Characterization of CDC14 and NAGK in model systems of development

Abstract

Cell division cycle 14 (Cdc14) is an evolutionarily conserved phosphatase originally identified in Saccharomyces cerevisiae as a cell cycle regulator. In Drosophila melanogaster, Cdc14 is encoded by a single gene, thus facilitating its study. I characterized a null mutation of cdc14 in D. melanogaster, and found that the animals were viable, with no obvious defects in the cell cycle or DNA damage repair. However, the cdc14 null mutants have defects in chemosensation, mechanosensation, lipid metabolism and resistance to starvation. Additionally, the cdc14 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 D. melanogaster and demonstrates several novel roles for Cdc14 in metazoans. N-acetyl-D-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-N-acetyl-D-glucosamine Pyrophosphorylase 1 (UAP1)), the first enzyme in the N-glycosylation pathway (Dolichyl-Phosphate (UDP-N-Acetylglucosamine) N-Acetylglucosaminephosphotransferase 1 (DPAGT1)) or injection of the UDP-GlcNAc salvage pathway sugars (N-acetyl-D-glucosamine (GlcNAc), GlcNAc-1-Phosphate (GlcNAc-1-P), GlcNAc-6-Phosphate (GlcNAc-6-P), and Uridine diphosphate GlcNAc (UDP-GlcNAc)) posteriorized Xenopus laevis embryos and inhibited eye formation in Danio rerio. Conversely, inhibition of NAGK, PGM3, UAP1, and DPAGT1 anteriorized X. laevis embryos and resulted in cyclopia in D. rerio. Injection of N-glycanase 1 (NGLY1) mRNA, which removes N-linked glycans from glycoproteins, anteriorized X. laevis embryos and resulted in cyclopia in D. rerio. 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.