Hong JY et al. (2012), Down's-syndrome-related kinase Dyrk1A modulates...

XB-ART-44923

J Cell Sci 2012 Feb 01;125Pt 3:561-9. doi: 10.1242/jcs.086173.

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Down's-syndrome-related kinase Dyrk1A modulates the p120-catenin-Kaiso trajectory of the Wnt signaling pathway.

Hong JY , Park JI , Lee M , Muñoz WA , Miller RK , Ji H , Gu D , Ezan J , Sokol SY , McCrea PD .

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The Wnt pathways contribute to many processes in cancer and development, with β-catenin being a key canonical component. p120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin-Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin-Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.

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Species referenced: Xenopus laevis
Genes referenced: actl6a arvcf cdh3 ctnnd1 dyrk1a.2 myc odc1 sia1 tbx2 wnt11 zbtb33
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	Fig. 1. Dyrk1A modulates p120-catenin levels and the intracellular localization of Kaiso. (A) Either Dyrk1A1 or -1A2 was co-injected with HA-tagged p120-catenin (0.25 ng) into Xenopus embryos at the one-cell stage, followed by immunoblotting for HA120-catenin (12CA5) or actin (negative control). (B) HA120-catenin (0.25 ng) was microinjected into Xenopus embryos with wild-type or kinase-dead (KD-K180R) Dyrk1A. Embryos were harvested as early gastrulas (stage 101) and immunoblotted for HA120-catenin, with actin serving as an internal loading control. (C) Increasing doses of HAyrk1A were transfected into 293T cells, and endogenous p120-catenin, β-catenin and GAPDH monitored by immunoblotting. (D) HEK293T cells were transfected with one or both Dyrk1A siRNAs (50 pmol), as indicated, for 48 hours. Endogenous p120-catenin, β-catenin, Dyrk1A and GAPDH levels were monitored by immunoblotting (pp120, BD Transduction; Dyrk1A, ab71464, Abcam). Representative outcomes of experiments repeated three or more times with consistent results are shown.
	Fig. 2. Association of Dyrk1A with p120-catenin. (A) Embryos were injected with HAyrk1A and harvested as early gastrulas (stage 10.5). Lysates were immunoprecipitated for HAyrk1A, and the association with endogenous p120-catenin resolved by immunoblotting. β-catenin and C-cadherin served negative controls. (B) HAyrk1A (0.5 ng) was co-injected with Myc-tagged β-catenin, C-cadherin, p120-catenin, ARVCF or δ-catenin (1 ng) into both blastomeres of two-cell embryos. HAyrk1A immunoprecipitates were immunoblotted with anti-Myc antibody to detect co-associating proteins. (C) Endogenous Dyrk1A was immunoprecipitated from HEK293T cells, and endogenous p120-catenin monitored using anti-p120-catenin antibody (6H11, Santa Cruz). (D) HAyrk1A was co-injected with Myc120-catenin, Myc#946;-catenin (β) or Mycaiso into early Xenopus embryos. Embryos lysates were immunoprecipitated for HAyrk1A. Anti-Myc or -HA immunoblotting was used to test association (versus none) of β-catenin, p120-catenin or Kaiso. (E) Nuclear (N) and cytoplasmic (C) extracts were made from HeLa cells, followed by Dyrk1A immunoprecipitation and immunoblotting using anti-p120-catenin or Dyrk1A antibodies. WCL, whole cell lysate. (F) Myc120-catenin, Mycaiso and HAyrk1A were in vitro transcribed as described previously (Hong et al., 2010). Differing combinations of the proteins were mixed as indicated, and immunoprecipitated for Dyrk1A (using anti-HA). Co-precipitating p120-catenin and Kaiso were then monitored (using anti-Myc). (G) Depiction of Myc120-catenin deletion constructs (a). Panels to the right show immunoblotting of the Myc120-catenin constructs (a; 0.5 ng mRNA), co-precipitating (versus not) with co-injected HAyrk1A1 (0.5 ng mRNA).
	Fig. 3. Dyrk1A modulates p120-cateninaiso-dependent gene expression. (A) The indicated amounts of Dyrk1A morpholino were injected into embryos at the 1-cell stage. Gastrula cDNA was assayed by real-time RT-PCR for Wnt11 and Siamois transcript levels. Embryos were harvested at stage 90, which proved best for tests involving Siamois expression. For Wnt 11, embryos were collected mainly at stage 10.52. Gene expression levels were normalized to ODC (ornithine decarboxylase). (B) Dyrk1A (5 pg) was injected into embryos at the 1-cell stage. Wnt11 and Siamois transcript levels were analyzed by real-time PCR as described in A. (C) Gastrulation (blastopore closure) failures followed the expression of Kaiso (0.5 ng), whereas the co-expression of Dyrk1A (5 pg or 10 pg), partially rescued the effects of Kaiso. Under similar experimental conditions, gastrula cDNA was assayed by real-time RT-PCR for wnt11 and siamois transcript levels, normalized to ODC (right panel). (D) Gastrulation failures resulting from Dyrk1A expression are rescued by co-expression of Kaiso. The coexpression of a third component, p120-catenin, once again results in increased gastrulation failures, presumably in part by relieving the repression and/or rescue conferred by Kaiso. (E) Dyrk1A (5 pg) or β-galactosidase (negative control) were co-injected or not with p120-catenin morpholino (20 ng). Gastrula cDNA was analyzed by real-time PCR for expression of wnt11 and siamois and normalized to ODC. (F) Standard or Dyrk1A morpholinos (10 ng) were microinjected into Xenopus embryos. To test for rescue of morpholino-directed Dyrk1A depletion, p120-catenin wild type, p120-catenin T47D or p120-catenin were co-injected. The gene expression levels of Wnt11 were monitored using real-time PCR.
	Fig. 4. Phosphorylation-dependent Dyrk1A in embryonic development. (A) Duplicate axis formation following the expression of a sub-maximal dose of β-catenin, is enhanced upon the co-expression of the indicated doses of Dyrk1A, as evaluated in tailbud embryos (stage 279). Kinase-dead (KD) Dyrk1A serves as a negative control. (B) Cross-species p120-catenin sequence alignment of the conserved predicted Dyrk1A phosphorylation-site region (box). (C) Expression of HA120-catenin wild type, versus the phosphomimic mutant (T47D), was detected by immunoblotting of Xenopus embryo lysates. (D) p120-catenin wild type (WT) or the p120-catenin point-mutant (T47D), was injected into Xenopus embryos at the one-cell stage, followed later by cDNA isolation and real-time PCR, to monitor increased wnt11 and siamois transcript levels. (E) Gastrulation failures were more severe in embryos expressing p120-cateninT47D relative to p120-catenin WT. (F) HA120-catenin WT or p120-cateninT47D was expressed in HeLa cells. Cells transfected with each construct were treated with cyclohexamide (CHX) for the times indicated. Each HA-tagged construct was detected by immunoblotting of the corresponding cell extracts (right panel), followed by densitometer quantification of the band intensities (left panel). These data were collected from two independent experiments. (G) Full-length p120-catenin (p120 FL), or an N-terminal-deleted construct of p120-catenin (delta-N), were transfected into HEK293T cells and increasing amounts of Dyrk1A co-transfected. The levels of p120-catenin FL and delta-N protein were monitored (using an anti-Myc antibody). β-tubulin3 and GAPDH served as negative controls.
	Fig. S1. Dyrk1A relocalizes Kaiso from the nucleus to the cytoplasm by increasing p120�s level in the nucleus. (A) HeLa cells were grown on glass coverslips, and transiently transfected with Myc-Kaiso or Myc-Kaiso plus HA-Dyrk1A as indicated. Cells were fixed with 4% PFA for 10 min, blocked with 5% goat serum in PBS and immuno-stained with anti-Myc antibody (9E10) (B) The experiment here performed as described in (A) with Myc-p120 or Myc-p120 plus HA-Dyrk1A. Red arrows indicates increased level of p120-catenin in the nucleus.
	Fig. S2. Effect of Dyrk1A depletion upon endogenous Dyrk1A, p120-catenin, β-catenin and C-cadherin levels, and the corresponding gross effect of the depletion of Dyrk1A upon gastrulation. (A) The expression of HA tagged Dyrk1A1, 1A1 KD and 1A2, which are in vitro transcribed were tested employing Xenopus embryos. (B) Dyrk1A morpholino (40 ng) was microinjected into both blastomeres at the two-cell. Embryos were harvested at stages 10-11 for immunoblotting of Dyrk1A, with actin serving as an internal loading control. (C) The efficiency of Dyrk1A siRNA was tested in 293T cells. siRNA were transfected in 293T cells for 72 hours and cells were harvested to monitor endogenous Dyrk1A. β-catenin and GAPDH served as internal loading controls. (D) Increasing total doses of Dyrk morpholino were microinjected into both blastomeres at the 2-cell stage. Embryos were collected at stage 12 and endogenous p120-catenin and β-catenin (etc.) levels, respectively, visualized via immunoblotting. (E) Gross gastrulation effects following Dyrk1A depletion in Xenopus embryos.
	Fig. S3. Dyrk1A phosphorylation of p120 in vitro. An amino-terminal region of p120-catenin (flag-tagged wild-type or T47D mutant; amino acids 1-280) (Hong et al., 2010), or full-length Dyrk1A (HA-tagged wild-type or kinase dead), were each in vitro transcribed and translated using a reticulocyte lysates system (Promega, L4611). Kinase (Dyrk1A) and substrate (p120-catenin) were incubated for 30 minutes at 30 as indicated in kinase reaction buffer (10 mM Hepes, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM EGTA, 1 mM dithiothreitol, 5 ATP, 10 mM NaF and 1 mM Na3VO4), along with 10 i γ-32P-ATP. Samples were then immunoprecipitated for 1 hour at 4 with anti-FLAG-M2 magnetic beads (Sigma) in the presence of 0.5% NP-40 buffer (25 mM Hepes pH 7.5, 150 mM KCl, 0.5% NP-40, 1.5 mM MgCl2, 10% glycerol, 5 mM β-mercaptoethanol). The precipitates were washed 3x and the samples then subjected to SDS-PAGE and autoradiography. Anti-HA antibody (3F10) and anti-flag antibody (M2) were employed to detect Dyrk1A and p120-catenin, respectively.
	Fig. S4. (A) Kaiso expression rescues the gastrulation delays resulting from Dyrk1A expression (left panels). At the molecular level, Wnt11 and Siamois transcript levels similarly reflect the rescue effected by Kaiso (right panels). (B) To compare Dyrk1A1 and 1A2, each RNA was microinjected into Xenopus embryos, followed by real-time PCR to monitor Wnt11 transcript levels.

References [+] :

Altafaj, Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down's syndrome. 2001, Pubmed