Protein O-GlcNAcylation is required for fibroblast growth factor signaling in Drosophila

Abstract

Glycosylation is essential for growth factor signaling through N-glycosylation of ligands and receptors and the biosynthesis of proteoglycans as co-receptors. Here, we show that protein O-GlcNAcylation is crucial for fibroblast growth factor (FGF) signaling in Drosophila. We found that nesthocker (nst) encodes a phosphoacetylglucosamine mutase and that nst mutant embryos exhibited low amounts of intracellular uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc), which disrupted protein O-GlcNAcylation. Nst was required for mitogen-activated protein kinase (MAPK) signaling downstream of FGF but not MAPK signaling activated by epidermal growth factor. nst was dispensable for the function of the FGF ligands and the FGF receptor's extracellular domain but was essential in the signal-receiving cells downstream of the FGF receptor. We identified the adaptor protein Downstream of FGF receptor (Dof), which interacts with the FGF receptor, as the relevant target for O-GlcNAcylation in the FGF pathway, suggesting that protein O-GlcNAcylation of the activated receptor complex is essential for FGF signal transduction.

Fig. 1

nst mutant phenotype and genetic interaction with htl. (A) Cross sections of embryos of the indicated genotypes at the extended germ band stage (stage 9). Antibodies recognizing Twi (black) mark mesoderm cell nuclei. WT, wild type. (B) Trachea of stage 15 embryos of the indicated genotypes stained with antibodies recognizing Verm; the dorsal trunk (DT) is marked by arrows. (C) Dorsal mesoderm cells of stage 11 embryos of the indicated genotypes stained with antibodies against Eve (arrowheads). (D) Quantification of Eve clusters for genotypes depicted in (C). Bars with the same symbol show values that are significantly different (P < 0.01; n = 35 embryos; table S1). (E) Stage 8 embryos stained with antibodies against Twi (green) and antibodies against dpERK (red); merged images to the right. Top row, WT embryos; bottom, nst^MZ embryos. Arrows indicate Htl-dependent MAPK activation in mesoderm. Arrowheads indicate EGFR-dependent MAPK activation in nst^MZ embryos (see also fig. S2). htl: htl^AB42 zygotic homozygote; nst^MZ: nst¹⁶⁹²³ maternal zygotic mutants; nst^Z: nst¹⁶⁹²³ zygotic homozygotes; htl^YY: htl^YY262 zygotic homozygotes; htl^YY, nst^Z: htl^YY262, nst¹⁶⁹²³ zygotic double mutants; nst^Df: Df(3L)4486 homozygotes.

Fig. 2

UDP-HexNAc nucleotide biosynthesis and glycosylation in nst mutants. (A) Relative amount of UDP-HexNAc in embryo (stages 9 to 11) extracts of the indicated genotypes (n = 2 experiments at 40 to 45 embryos per sample; table S3). (B) Relative amount of UDP-HexNAc in mmy and nst^MZ mutants (n = 2 experiments; table S3). (C) Eve staining of dorsal mesoderm in stage 11 embryos of the indicated genotypes. (D) Verm staining of the trachea in stage 15 embryos of the indicated genotypes. (E) Wg signaling was detected in stage 10 embryos of the indicated genotypes with antibodies recognizing Wg (left) or En (right). (F) GPI-anchored GFP (gpi-GFP) in the indicated nst embryos. Scale bar, 5 μm. (G) Eastern blot of protein lysates of stage 9 to 11 WT and nst^MZ embryos with concanavalin A (Con A) or jacalin (Jac). Where indicated, lectins were preincubated with either 0.5 M α-methylmannopyranoside (MMP) for Con A or 0.8 M galactose (Gal) for Jac. A nonspecific band reactive with ExtrAvidin HRP (2°) was used as loading control (arrow). Black lines indicate separation of differently treated nitrocellulose filters originating from the same SDS–polyacrylamide gel electrophoresis (SDS-PAGE). (H) Western blot of protein lysates from stage 9 to 11 WT or nst^MZ embryos probed with antibodies recognizing O-GlcNAc (anti–O-GlcNAc monoclonal antibody RL-2). Where indicated, antibodies were preincubated with 0.5 M GlcNAc. α-Tubulin (αtub) served as loading control. Data in (G) and (H) are representative of three experiments. nst^Z, zygotic nst¹⁶⁹²³ homozygotes; nst^MZ hs>CG10627, nst¹⁶⁹²³ maternal zygotic mutants expressing the rescue construct phsp70::CG10627; mmy^Z, zygotic mmy^IK63 homozygotes; wg, wg^CE7 homozygotes; nst^M, nst¹⁶⁹²³ maternal mutant with WT nst gene expressed from paternal chromosome. Molecular masses are indicated in kilodaltons.

Fig. 3

Epistatic relationship between nst and htl. (A) Expression of UAS::Nst rescues nst^MZ mutant cells autonomously. Embryos of the indicated genotypes were stained with antibodies recognizing either Eve (stage 11, top row) or Verm (stage 15, bottom row). (B) Quantification of Eve-positive hemisegments in embryos of indicated genotypes (n = 35 or 50 embryos; table S4). (C) Percentage of embryos with either intact dorsal trunk (DT) or the presence (Verm+) or absence (Verm−) of Verm staining (n = 100 embryos) for the indicated genotypes. The mutase-dead Nst[S68A] mutant served as a control. (D) Expression of constitutively active Htl (λHtl) in the mesoderm of nst mutant embryos stained with antibodies recognizing Eve. Arrows mark enlarged Eve cell clusters caused by expression of λHtl in maternal nst (nst^M) mutants, which are lacking in the nst^MZ mutants expressing λHtl. (E) Quantification of the Eve-positive hemisegments of embryos expressing λHtl in nst^MZ mutants (n = 35 embryos; see table S5). (F) MAPK activation in the mesoderm was detected with antibodies against dpERK (red) in stage 8 embryos with λHtl in maternal nst^M mutants (top row) and nst^MZ mutants expressing λHtl (bottom row). The mesoderm nuclei were stained with antibodies recognizing Twi (green) and the presence of the paternal balancer chromosome by anti-βGal (green). twi>>Nst: twi::Gal4,UAS::Nst; btl>>Nst: btl::Gal4,UAS::Nst; twi>>NstS68A: twi::Gal4,UAS::Nst[S86A]; btl>>NstS68A: btl::Gal4, UAS:: Nst[S86A]; twi>>λHtl: twi::Gal4,UAS::λHtl.

Fig. 4

FGF signaling in nst embryos upon altered protein O-GlcNAcylation. (A) Embryos of the indicated genotypes were injected at syncytial stages with GlcNAcstatin C (GC) or DMSO (DM) as a control, fixed at stage 11, and stained with antibodies against Eve. Eve-positive hemisegments were quantified for the indicated genotypes and injection protocols (n = 10 to 19 embryos; table S6). (B) Quantification of Eve-positive hemisegments in nst^MZ embryos overexpressing UAS::OGT or UAS::OGA in the mesoderm (n = 35 embryos; table S7). (C) Activation of MAPK in response to the expression of chimeric receptor tyrosine kinases in the mesoderm of nst^MZ embryos was detected in fixed stage 8 embryos immunolabeled with Twi (green) and dpERK (red) antibodies. Arrows mark nst-independent activation of MAPK in the ectoderm, and arrowheads mark MAPK activation in the mesoderm (positive for Twi). btl-htl: UAS::btl-htl, fusion of the extracellular domain of the Btl FGFR and the intracellular domain of Htl; htl-tor: UAS::htl-tor, fusion of the extracellular domain of Htl and the cytoplasmic domain of Tor; twi>>OGT: twi::Gal4,UAS::OGT; twi>>OGA: twi::Gal4,UAS::OGA.

Fig. 5

OGT-dependent glycosylation of Dof. Lysates of FLAG-Dof–expressing S2 cells were subjected to sWGA affinity binding (IP: sWGA) followed by immunoblotting with antibodies recognizing the FLAG epitope (WB: anti-FLAG) (A to D) or were subjected to immunoprecipitation using antibody against FLAG (IP: anti-FLAG) followed by Eastern blot using sWGA (EB: sWGA) (E). Input lanes show lysates of cells with and without transfection of FLAG-Dof and arrowheads mark full-length FLAG-Dof. (A) Precipitation of FLAG-Dof from S2 cell lysate by sWGA beads. GlcNAc: control of FLAG-Dof binding to sWGA by preabsorbing sWGA beads with 250 mM GlcNAc. (B) Effect of RNAi (dsRNA) against OGT on FLAG-Dof binding to sWGA. Cells treated with dsRNA directed against OGT or GFP (control). (C) Coexpression of FLAG-Dof with OGT-HA. Global protein O-GlcNAcylation in S2 cells upon expression of OGT-HA is depicted in fig. S5B. (D) Effect of GlcNAcstatin C on FLAG-Dof O-GlcNAcylation. S2 cells overexpressing FLAG-Dof were treated with 1 μM GlcNAcstatin C or DMSO as control. Global protein O-GlcNAcylation in S2 cells after treatment with GlcNAcstatin C is depicted in fig. S5C. (E) Anti-FLAG immunoprecipitates from lysates of FLAG-Dof–expressing S2 cells analyzed by Eastern blotting (EB) using sWGA. Molecular masses are indicated in kilodaltons. Data are representative of three separate experiments.

Fig. 6

Model for O-GlcNAcylation in FGF signaling. The model shows the requirement of protein O-GlcNAcylation for Htl-dependent mesoderm spreading mediated by the Pebble (Pbl)–Rac pathway and mesoderm differentiation mediated by MAPK activation. The HBP converts glucose (blue circle) into UDP-GlcNAc (blue squares). With normal amounts of UDP-GlcNAc, OGT transfers O-GlcNAc to the adaptor protein Dof. O-GlcNAcylated Dof may be phosphorylated (red circles) upon activation of Htl by FGF8-like ligands and recruits effector proteins, such as the phosphatase Corkscrew (Csw), to trigger FGF-dependent MAPK activation and cell shape changes. This model does not exclude the possibility that OGT might also O-GlcNAcylate yet unknown protein(s) (X), which may be indirectly required for FGF signaling (transparent brown arrow).