Regulation Of Developmental Gene Expression

Diabetes, Digestive, Kidney Diseases

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Abstract

Our laboratory is investigating molecular processes critical for developing a terminally differentiated organism from a homogeneous population of totipotent cells. We are using molecular, genetic, and biochemical techniques and the model eukaryotic system Dictyostelium to define cell autonomous and non-autonomous signal transduction pathways that specify cell fate and pattern formation. In Dictyostelium, stimulation of a family of 7-transmembrane (7-TM) domain receptors by its ligand, the secreted chemoattractant-morphogen cAMP, establishes the fundamental developmental organization, an anterior (prestalk)/posterior (prespore) axis. As a chemoattractant, cAMP activates cAMP receptor/G protein signaling to mobilize individual cells to form multicellular aggregates; later in development, cAMP receptor signaling regulates cell fate specification. We focus on signaling pathways, both common and unique to the family of cAMP receptors (CARs), to elucidate mechanisms and circuits that may be conserved in more complex developmental programs. GSK3 acts as developmental switch to establish cell fates in the metazoa. In Dictyostelium, activated GSK3 stimulates prespore/spore pathways, while suppressing prestalk/stalk differentiation. Activation/de-activation of GSK3 is mediated by tyrosine kinase ZAK1 and a CAR4-dependent PTPase, respectively. Although zak1-nulls share many of the phenotypes of gsk3-nulls, their prestalk patterns are not identical. Further, residual tyrosine phosphorylation and activation of GSK3 is observed in the absence of ZAK1. We identified ZAK2, the other tyrosine kinase in the GSK3 activation pathway; no additional family members exist. Like ZAK1, ZAK2 will phosphorylate and activate GSK3, but while both act via GSK3, their roles in cellular differentiation are distinct. ZAK2 and GSK3 repress the differentiation the prestalk A sub-population autonomously. ZAK1 has no regulatory role in these cells. Conversely, ZAK1 and GSK3, but not ZAK2, antagonize the differentiation of a different prestalk class, prestalk B cells. Further, ZAK2/GSK3 is required non-autonomously, while ZAK1/GSK3 acts autonomously, to promote prespore/spore cell fates. Finally, we show that tyrosine phosphorylation/activation of GSK3 is required for cell polarity and chemotaxis during development. We propose that combinatorial regulation of GSK3 can differentially guide cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. Cells move directionally in chemoattractant gradients that differ by less than 2% across the cell body. These shallow extracellular gradients are amplified into very steep intracellular gradients, with signaling components localized specifically to the leading edge or rear of moving cells. This occurs despite uniform receptor distribution and proportionate activation. Complex feedback loops that are downstream of receptor signaling and that integrate activating and inhibiting pathways are suggested to regulate this steep intracellular gradient. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Ga9) that antagonizes chemotactic response. Most dramatically, cells lacking Ga9 are hyperpolarized and hyperchemotactic, whereas cells expressing constitutively activated Ga9 exhibit a reciprocal phenotype. Mechanistically, Ga9 functions very rapidly following receptor stimulation to negatively regulate multiple downstream pathways that ultimately establish the asymmetric mobilizations of actin and myosin. We suggest that functionally similar Ga-mediated inhibitory signaling may modulate chemoattractant responses in most eukayotic cells. Secreted factors, such as hormones and cytokines, are essential for many developmental processes. We have now identified a novel, secreted factor APF (aggregation promotion factor) that augments early Dictyostelium development. Using a bioassay that rescues the developmental defect of cells placed at very low cell density, we purified APF to homogeneity. APF activity is distinct from other known factors. It is a glycosylated, 250 kDa complex and each protein of the complex was identified by mass spectrometry. The complex includes a novel 150 kDa protein (p150), a cysteine protease, a novel oxidase-related protein (OxyA), and PDE. APF purified from pde-nulls is fully active, but fractionates with a molecular mass of 150 kDa. OxyA and the cysteine protease do not co-fractionate with APF activity in pde-nulls. We disrupted the gene encoding OxyA and confirmed that OxyA does not contribute to APF activity. We isolated the full-length p150 gene and showed that overexpression of p150 leads to an overexpression of APF activity. p150 is synthesized as a single pass, integral membrane precursor protein and cleavage at or within the membrane releases active APF. In a separate project we study lipolysis in adipocytes that governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by protein kinase A (PKA) activation, hormone-sensitive lipase (HSL), a cytosolic enzyme, and Perilipin, which coats the lipid droplet surface in adipocytes. A key step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. We have demonstrated that PKA phosphorylation at either serine 659 or 660 within HSL, is required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic serine 423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. To assess whether the Perilipins participate directly in PKA-mediated lipolysis, we expressed constructs coding for native and mutated forms of the two major splice variants of the Perilipin gene, Perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of Perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of Perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of Perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of Perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. We showed that HSL is basally associated with lipid droplet surfaces at a low level in perilipin-nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without Perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable Perilipin A, confirming that Perilipin is required to elicit the HSL translocation reaction.