Characterization of CDC14 and NAGK in model systems of development

Abstract

Cell division cycle 14 (Cdc14) is an evolutionarily conserved phosphatase originally identified in <em>Saccharomyces cerevisiae</em> as a cell cycle regulator. In <em>Drosophila melanogaster</em>, Cdc14 is encoded by a single gene, thus facilitating its study. I characterized a null mutation of <em>cdc14</em> in <em>D. melanogaster</em>, and found that the animals were viable, with no obvious defects in the cell cycle or DNA damage repair. However, the <em>cdc14</em> null mutants have defects in chemosensation, mechanosensation, lipid metabolism and resistance to starvation. Additionally, the <em>cdc14</em> null males exhibit decreased sperm competitiveness, which was not the result of decreased mating behavior from motor defects or decreased fertility. This is the first characterization of Cdc14 in <em>D. melanogaster</em> and demonstrates several novel roles for Cdc14 in metazoans. <em>N</em>-acetyl-<em>D</em>-glucosamine kinase (NAGK) is a sugar kinase and first step in the UDP-GlcNAc salvage pathway of glycosylation. The UDP-GlcNAc salvage pathway is responsible for approximatly 80% of the free UDP-GlcNAc pool that is required for protein glycosylation. I demonstrate that overexpression of NAGK, the UDP-GlcNAc salvage pathway enzymes (Phosphoglucomutase 3 (PGM3) and UDP-<em>N</em>-acetyl-<em>D</em>-glucosamine Pyrophosphorylase 1 (UAP1)), the first enzyme in the <em>N</em>-glycosylation pathway (Dolichyl-Phosphate (UDP-<em>N</em>-Acetylglucosamine) <em>N</em>-Acetylglucosaminephosphotransferase 1 (DPAGT1)) or injection of the UDP-GlcNAc salvage pathway sugars (<em>N</em>-acetyl-<em>D</em>-glucosamine (GlcNAc), GlcNAc-1-Phosphate (GlcNAc-1-P), GlcNAc-6-Phosphate (GlcNAc-6-P), and Uridine diphosphate GlcNAc (UDP-GlcNAc)) posteriorized <em>Xenopus laevis</em> embryos and inhibited eye formation in <em>Danio rerio</em>. Conversely, inhibition of NAGK, PGM3, UAP1, and DPAGT1 anteriorized <em>X. laevis</em> embryos and resulted in cyclopia in <em>D. rerio</em>. Injection of <em>N</em>-<em>glycanase 1</em> (NGLY1) mRNA, which removes <em>N</em>-linked glycans from glycoproteins, anteriorized <em>X. laevis</em> embryos and resulted in cyclopia in <em>D. rerio</em>. qRT-PCR data suggests NAGK, PGM3, UAP1, and DPAGT1 specifically affect Wnt signaling in the early embryo, likely through the control of the Wnt ligand and/or the receptors Frizzled (Fz) and Low-density lipoprotein 6 (LRP6). This is the first work to link NAGK, PGM3, UAP1, and DPAGT1 to Wnt signaling.