeneous population of ?totipotent? cells. Our research serves to identify signaling cascades central to multicellular differentiation. We use molecular, genetic, cellular, and biochemical techniques, and the model eukaryote Dictyostelium to define cell autonomous and non-autonomous signal transduction pathways that regulate developmental organization. Dictyostelium has a small, haploid genome that is particularly amenable to genetic manipulation. Dictyostelium grow as individual cells in enriched media, but develop multicellularly upon nutrient depletion. Growth and development are separate, allowing the isolation and propagation of mutants deficient only in developmental pathways. We study a variety of signaling pathways to better understand mechanisms and circuits conserved in more complex developmental systems. In certain instances, we extend studies to mammalian models. Many receptor-mediated pathways in Dictyostelium and other cells adapt/deactivate to persistent stimulation. While MAP kinase ERK2 in Dictyostelium has been assumed to adapt to continuous CAR engagement, we have shown, to the contrary, that ERK2 remains active under such conditions. The upstream phosphorylation pathway, which is responsible for ERK2 activation, transiently responds to CAR stimulation, whereas ERK2 de-phosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result is persistently active ERK2 when cAMP concentration is constant and that the oscillating production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also showed that CAR-dependent pathways that control ERK2 activation/deactivation function independently of G proteins and of ligand-induced production of intracellular cAMP and the consequent activation of PKA. This regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted cAMP increases during later development. This work redefines mechanisms of ERK2 regulation by CAR signaling in Dictyostelium and establishes new implications for control of signal-relay during chemotaxis. 7-TMRs activate multiple downstream signaling cascades via heterotrimeric G protein-dependent and -independent pathways and control a wide range of biological processes. Upon ligand binding, 7-TMRs become phosphorylated at cytoplasmic serine and threonine residues by specific receptor kinases, functioning to uncouple heterotrimeric G protein pathways from receptor signaling and to activate G protein-independent pathways. In Dictyostelium, CAR signaling regulates chemotaxis and both G protein-dependent and -independent signaling cascades. Since the CARs become phosphorylated upon cAMP binding, we have expressed several non-phosphorylatable variants to further study the function of receptor phosphorylation. Cells expressing the CAR variants are defective in adenylyl cyclase adaptation and in cell polarization and chemotaxis. While receptor phosphorylation is known to uncouple certain G protein-mediated signaling pathways from 7-TMRs in mammalian cells, our studies are the first to show that such mechanisms also function in pathways that regulate chemotaxis. We identified two Dictyostelium LIM domain proteins, LimF and ChLim, that interact with each other and with the small, Rab5-related Rab21 GTPase to collectively regulate actin-mediated phagocytosis. We have shown that overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases phagocytosis above that of wild-type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Studies using cells with multiple mutations in these genes show that ChLim antagonizes the activating function of Rab21-GTP; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrated that ChLim and LimF localize to phagocytic cups and phago-lysosomal vesicles and suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytosis. Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for eukaryotic cellular function. The COP9 signalosome (CSN) regulates cullin within SCF in all eukaryotes, but there is extreme sequence divergence of CSN subunits of the yeasts compared to plants and animals. Using the yeast two-hybrid system, we identified the CSN5 subunit as a potential interacting partner of a CAR. We further identified and characterized all 8 CSN subunits in Dictyostelium. Remarkably, despite the ancient origin of Dictyostelium, these CSN protein sequences cluster very closely with their plant and animal counterparts. We additionally showed that the Dictyostelium subunits, like those of other systems, are capable of multi-protein interactions within the CSN complex and that CSN5 and CSN2 are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from the yeasts. 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 and that efficient polarization and chemotaxis 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. Our interest in 7-TM receptor regulation of GSK3 in development, led us to investigate the role of G proteins in the mammalian pathway. In mouse cells cultured without Wnt, the transcriptional cofactor beta-catenin is destabilized via phosphorylation by protein kinase GSK3-beta in complex with Axin proteins. In the "canonical" pathway, Wnt functionally disrupts GSK3-beta/Axin complexes and thereby stabilizes beta-catenin. Wnt regulation of GSK3 and stabilization of beta-catenin are still not fully understood. We investigated if signaling via Frizzled (Fz), the 7-TM receptor for Wnt, was mediated by G proteins. We demonstrated that depletion of Galphao or Galphaq suppressed, respectively, Wnt-induced disruption of GSK3-beta/Axin2 and GSK3-beta/Axin complexes and diminished Wnt stabilization of beta-catenin. We also showed that direct activation of G proteins in vivo with GTPgammaS in the absence of exogenous Wnt disrupted GSK3-beta/Axin2 complexes and stabilized beta-catenin. We examined time-dependent changes in protein-protein interactions that occur in response to Wnt treatment. The GSK3-beta/Axin complexes are rapidly disrupted upon Wnt stimulation and the changes in GSK3-beta/Axin associations precede both beta-catenin stabilization and Axin degradation. Finally, we demonstrated an association of Galphao with Fz that is also very rapidly perturbed upon Wnt-3a stimulation and that Wnt-dependent effects on both GSK3-beta/Axin2 and Galphao/Fz are pertussis toxin (PTX) sensitive. We conclude that Galphao and Galphaq signaling functions downstream of Fz and contributes to the rapid Wnt-mediated disruption of GSK3-beta/Axin interactions that ultimately stabilize beta-catenin.