Creyghton MP et al. (2006), PR130 is a modulator of the Wnt-signaling casca...

XB-ART-534

Proc Natl Acad Sci U S A 2006 Apr 04;10314:5397-402. doi: 10.1073/pnas.0507237103.

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PR130 is a modulator of the Wnt-signaling cascade that counters repression of the antagonist Naked cuticle.

Creyghton MP , Roël G , Eichhorn PJ , Vredeveld LC , Destrée O , Bernards R .

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The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. We have recently shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. In the present study, we show that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. We find that PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. Our data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit.

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Species referenced: Xenopus
Genes referenced: cep43 dvl1 dvl2 myod1 nkd1 odc1 ppp2ca ppp2r3a ptpa
???displayArticle.antibodies??? dvl1 Ab1 dvl2 Ab4 dvl3 Ab3
???displayArticle.morpholinos??? ppp2r3a MO5 ppp2r3a MO6 ppp2r3a MO7

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	Fig. 1. PR130 can interact with Nkd. (A) PR72 has an N-terminal stretch of 44 specific amino acids (light speckled), whereas PR130 has a 665-N-terminal-specific stretch (dark speckled). (B) IP of PR130 and Nkd. Extracts from HEK 293 ectopically expressing flag- tagged hNkd1, HA-tagged hPR130, or both were subjected to co-IP with anti-flag and anti-HA antibodies as indicated. Flag-antibodies did not precipitate HA-PR130 (lane 1), and the HA-antibody did not precipitate flag-hNkd1 (lane 3); when coexpressed, both were coprecipitated with either anti-HA or ant-flag (lanes 2 and 4). (Lower) Shown is 10% of the input in either lane. (C) IP of PR130 and Nkd containing the PP2A holoenzyme. Extracts from HEK 293 cells expressing flag-Nkd1, HA-PP2Ac, and HA-PR65 (input, Left) were cotransfected with either HA- PR72 (lane 2) or HA-PR130 (lane 3). IP anti- flag (Right) pulled down flag-Nkd1 (lane 1). PP2Ac and PR65 were pulled down only in the presence of PR72 (lane 2) or PR130 (lane 3) indicated with arrowheads. (D) IP of endogenous PR130 using an anti-Nkd antibody able to precipitate flag-Nkd1 (Left). PI, pre- immune serum. (Right) IP on U2OS lysate using PI, anti-Nkd, or anti-PR130 antibody. PR130 is shown by arrowhead.
	Fig. 2. PR130 is an activator of the classical Wnt signaling pathway. TCF containing Top-Glow or Fop- Glow (containing mutated TCF sites) luciferase reporter, CMV Renilla luciferase, and Wnt-1 was cotransfected in HEK 293 cells (A) with or without HA-PR72 or HA-PR130 as indicated (C) or with pS-PR72 or pRS-PR130. The bars represent relative luciferase activity compared with the first bar in the graph. (B Lower) Western blot showing extracts from HEK 293 cells coexpressing HA-PR130 (lanes 2–4) and GFP (all lanes) as a loading control. pRS-PR130.1 (lane 3), pRS- PR130.2 (lane 4), or pRS (lanes 1 and 2) was cotransfected. (B Upper) QRT-PCR showing relative amount of mRNA levels of endogenous PR130 in the presence of indicated PR130 targeting pRetroSuper vectors or a control vector. (D) Top-Glow reporter assay as described for Fig. 2 A and C with either pS-PR72 or CMV HA-PR130 or a combination of the two as indicated. (E) IP of flag-Nkd pulling down dishevelled in the presence (lane 4) or absence (lane 3) of ectopic PR130. Lanes 1 and 2 show the corresponding inputs.
	Fig. 3. PR130 depletion affects somite organization and tail development. (A) RT- PCR analysis of X. tropicalis total RNA from different developmental stages. Xpr130 is 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 loading control. (A Lower ) Whole-mount ISH for Xpr130 in embryos showing predominant staining of somites from the earliest stage detectable. (B) ISH of tailbud stage embryo with an Xnkd-specific probe. (C) Control embryo shows organized somites in a chevron-like shape as visualized by whole-mount ISH by using XmyoD as a marker (Left). PR130-depleted em- bryos lack chevron-like shaped somites (Right). Arrowheads show somitic borders. (D) Similar to B but without XmyoD. Shown are embryos injected with control MO (CoMO) (Left), PR130 MO (Center), and PR130 MO plus hPR130 RNA (Right). (Lower) Sagittal sections of the somites.
	Fig. 4. PR130 depletion enhances the inhibitory function of Nkd on the Wnt-signaling cascade. (A) Embryos shown as representative for C injected as indicated. (B) PR130 depletion inhibits the ectopic Wnt signal but not in the absence of Nkd. The effect is also rescued by add-back of ectopic hPR130. (C) Nkd antagonizes an ectopic Wnt signal but not in the presence of ectopic PR130. (D) Loss of PR72 abrogates Nkd- mediated inhibition of the Wnt signal. (E) In contrast, injection of suboptimal amounts of Nkd RNA demonstrates that loss of PR130 enhances the antagonizing function of Nkd.

References [+] :

Agathon, The molecular nature of the zebrafish tail organizer. 2003, Pubmed