Creyghton MP et al. (2005), PR72, a novel regulator of Wnt signaling requir...

XB-ART-2383

Genes Dev 2005 Feb 01;193:376-86. doi: 10.1101/gad.328905.

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PR72, a novel regulator of Wnt signaling required for Naked cuticle function.

Creyghton MP , Roël G , Eichhorn PJ , Hijmans EM , Maurer I , Destrée O , Bernards R .

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The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required for early segmentation. In addition, Naked cuticle is proposed to function as a switch, acting to restrict classical Wnt signaling and to activate a second Wnt signaling pathway that controls planar cell polarity during gastrulation movements in vertebrates. Little is known about the biochemical function of Naked cuticle or its regulation. Here we report that PR72, a Protein Phosphatase type 2A regulatory subunit of unknown function, interacts both physically and functionally with Naked cuticle. We show that PR72, like Naked cuticle, acts as a negative regulator of the classical Wnt signaling cascade, establishing PR72 as a novel regulator of the Wnt signaling pathway. Our data provide evidence that the inhibitory effect of Naked cuticle on Wnt signaling depends on the presence of PR72, both in mammalian cell culture and in Xenopus embryos. Moreover, PR72 is required during early embryonic development to regulate cell morphogenetic movements during body axis formation.

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Species referenced: Xenopus
Genes referenced: acvr1 apc axin1 cep43 ctnnb1 dvl2 gsk3b lef1 lgals4.2 mst1 myod1 nkd1 odc1 ppp2ca ppp2r3a rbl1
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	Figure 1. PR72 interacts with Naked cuticle. (A) A yeast two hybrid showing Î²-galactosidase staining of yeast colonies containing Gal4-coupled PR72 and transactivation domain (TA)- coupled Naked cuticle. (Lower right) Negative controls Gal4-PR72 and vector-TA or Gal4-vector and Naked-TA do not stain positive. (Left two panels) The positive control is Gal4-p107 and adenovirus E1A-TA. (B) Immune precipitation of PR72 and Naked cuticle. (Upper right) Extracts from HEK 293 cells ectopically expressing Flag-tagged hNkd1 or HA-tagged hPR72 or both were subjected to immunoprecipitation with anti-Flag and anti-HA antibodies as indicated. HA-antibodies did not precipitate Flag-Naked (first lane) and the Flag antibody did not precipitate HA-PR72 (third lane); when coexpressed both were coprecipitated with either anti-HA or ant-Flag (second and fourth lanes). The background in the upper section of the Flag-Naked IP is the Ig heavy chain running at the same height as Flag-Naked. The lower panel shows 10% of the total lysate used in each of the immunoprecipitations. (C, right two panels) Immune precipitation of Naked cuticle and the PP2Ac/PR65 core dimer in HEK 293 cells in the presence (right) or absence (left) of HA-PR72. The left two panels show 10% of the total lysate.
	Figure 2. PR72 is a repressor of the canonical Wnt pathway. (A) One-hundred nanograms of TCF containing Top-Glow or Fop-Glow (containing mutated TCF sites) luciferase reporter, 50 ng of CMV Renilla luciferase, and 5 ng of CMV-Wnt-1 was cotransfected in HEK 293 cells with or without 0.5 Î¼g of CMV HA-PR72 or CMV HA-PR55Î³ as indicated. (B) As in A, in the presence of either 100 ng of empty vector or phospho-site mutant Î²-catenin as indicated. Luciferase activities were determined. (C) Western blot showing lysates of non-Wnt-1-stimulated HEK 293 cells transfected with or without HA-PR72 (lanes 1,2) and repeated after overnight treatment with 10 mM of LiCl (lanes 3,4). Blots were probed with anti-HA (upper panel) or anti-Î²-catenin (lower panel). (D) Efficiency of the knock-down constructs was determined by cotransfection of 5 Î¼g of the shRNA vectors with a mix of 2 Î¼g of CMV HA-PR72, CMV HA-PR55Î³, and CMV-GFP in a ratio of 1:1:1, one of which was the target for suppression. Forty-eight to seventy-two hours post-transfection, lysates were subjected to Western blot analysis with either anti-HA or anti-Flag and then reprobed with anti-GFP antibodies (l.c.) to determine protein levels. (E) One-hundred nanograms of TCF containing Top-Glow or Fop-Glow (containing mutated TCF sites) luciferase reporter, 50 ng of CMV Renilla luciferase, and 5 ng of CMV-Wnt-1 was cotransfected in HEK 293 cells with 2 Î¼g of either an empty pSUPER (pS) or one containing a PR72 targeting shRNA (pS-PR72) or a PR55 targeting shRNA, as indicated.
	Figure 3. Naked requires PR72 for its function as a Wnt antagonist. (A) Efficiency of knock-down constructs was determined by cotransfection of 5 Î¼g of the shRNA vectors with a mix of 2 Î¼g of CMV Flag-hNkd1, CMV Flag-hNkd2, or CMV-GFP in a ratio of 1:1:1. Lysates were subjected to Western blot analysis with either anti-HA or anti-Flag and then reprobed with anti-GFP antibodies (l.c.) to determine protein levels. (B) One-hundred nanograms of TCF containing Top-Glow or Fop-Glow (containing mutated TCF sites) luciferase reporter, 50 ng of CMV Renilla luciferase and 5 ng of CMV-Wnt-1 was cotransfected in HEK 293 cells with or without 1 Î¼g of pRS-hNkd1 and 1 Î¼g of pRS-hNkd2 either in the presence or absence of pS-PR72. (C) In a similar experiment, 0, 0.2, or 0.5 Î¼g of CMV-hNkd1 was cotransfected with the Top-Glow reporter in the presence of 2 Î¼g of pS or pS-PR72. (D) Immunoprecipitation for Flag-tagged Naked was done on cell lysates of HEK 293 cells transfected with HA-PR65, HA-PP2Ac, and mDsh1 in the absence or presence of HA-PR72 (every second lane) or Flag-hNkd1 (last four lanes). (E) Cell lysates of HEK 293 cells containing HA-PR72 and mDsh1 with (last two lanes) or without proteasome inhibitors were subjected to Western blot analysis and probed with antibodies targeting dishevelled. PR72 levels were detected using an anti-HA reprobe. (F) Cell lysates of HEK 293 cells containing HA-PR72 with (last two lanes) or without pRs-Nkd1 were subjected to Western blot analysis and probed with antibodies targeting dishevelled.
	Figure 4. PR72 is required for cell morphogenetic movements during gastrulation. (A) RT-PCR analysis of X. tropicalis total RNA from different developmental stages. XPR72 and Xnkd are expressed maternally (stage 7) and throughout early development (stages 10, 18, 22, and 30). (-RT) RT-PCR without reverse transcriptase. ODC1 expression was used as a loading control. (Bottom panel) Whole-mount in situ hybridization for XPR72 in embryos showing predominant staining of somites. (B) Two sets of three nonoverlapping PR72 MOs (1, 4, 5 or 1, 2, 3) both show similar phenotypes (bottom two panels) when injected dorsally at the four-cell stage at equal amounts (10 ng total) compared to control MO-injected (top panel). Phenotypes include defective cell morphogenetic movements during gastrulation, defect in eye development, incomplete neural tube closure (arrows), and incomplete closure of the blastopore during late gastrulation (arrowheads). (C) Injection of PR72 MO in either the left half (middle panel) or right half (right panel) results in a bent axis (black arrow) and underdevelopment of the eye (white arrow) compared to control MO-injected embryos (left panel). (D) Coinjection of 20 pg of human PR72 RNA rescues the developmental defect (graph).
	Figure 5. PR72-depleted embryos show defective somitogenesis and resistance to the effect of hNkd1 in a second axis assay. (A) Whole-mount in situ hybridization for somite marker MyoD in embryos injected with controlMO(left panel) or equal amounts of PR72 MO (right panel). (B) Whole-mount in situ hybridization for somite marker MyoD in embryos injected with control MO (upper panel) or low amounts of PR72 MO combined with Xnkd MO (lower panel). (C) Wnt-1-induced second axis assay in Xenopus. (Upper panel) Wild-type embryos showing normal embryonic development. (Middle panels) Wnt-1 RNA injection at the ventral side results in embryos with a partial secondary body axis (asterisk). (Bottom panel) The induction of a second axis can be blocked by coinjection of hNkd RNA. (D) When XPR72 MO was coinjected, hNkd1 lost its ability to antagonize the Wnt signaling pathway. Restoring PR72 levels with hPR72 RNA restored the antagonizing function of hNkd1.
	ppp2r3a (protein phosphatase 2 regulatory subunit B'', alpha) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.

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Aberle, beta-catenin is a target for the ubiquitin-proteasome pathway. 1997, Pubmed