Pfister AS et al. (2012), Amer2 protein is a novel negative regulator of ...

XB-ART-44494

J Biol Chem 2012 Jan 13;2873:1734-41. doi: 10.1074/jbc.M111.308650.

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Amer2 protein is a novel negative regulator of Wnt/β-catenin signaling involved in neuroectodermal patterning.

Pfister AS , Tanneberger K , Schambony A , Behrens J .

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Wnt/β-catenin signaling is negatively controlled by the adenomatous polyposis coli (APC) tumor suppressor, which induces proteasomal degradation of β-catenin as part of the β-catenin destruction complex. Amer2 (APC membrane recruitment 2; FAM123A) is a direct interaction partner of APC, related to the tumor suppressor Amer1/WTX, but its function in Wnt signaling is not known. Here, we show that Amer2 recruits APC to the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate lipids via lysine-rich motifs and that APC links β-catenin and the destruction complex components axin and conductin to Amer2. Knockdown of Amer2 increased Wnt target gene expression and reporter activity in cell lines, and overexpression reduced reporter activity, which required membrane association of Amer2. In Xenopus embryos, Amer2 is expressed mainly in the dorsal neuroectoderm and neural tissues. Down-regulation of Amer2 by specific morpholino oligonucleotides altered neuroectodermal patterning, which could be rescued by expression of a dominant-negative mutant of Lef1 that interferes with β-catenin-dependent transcription. Our data characterize Amer2 for the first time as a negative regulator of Wnt signaling both in cell lines and in vivo and define Amer proteins as a novel family of Wnt pathway regulators.

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Species referenced: Xenopus laevis
Genes referenced: amer2 axin2l ctnnb1 egr2 en2 lef1 nodal3.1 rax sox2

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	Figure S4. Amer2 affects neuroectodermal patterning. A, The xAmer2-MO sequence specifically targets xAmer2. Embryos were injected in both blastomeres at the 2-cell-stage with 0.8 pmol of xAmer2-MO or control-MO along with either 100 pg RNA of the Amer2 pCS-MOsite-EGFP construct or pCS-EGFP alone. The Amer2-MOsite construct contains the xAmer2 sequence targeted by the Amer2-MO fused to EGFP. Injected embryos and uninjected controls were analyzed by western blotting at NF stage 12.5 as indicated. B, Depletion of xAmer2 by Amer2-MO injection affects anterior-posterior patterning of the neuroectoderm as shown by expression of En-2 and krox-20. Embryos were injected in one anterior-dorsal blastomere at the 8-cell-stage with 100pg LacZ DNA plus 0.2 or 0.4 pmol of Amer2-MO or control-MO. For rescue experiments 500 pg of LefδBD RNA were co-injected as indicated below the panels. Embryos were stained for LacZ to label the injected side, probed by in situ hybridization for expression of En-2 and krox-20 at NF stage 20 and scored for reduced or expanded expression of the markers on the injected side as compared to the uninjected side of the same embryo. Images on the top show embryos representative for observed phenotypes, injected sides are oriented to the right in all images. Images on the top show embryos representative for observed phenotypes. The colored insets represent phenotypes as following: blue=enhanced/posteriorly shifted markers at injected side, green=equal/normal expression of markers, yellow=markers reduced/anteriorly shifted at injected side, orange=markers lost at injected side, red=markers lost at injected and uninjected side of the embryo. The graph shows the statistics of at least three independent experiments. Numbers below the graph represent analyzed embryos. C, Depletion of xAmer2 by Amer2-MO injection does not affect neural induction as revealed by Sox2. Whole mount in situ hybridization with the pan-neural marker Sox2 at NF stage 20 as indicated. Embryos were injected in both dorsal blastomeres at the 4-cell-stage with 0.8 pmol of xAmer2-MO or control-MO and 100 pg LacZ DNA.
	FIGURE 4. xAmer2 negatively regulates Wnt signaling in vivo. A, depletion of xAmer2 by xAmer2 MO (cf. supplemental Fig. 4A) increased expression of the Wnt target gene Xnr3. Xenopus embryos were injected in both dorsal blastomeres at the four-cell stage with 0.4 pmol of xAmer2 MO or control MO or with 100 pg of β-catenin RNA as a positive control, and Xnr3 mRNA levels were determined by RT-PCR at NF stage 10.5. Ornithine decarboxylase (ODC) RT-PCR was used for normalization. Numbers between the panels represent the levels of Xnr3 normalized to ornithine decarboxylase as determined by densitometry. B, depletion of xAmer2 by xAmer2 MO affected anterior-posterior patterning of the neuroectoderm, consistent with a negative function in Wnt signaling. Embryos were injected in one anterior-dorsal blastomere at the eight-cell stage with 100 pg of lacZ DNA plus 0.2 or 0.4 pmol of Amer2 MO or control MO without or with 500 pg of LefδBD RNA as indicated. Embryos were stained for lacZ to label the injected side, probed by in situ hybridization for expression of Rx1 and En-2 at NF stage 20, and scored for reduced or expanded expression of the markers on the injected side compared with the uninjected side of the same embryo. Images show embryos representative of the observed phenotypes. Injected sides are oriented to the right in all images. The colored insets represent phenotypes as follows: blue, enhanced Rx1 and En-2; green, equal/normal expression of markers; yellow, markers reduced/anteriorly shifted at the injected side; orange, markers lost at the injected side; red, markers lost at both the injected and uninjected sides of the embryo. The graph shows the statistics of at least three independent experiments. Numbers below the graph represent analyzed embryos.
	FIGURE 3. Expression pattern of xAmer2 in Xenopus embryos by whole-mount in situ hybridization. A, xAmer2 expression in Xenopus embryos at NF stage 10.5 in the animal hemisphere (lateral view, animal pole to the top). B, xAmer2 expression in the anterior neural plate at NF stage 16 (dorsal view, anterior to the top). C, xAmer2 expression in the central nervous system, otic vesicle (o), eye (e), and cranial ganglia and nerves (arrowheads; V, trigeminal; VII, facial; IX, glossopharyngeal; X, vagal) at NF stage 35 (lateral view, anterior to the left). C′, cross-section through the head region of an NF stage 35 embryo (anterior to the left). xAmer2 expression was clearly seen in the forebrain (fb), midbrain (mb), and hindbrain (hb) but not the boundaries between these domains (arrowheads).

References [+] :

Behrens, Functional interaction of beta-catenin with the transcription factor LEF-1. 1996, Pubmed, Xenbase