The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper 2 . The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells 3 , developmentally pruned axons 4,5 and dendrites 6 , and axons undergoing Wallerian degeneration 7 . Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells 8,9 . Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases 10-13 . In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis 1 . CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains 1 . The PTB domain adaptor protein CED-6 can bind the NPXY motif of , is required for cell corpse Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning 19,20 or during Wallerian degeneration in the adult brain 21 . In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1 (refs 2 , 4-6 ). Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY an...

cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKAindependent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.cAMP stimulates proliferation in several model systems (1). Particularly in endocrine cells, in vitro and in vivo studies demonstrated a role for cAMP in mitogenesis (2), a concept further supported by the identification of mutant receptors and G-proteins causally linking constitutive cAMP signaling with hyperproliferative states (3, 4). For many years, PKA 2 represented the only known cAMP effector (5); however, although its activity is necessary, it is not sufficient for cAMP mitogenic action (6, 7). These studies indicated the existence of PKA independent effectors involved in cAMP-mediated proliferation.Epac (exchange protein activated by cAMP) is a new cAMPdependent, PKA-independent guanine nucleotide-exchange factor (GEF) for the small G-protein Rap (8,9). Newly developed cAMP analogs (10) capable of discriminating between Epac and PKA are starting to unravel Epac's role in diverse biological responses (11, 12). Epac's N-terminal regulatory domain includes a DEP module (Disheveled, Egl10, Pleckstrin) responsible for its membrane localization (13) and a cAMPbinding domain (CBD) that directly binds cAMP (K d ϳ 4 M) (13). The catalytic domain consists of a Ras exchange motif (REM), and the CDC25-like catalytic core, sufficient for GEF action (8). Biochemical (13-16) and crystallographic studies (17) unmasked a role for the regulatory domain in maintaining Epac in a basal autoinhibited state; deletion of its N terminus converted REM-cdc25-Epac to a constitutively active (cAMPindependent) Rap GEF. The addition of the N terminus in trans to the active C terminus was able to inhibit its GEF activity, and this inhibition could be relieved by cAMP. Mutation analysis indicated a role for the N terminus sequence 321 VLVLE 325 (as in Epac1) as part of this inhibitory domain, and accordingly, disruption of this domain by conversion of this sequence to 321 AAAAA 325...

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.

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LPG2 (a gene involved in lipophosphoglycan assembly) encodes the Golgi GDP-Man transporter of the protozoan parasite Leishmania and is a defining member of a new family of eukaryotic nucleotide-sugar transporters (NSTs). Although NST activities are widespread, mammalian cells lack a GDP-Man NST, thereby providing an ideal heterologous system for probing the LPG2 structure and activity. LPG2 expression constructs introduced into either mammalian cells or a Leishmania lpg2(-) mutant conferred GDP-Man, GDP-Ara, and GDP-Fuc (in Leishmania only) uptake in isolated microsomes. LPG2 is the first NST to be associated with multiple substrate specificities. Uptake activity showed latency, exhibited an antiport mechanism of transport with GMP, and was susceptible to the anion transport inhibitor DIDS. The apparent K(m) for GDP-Man uptake was similar in transfected mammalian cells (12.2 microM) or Leishmania (6.9 microM). Given the evolutionary distance between protozoans and vertebrates, these data suggest that LPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, we showed the existence of hexameric LPG2 complexes by immunoprecipitation experiments, glycerol gradient centrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strong biochemical evidence for a multimeric complex of NSTs, a finding with important implications to the structure and specificity of NSTs in both Leishmania and other organisms. Inhibition of essential GDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.