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. 2009 Jul 21;106(29):11989-94.
doi: 10.1073/pnas.0811540106. Epub 2009 Jul 2.

Processing and phosphorylation of the Fat receptor

Yongqiang Feng  1 , Kenneth D Irvine
Affiliations

Processing and phosphorylation of the Fat receptor

Yongqiang Feng et al. Proc Natl Acad Sci U S A. .

Abstract

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. We show here that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. Our observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance our understanding of the requirement for Dco in Fat signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Processing of Fat. Western blot analysis of Fat and Fat constructs, the approximate positions of markers, and inferred identity of Fat bands, are indicated. (A) Lysates of wild-type and fatG-rv wing discs. (B) Lysates of S2 cells expressing V5:Fat or Fat:FVH. V5 and Fat were detected simultaneously by immunofluorescense, and are shown together (Left) and separately (Center and Right). (C) Lysates of wild-type, fatG-rv, and fatG-rv/fat8 attB-P[acmanV5:fat+] wing discs. (D) Lysates of S2 cells expressing Fat:FVH or truncated isoforms. (E) Left 2 gels show lysates of S2 cells expressing V5:Fat or Fat:FVH whereas the right 2 gels show material immunoprecipitated after anti-V5 antibodies were incubated with intact cells.
Fig. 2.
Fig. 2.
Ds-promoted phosphorylation of Fat. Western blot analyses of lysates of wing discs from ds36D/Df(2L) ds, fjd1, fjd1 ds36D/fjd1 Df(2L) ds, tub-Gal4 UAS-ds, tub-Gal4 UAS-fj, tub-Gal4 UAS-ds UAS-fj, wild-type, dco3, and ftG-rv as indicated. (A) Ft-95 mobility in different genotypes; Wts levels indicate relative Fat activity and Act (actin) is a loading control. (B) Comparison of lysates treated with CIP versus untreated lysates. (C) Lysates from wing discs of the indicated genotypes shows that dco3 reverses the promotion of Ft-95 phosphorylation by ds.
Fig. 3.
Fig. 3.
Dco-mediated phosphorylation of Fat. Western blot analysis of Fat and Fat constructs (detected using anti-FLAG). Where indicated by labelling above gels, Fat constructs were cotransfected with Dco or CKI constructs (tagged with V5 or HA epitopes), or treated with phosphatase (CIP). (A) Fat:FVH in S2 cells. (B) Fat-STI-4:FVH in S2 cells. (C) wild-type (+), dco3, and fatG-rv wing disc lysates. (D) Fat-STI:FVH and Fat-STI-4:FVH in S2 cells. CKIαKN:HA is a mutant form of CKIα that lacks kinase activity, and serves as a negative control. (E) Lysates from wild-type (+), dco3, tub-Gal4 UAS-CKIα; dco3, tub-Gal4 UAS-Dco-KD (kinase domain), dco3, tub-Gal4 UAS-CKIα, and tub-Gal4 UAS-Dco-KD wing discs as indicated.
Fig. 4.
Fig. 4.
Dominant negative activity of Dco3. (A) Dco-dependent destabilization of Per. (Upper) Western blot analysis of Per in lysates of transfected S2 cells shows that Dco and Dco3 promote Per degradation. (Lower) Western blot analysis of Dco:V5 shows that similar amounts of each isoform were expressed; GFP:V5 is a transfection and loading control. (B) Western blot analyses (anti-V5) on S2 cells cotransfected with plasmids expressing (lane 1) Dco:V5, (2) Dco3:V5, (3)Dco:V5 + Fat-STI-4:FVH, (4) Dco3:V5 + Fat-STI-4:FVH, and (5) Fat-STI-4:FVH. (Upper) Shown are blot of lysates (Input). (Lower) Shown are blots of material precipitated with anti-FLAG beads. (C) Adult wing showing that overexpression of dco3 under en-Gal4 control induces overgrowth of the posterior compartment. (D) wild-type wing. (E) Wing imaginal disc expressing of dco3 under en-Gal4. The posterior compartment (marked by GFP, green) is enlarged and WG expression (red) is broadened. (F) WG expression in a disc without dco3 expression.
Fig. 5.
Fig. 5.
dco3 specifically affects the Fat tumor suppressor pathway. (A) Wild-type fly. (B) dco3 fly rescued by overexpression of wts. (C–E) Clones of cells expressing Dachs:V5 (white); in D and E the MARCM technique was used to make these clones mutant for fat8 (D) or dco3 (E). Arrows point to clone edges with accumulation of Dachs:V5, arrowheads point to clone edges where Dachs:V5 is low. (F–H) Wings from wild-type (F), ft8/ftG-rv rescued by overexpression of Wts (G), and dco3 rescued by overexpression of Wts (H). Arrowheads point to the cross-veins, arrow points to extra vein material. cross-vein spacing is rescued for dco3, but not for fat. (I–K) Portions of adult abdomens from ft8/ftG-rv rescued by overexpression of Wts (I), dco3 rescued by overexpression of Wts (J), an animal with dco3 mutant clones, marked by y+ (dark patches). A PCP phenotype is observed in I, but not in J or K.
See this image and copyright information in PMC

References

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