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. 2009 Jul 14;19(13):1112-7.
doi: 10.1016/j.cub.2009.05.049. Epub 2009 Jun 18.

Phosphorylation of the tumor suppressor fat is regulated by its ligand Dachsous and the kinase discs overgrown

Richelle Sopko  1 , Elizabeth Silva, Lesley Clayton, Laura Gardano, Miriam Barrios-Rodiles, Jeff Wrana, Xaralabos Varelas, Natalia I Arbouzova, Sanjeev Shaw, Sakura Saburi, Hitoshi Matakatsu, Seth Blair, Helen McNeill
Affiliations

Phosphorylation of the tumor suppressor fat is regulated by its ligand Dachsous and the kinase discs overgrown

Richelle Sopko et al. Curr Biol. .

Abstract

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

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Figures

Figure 1
Figure 1. Fat processing generates a 110kDa form, which displays altered electrophoretic mobility in dco mutants
A) Larval extracts from yw, fatalb, fatfd, fatfd/fatG-rv, fatfd/fatx13 were subjected to SDS-PAGE and analyzed by Western blotting with α-Fat (IC) antibody. 560kDa and 110kDa bands (indicated by arrows) recognized in yw (wildtype) extracts by the α-Fat (IC) antibody are absent from all fat extracts. B) In vivo pulse chase analysis of FatHA. Larvae bearing a C terminally HA-tagged UAS-Fat transgene and hs-gal4 driver (hs-Gal4; UAS-FatHA) were subjected to a brief heat shock at 37°C and then returned to 18°C. Western blotting with α-HA of larval extracts from these animals at the indicated time points following heat shock revealed that Fat is first produced as a 560kDa protein (Fat560; top arrow). A band migrating at 110kDa (Fat110; lower arrow) however appears 4–6 hours post heat shock, and this band predominates after 10hrs (further emphasized by quantitation of the ratio of Fat560 to Fat110 below blot). Immunoblotting with α-lamin (lower panel) serves as a loading control. A 70kDa band (indicated with an asterisk) is also inconsistently detected with α-Fat (IC) antibody. C) Larval extracts from yw, fatfd, and hs-Gal4;UAS-fatHA animals were subjected to SDS-PAGE and analyzed by Western blotting with α-Fat (N) antibody. This antibody detected a 450kDa band (Fat450; top arrow, right panel), while α-HA recognized a 560kDa band (Fat560; top arrow, left panel) and a 110kDa band (Fat110; bottom arrow, left panel). A non-specific band in all extracts (*ns) is also detected with α-Fat (N). D) Schematic of Fat protein generated from expression of the UAS-fatHA transgene. The transmembrane domain (TM), C-terminal HA tag, and fragments generated upon cleavage of full length Fat (Fat560) are indicated. E) Extracts from Drosophila S2 cells expressing HA-tagged full length Fat (lane 3), the Fat intracellular domain (ICD, lane 5), or versions of Fat encompassing both the transmembrane and intracellular domains (FatB and FatΔECD which vary in their extracellular sequence, lanes 4 and 6 respectively) were subjected to SDS-PAGE and analyzed by Western blotting with α-HA antibody. Expression of full-length Fat-HA generates Fat110, as well as Fat560 (not shown on this portion of the gel) while Fat ICD migrates at ~70kDa and FatB and FatΔECD at ~110kDa, suggesting Fat110 includes the transmembrane domain. F) Lysates from Drosophila S2 cells expressing FatΔECD with or without HA-tagged Dco were divided. One half was treated with lambda phosphatase and the other mock treated. Samples were subjected to SDS-PAGE and Western blotting using α-HA or α-Fat (IC) antibodies. G) Larval extracts from yw, dco3/dcoi3-193, fatAlb/fatx13, dcoK38R expressing, dco and fatHA co-expressing or dcoK38R and fatHA co-expressing larvae were analyzed by immunoblotting with α-Fat (IC) antibody. The Fat110 doublet is reduced to a single band in extracts from dco3/dcoi3-193 larvae and larvae expressing dcoK38A while overexpression of dco causes a decrease in the mobility of co-overexpressed Fat, visualized as an increase in the slower migrating form of the doublet. Immunoblotting with a-lamin (lower panel) serves as a loading control.
Figure 2
Figure 2. Dco interacts with the cytoplasmic domain of Fat
Lysates from HEK293T cells expressing HA-tagged Dco together with 3FLAG-tagged variants of FatΔECD were subjected to immunoprecipitation with α-FLAG antibody and analyzed by immunoblotting with the indicated antibodies. A) Dco was detected in immunoprecipitates of full-length FatΔECD, comprised of the transmembrane and entire intracellular portion of Fat, and in immunoprecipitates from versions of FatΔECD lacking 55 and 154 C-terminal amino acids (CΔ55 and CΔ154). Versions of FatΔECD lacking more than 203 amino acids (CΔ203) failed to pull down Dco. B) While FatΔECD lacking 154 C-terminal amino acids (CΔ154) can strongly interact with Dco, deletion of 183 amino acids (CΔ183) weakens the Dco interaction, and deletion of 203 amino acids (CΔ203) completely abolishes the interaction. C) Internal deletions of 39 amino acids (Δ513–552) from the cytoplasmic portion of FatΔECD eliminates interaction with Dco, while removal of 19 of these internal residues (Δ533–552) weakens but does not abolish the interaction. Mutation of conserved residues within this region (AAGG or KAEG) does not block Fat interaction with Dco.
Figure 3
Figure 3. CKIε phosphorylates Fat and binding to Fat is not affected by the Dco3 mutation
A) Recombinant GST-Fat fusion proteins (100ng) were incubated with human CKIε (50ng) and γ-32P-ATP. Casein was included as a positive control (lane 1). Phosphorylation of Fat fragments was analyzed by SDS-PAGE and autoradiography. The position of migration of input proteins is indicated with asterisks. Auto-phosphorylated CKIε migrates at ~55kDa. All fragments except Fat5 and GST (lanes 7 and 10 respectively, indicated with blue asterisks) showed robust phosphorylation, indicating multiple sites are phosphorylated by CKIε. B) Both Dco and Dco3 interact with Fat. Lysates from Drosophila S2 cells expressing HA-tagged Dco or Dco3 together with FLAG-tagged FatΔECD were subjected to immunoprecipitation with anti-HA antibody and immunoprecipitates analyzed by immunoblotting with the indicated antibodies.
Figure 4
Figure 4. Fat110 is altered in ds mutants, and Fat can form dimers or oligomers
A) Extracts from ey,GMR-gal4 UAS atrophin RNAi (lane 1), yw (lane 2), fat (lane 3), dco (lane 4), ds (lane 5) mutant and tub-gal4 UAS-ds (lane 6), tub-gal4 UAS-fj (lane 7), and tub-gal4 UAS-fj UAS-ds (lane 8) larvae were subjected to SDS-PAGE and analyzed by immunoblotting with α-Fat (IC) antibody. Fat110 mobility is altered in dco and ds mutants (lanes 4 and 5). B) Lysates from HEK293T cells expressing HA-tagged FatB and/or 3FLAG-tagged FatΔECD together with Dco-HA or Dco3-HA were subjected to immunoprecipitation with α-FLAG antibody and analyzed by immunoblotting with the indicated antibodies. FatB was detected in immunoprecipitates of FatΔECD, along with Dco or Dco3, when co-expressed.
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References

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