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Biochem Biophys Res Commun
2021 Jul 23;563:31-39. doi: 10.1016/j.bbrc.2021.05.043.
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A catenin of the plakophilin-subfamily, Pkp3, responds to canonical-Wnt pathway components and signals.
Hong JY
,
Zapata J
,
Blackburn A
,
Baumert R
,
Bae SM
,
Ji H
,
Nam HJ
,
Miller RK
,
McCrea PD
.
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Vertebrate beta-catenin plays a key role as a transducer of canonical-Wnt signals. We earlier reported that, similar to beta-catenin, the cytoplasmic signaling pool of p120-catenin-isoform1 is stabilized in response to canonical-Wnt signals. To obtain a yet broader view of the Wnt-pathway's impact upon catenin proteins, we focused upon plakophilin3 (plakophilin-3; Pkp3) as a representative of the plakophilin-catenin subfamily. Promoting tissue integrity, the plakophilins assist in linking desmosomal cadherins to intermediate filaments at desmosome junctions, and in common with other catenins they perform additional functions including in the nucleus. In this report, we test whether canonical-Wnt pathway components modulate Pkp3 protein levels. We find that in common with beta-catenin and p120-catenin-isoform1, Pkp3 is stabilized in the presence of a Wnt-ligand or a dominant-active form of the LRP6 receptor. Pkp3's levels are conversely lowered upon expressing destruction-complex components such as GSK3β and Axin, and in further likeness to beta-catenin and p120-isoform1, Pkp3 associates with GSK3beta and Axin. Finally, we note that Pkp3-catenin trans-localizes into the nucleus in response to Wnt-ligand and its exogenous expression stimulates an accepted Wnt reporter. These findings fit an expanded model where context-dependent Wnt-signals or pathway components modulate Pkp3-catenin levels. Future studies will be needed to assess potential gene regulatory, cell adhesive, or cytoskeletal effects.
Fig. 1. Pkp3-catenin responds to components of the canonical-Wnt pathway. (A) In vitro transcribed mRNA encoding Myc-tagged Pkp3-catenin (1 ng) or Myc-tagged δ-catenin (1 ng) microinjected with mRNAs encoding Wnt8 (1 ng) or β-gal (1 ng; negative control) into one-cell stage embryos. Gastrulating embryos (stages 10–11) were harvested followed by immuno-blotting of their lysates. Catenin-protein detection occurred using anti-Myc antibodies, with GAPDH serving as an internal loading control. (B) Myc-pkp3 mRNA (500 pg) was microinjected into each blastomere of one-cell stage embryos with the indicated mRNA levels of wnt8, LRP6△E1-4 or β -gal (500 pg; negative control) varying from 100 to 500 pg. Statistical significance was found when comparing β-gal (lane 2) to LRP6△E1-4 at 200 pg p = 0.01 (lane 4), and wnt8 at both 200 pg p = 0.02 (lane 7), and 500 pg p = 0.007 (lane 8). GAPDH was used as a loading control. Bars along the sides of immuno-blots refers to the observed SDS-PAGE mobility of the corresponding molecular-weight standards. Statistical significance was established using a two-tailed T-test. Myc-Pkp3 and GAPDH were visualized using antibodies directed against Myc or GAPDH. (C) HaCaT and 293T cells were seeded in six-well plates, followed by the combined siRNA mediated depletion of Axin1 and Axin2. NC denotes negative-control siRNA. Endogenous Pkp3 was monitored using the 23E3/4 monoclonal antibody. Lower panel: employing densitometry and ImageJ software, endogenous Pkp3 levels were quantitated and normalized relative to Actin loading control. (D) HaCaT and 293T cells were transfected (48 h) with siRNAs [15] to deplete LRP5 and LRP6. Endogenous Pkp3 was monitored using the 23E3/4 monoclonal antibody. Lower panel: using densitometry and ImageJ software, endogenous Pkp3 levels were quantitated and normalized to the actin loading control. Experiments were repeated at least three times with consistent results.
Fig. 2. GSK3β and Axin modulate Pkp3-catenin protein levels. (A) beta-Catenin mRNA (1 ng) was co-injected with GSK3beta (500 pg) into embryos at the one-to-two-cell stage. Gastrulae extracts (stages 11–12) were immuno-blotted with anti-Myc antibody. Actin serves as an internal loading control. (B) HaCaT cells were transfected with the indicated shRNA constructs directed against transcripts of GSK. Endogenous Pkp3 and GAPDH levels were assessed via immuno-blotting. (C) HaCaT and MDA-231 cells were incubated with LiCl for 6 h at the indicated concentrations, followed by immuno-blotting for endogenous Pkp3 (23E3/4 mA b). Endogenous Pkp3 levels are normalized to Actin in the lower panel. (D) Myc-pkp3 mRNA (500 pg) was microinjected into each blastomere of one-cell stage embryos with the indicated mRNA levels of gsk3β, axin1, or β-gal (500 pg; negative control), varying from 5 to 500 pg. Although no significance was found when compared to the negative-control β-gal (lane 2), there is a general trend of loss of Pkp3 expression upon increasing doses of negative regulators of the Wnt pathway, gsk3β or axin1. GAPDH was used as a loading control. Bars along the sides of immuno-blots refers to the observed SDS-PAGE mobility of the corresponding molecular-weight standard(s). Statistical significance was established using a two-tailed T-test.
Fig. 3. Pkp3-catenin associates with components of the canonical-Wnt destruction complex. (A) 293T cells in 100 mm dishes were transfected with Flag-Pkp3 (8 μg) and HA-GSK3beta (5 μg). The Pkp3:GSK3β association was resolved via anti-HA or anti-Flag immuno-precipitation/immuno-blot. (B) Flag-Pkp3 (2 μg) and Myc-Axin (2 μg) were co-transfected in 293T cells. Immuno-precipitates (IP) of Flag-Pkp3 were immuno-blotted [19] with anti-Flag or -Myc antibodies to detect Pkp3 or Axin. (C) Flag-Pkp3 (3 μg) and Myc-β-TrCP (3 μg) were co-transfected into HaCaT cells. IPs were performed with anti-Myc antibody to precipitate β-TrCP, followed by immuno-blotting with anti-Flag antibody. (D) Endogenous Pkp3 co-precipitates with Myc-β-TrCP from HaCaT-cell extracts.
Fig. 4. Mapping of Pkp3-catenin in relation to GSK3beta. (A) Cartoon depicting Flag-tagged deletion constructs of Pkp3-isoformA (a–d). The chart summarizes construct association with GSK3beta as well as response to GSK3beta based upon data from panels B and C and similar experiments. (B) Deletion constructs of Flag-tagged Pkp3-catenin were co-transfected with HA-GSK3β in HaCaT cells. Immuno-precipitation (IP) of HA-GSK3beta was followed by anti-Flag immuno-blotting to resolve co-associated Pkp3. (C) Flag-tagged Pkp3 constructs were expressed in A431 cells, and their response monitored upon treatment with lithium chloride. The SDS-PAGE mobilities for constructs (a–c) are shown in Panel B, while (d) not shown in Panel B, migrates at ∼65 kDa). (D) Nuclear localization of Flag-tagged Pkp3 in HEK293 cells following treatment with Wnt3a peptide (50 μg/ml) for 24 h. The nuclear:cytosolic ratio of Pkp3-catenin notably increased relative to that of untreated controls (1.373 ± 0.041 versus 0.5778 ± 0.020; n = 30 cells/condition; ∗∗∗ = p < 0.0001). (E) Exogenous expression of Pkp3 increases activity of the TOP-/FOP-flash canonical-Wnt reporter system. Relative to controls, HEK293 cells expressing Flag-tagged Pkp3 produced a 6.67-fold (±1.62) increase in luciferase activity (p = 0.0006). Significance was determined using a One-way ANOVA. Error bars represent SEM.
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