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. 2021 Jul 15;32(15):1347–1358. doi: 10.1091/mbc.E20-07-0470

Tight junction protein ZO-2 modulates the nuclear accumulation of transcription factor TEAD

Helios Gallego-Gutiérrez a, Laura González-González a, Leticia Ramírez-Martínez b, Esther López-Bayghen b, Lorenza González-Mariscal a,*
Editor: Keith Mostovc
PMCID: PMC8694039  PMID: 34010016

Abstract

The presence of tight junction protein zonula occludens 2 (ZO-2) at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed whether the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knockdown Madin–Darby canine kidney cells, nuclear TEAD was diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation, and pull-down assays, TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated, and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals induces no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKCδ and ε and provide a novel role of ZO-2 as a nuclear translocator of TEAD.

INTRODUCTION

Zonula occludens 2 (ZO-2) is a tight junction (TJ) protein member of the MAGUK (membrane-associated guanylate kinase homologue) protein family that translocates to the nucleus in response to low cell density (Islas et al., 2002) and stress induced by chemicals (CdCl2) and heat shock (Traweger et al., 2003). In sparse cultures, newly synthesized ZO-2 temporarily localizes to the nucleus before reaching the TJs at the cell borders (Chamorro et al., 2009). In contrast, in confluent monolayers, ZO-2 goes directly to the cell borders (Quiros et al., 2013). Movement of ZO-2 from the cytoplasm to the plasma membrane involves activation of the calcium-sensing receptor and the novel protein kinase Cε (nPKCε)/with no lysine kinase-4 (WNK4) downstream signaling cascade, which leads to ZO-2 serine phosphorylation, its release from 14-3-3 protein in the cytoplasm, and incorporation to TJs (Amaya et al., 2019). ZO-2 moves in and out of the nucleus with nuclear localization and exportation signals (NLS and NES) (Lopez-Bayghen et al., 2006). In canine ZO-2 (cZO-2), one monopartite and two bipartite (bp) NLS are present at the U2 segment located at the amino portion of the molecule between PDZ1 and PDZ2 domains (Quiros et al., 2013). In addition, 16 serine–arginine (SR) repeats are found at this segment, whose phosphorylation by the kinase SRPK induces protein entrance to the nucleus and its localization at nuclear speckles (Quiros et al., 2013). cZO-2 has four NES, two located at the PDZ2 domain and the other two at the guanylate kinase (GuK) module (Gonzalez-Mariscal et al., 2006).

ZO-2 has multiple protein-binding domains and motifs that allow it to function as a scaffold that concentrates a wide variety of proteins at both the TJ and the nucleus (for reviews, see Gonzalez-Mariscal et al., 2017, 2019). At the plasma membrane, ZO-2 and ZO-1 act as a platform for the polymerization of claudins into TJ strands (Umeda et al., 2006). In contrast, at the nucleus, even though ZO-2 has no DNA-binding sites, it inhibits the transcription of several genes regulated by AP-1 (activator protein 1) (Betanzos et al., 2004), TEAD (transcriptional enhanced associated domain) (Dominguez-Calderon et al., 2016) and TCF (T cell factor)/LEF (lymphoid enhancer family) (Tapia et al., 2009; Wetzel et al., 2017) transcription factors. Here we have studied if ZO-2 plays a role as a nuclear translocator of TEAD transcription factor.

Mammals express four TEAD genes, and the particular function of each one remains controversial. Thus, Tead1 null mice are embryonic lethal due to defective cardiac development (Chen et al., 1994); Tead2 knockout (KO) mice either show no phenotype (Sawada et al., 2008) or have defects in neural tube closure (Kaneko et al., 2007); Tead4 null mice are lethal due to embryo implantation failure (Yagi et al., 2007), and a KO of both Tead1 and Tead2 is embryonic lethal due to defects in neural tube closure (Sawada et al., 2008). In humans, an inactivation mutation of TEAD1 is associated with degeneration of the choroid and retina or Sveinsson’s chorioretinal atrophy (Fossdal et al., 2004).

Regulation of TEAD involves YAP (Yes-associated protein) and its paralogue TAZ (transcriptional activator with PDZ-binding domain). Thus, when YAP/TAZ are phosphorylated by the kinases of the Hippo pathway LATS1/2 (large tumor suppresor kinase 1/2), they remain in the cytoplasm by sequestration to 14-3-3 or are ubiquitinated and degraded. Instead, unphosphorylated YAP/TAZ move into the nucleus and bind to TEAD, driving the transcription of target genes (for review see Zhou et al., 2016).

Hippo-independent coactivators can also regulate TEAD. Such is the case of vestigial-like (VGLL) protein that competes with YAP/TAZ for TEAD binding (Pobbati et al., 2012; Jiao et al., 2014); of TCF4 (transcription factor 4), whose interaction with TEAD facilitates the expression of Wnt target genes (Jiao et al., 2017); of the p160 family of steroid receptor coactivators that potentiate TEAD transcriptional activity (Belandia and Parker, 2000); and of AP-1 that occupies the same chromatin sites as TEAD and is necessary for the activation of TEAD target genes (Zanconato et al., 2015; Liu et al., 2016).

Although TEAD function can also be regulated by posttranslational modifications, including palmitoylation (Chan et al., 2016) and phosphorylation by PKA (Gupta et al., 2000) and PKC (Jiang et al., 2001), it is noteworthy that TEAD subcellular localization regulates transcriptional programs that determine trophectoderm versus inner cell mass (ICM) lineage differentiation in embryos. Thus, in mouse embryonic stem cells (mESCs), TEAD4 localized exclusively in the cytoplasm, while in mouse trophoblast stem cells (mTSCs) TEAD4 was enriched in the nucleus, regulating a trophectoderm-specific transcriptional program (Home et al., 2012). Hyperosmotic stress also induces TEAD cytoplasmic localization through a mechanism where p38 binds to TEAD and subsequently translocates the transcription factor to the cytoplasm (Lin et al., 2017).

Previously we demonstrated that ZO-2 silencing in renal epithelial Madin-Darby canine kidney (MDCK) cells induced the nuclear accumulation of YAP and increased the activity of an artificial promoter driven by TEAD-binding sites. In contrast, the overexpression of ZO-2 abolished this activity (Dominguez-Calderon et al., 2016). Moreover, we showed that the promoter for the connective tissue growth factor (CTGF), regulated by TEAD-binding sites (Zhao et al., 2008), is more active in ZO-2 KD cells and that these changes correlated with increased CTGF mRNA (Dominguez-Calderon et al., 2016). These effects, in turn, were obliterated upon ZO-2 transfection (Dominguez-Calderon et al., 2016). These observations prompted us to explore whether the expression and subcellular localization of ZO-2 modulated the nuclear accumulation of TEAD.

Here, in epithelial cells we have found that the entry and departure of ZO-2 from the nucleus modulate TEAD intracellular traffic and that TEAD and ZO-2 form a complex that is negatively regulated by nPKCδ, which facilitates the nuclear importation of TEAD.

RESULTS

ZO-2 facilitates the entry of TEAD into the nucleus of epithelial cells

We first explored by immunofluorescence the subcellular localization of TEAD in parental and ZO-2 KD MDCK cells. For this purpose, we employed sparse and confluent cultures as we had previously shown that in the former, ZO-2 accumulates at the nucleus. In contrast, in confluent monolayers, the amount of nuclear ZO-2 is minimal (Islas et al., 2002). Figure 1A shows that the absence of ZO-2 induces a decreased expression of TEAD in the nuclei of sparse cultures. In contrast, in confluent cultures, an increased expression of nuclear TEAD is observed compared with parental cultures. A Western blot then revealed that in total cellular extracts, the amount of TEAD was diminished in sparse cultures in a ZO-2–dependent manner because the decrease in TEAD was reversed upon transfection with a human full-length ZO-2 construct with an altered short hairpin RNA (shRNA)-binding site (hZO-2). Instead, in confluent cultures, the cellular content of TEAD increased with the lack of ZO-2 and was diminished upon ZO-2 transfection (Figure 1B).

FIGURE 1:

FIGURE 1:

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ZO-2 facilitates the nuclear accumulation of TEAD. (A) In ZO-2 KD cells nuclear TEAD was diminished in sparse cultures, while cytoplasmic TEAD increased in confluent monolayers. Sparse and confluent cultures of parental and ZO-2 KD MDCK cells were processed for immunofluorescence with antibodies against TEAD. Left, representative images; right, quantitative analysis. The nuclei of 100 cells per condition derived from three independent experiments were quantitated. Statistical analysis done with Kruskal–Wallis test followed by Dunn’s multiple comparisons test. AU, arbitrary units. ***p < 0.001; ****p < 0.0001. (B) In ZO-2 KD cells, the amount of TEAD decreased in sparse cultures and increased in confluent cultures. Western blot of total cellular extracts derived from sparse and confluent cultures of parental and ZO-2 KD cells transfected or not with hZO-2. GADPH was employed as loading control. Top panel, representative image of three independent experiments; bottom panel, densitometric analysis. Statistics done with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, **p < 0.01; ***p < 0.001. (C) The lack of ZO-2 diminished the amount of TEAD at the nucleus of sparse cells and increased cytoplasmic TEAD in confluent monolayers. Western blot detection of TEAD in cytoplasmic (Cyt) and nuclear (Nuc) fractions derived from sparse and confluent cultures of parental and ZO-2 KD cells transfected or not with hZO-2. Lamin B1 and GAPDH were employed as markers of nuclear and cytosolic fractions, respectively. Top panels, representative images of three independent experiments; bottom panels, quantitative analysis of TEAD/lamin B1 in the nuclear fractions and of TEAD/GAPDH in the cytoplasmic fractions. Statistics done with two-way ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Nuclear TEAD has a higher molecular weight than cytoplasmic TEAD due to phosphorylation. Left panel, Western blot detection of TEAD in cytoplasmic (Cyt) and nuclear (Nuc) fractions treated or not with alkaline phosphatase (AP). Antibodies against lamin B1 and GAPDH were employed as markers of nuclear and cytoplasmic fractions, respectively. Representative image of two independent experiments. Filled arrowhead, 49 kDa band; empty arrowhead, 51 kDa band. Right panel, nuclear (Nuc) fractions treated or not with AP were run on an SDS–PAGE with acrylamide-pendant Phos-tag, and blotted with antibodies against TEAD.

Next, we analyzed by Western blot the amount of TEAD present in nuclear and cytoplasmic fractions. Figure 1C reveals that nuclear TEAD was diminished in ZO-2 KD cells in sparse cultures and that this effect was reversed by hZO-2 transfection. However, the decrease of nuclear TEAD in ZO-2 KD cells was not accompanied by an increase in cytoplasmic TEAD. Instead, in confluent cultures, the lack of ZO-2 augments the cytoplasmic content of TEAD, and the transfection of hZO-2 in ZO-2 KD cells abolishes the presence of nuclear TEAD. These results suggest that ZO-2 regulates the entry of TEAD into the nucleus.

To further test this point, we next analyzed the expression of TEAD in the nucleus of sparse parental cells transfected with Flag-hZO-2 wild type (WT) or a construct lacking the NLS of the molecule (Flag-hZO-2 ΔNLS) present at the U2 segment located between the PDZ1 and PDZ2 domains. Figure 2 shows that the nuclear staining for TEAD is reduced in cells transfected with a ZO-2 that, as previously reported, cannot localize at the nucleus due to the lack of NLS (Jaramillo et al., 2004; Oka et al., 2010; Amaya et al., 2019). However, because the reduction in nuclear TEAD is small in cells transfected with Flag-hZO-2 ΔNLS, our results suggest that the nuclear importation of ZO-2 only favors the movement of TEAD into the nucleus but that other mechanism might also be involved, including signaling by the bpNLS present in TEAD (Magico and Bell, 2011).

FIGURE 2:

FIGURE 2:

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The abundance of nuclear TEAD is diminished in cells transfected with a ZO-2 construct lacking the NLS. (A) Representative image of parental MDCK cells 24 h after transfection with full-length Flag-hZO-2 (WT) or Flag-hZO-2 ΔNLS. Cells were fixed and processed for immunofluorescence with antibodies against Flag and TEAD. Nuclei were stained with DAPI. Arrows, nucleus of transfected cells. (B) Quantitative analysis of TEAD nuclear immunofluorescence in Flag-hZO-2 or Flag-hZO-2 ΔNLS transfected cells. The nuclei of 30 transfected cells per condition derived from three independent experiments were quantitated. AU, arbitrary units. Statistical analysis done with Student’s t test, *p < 0.05. Each bar shows the mean ± SEM.

Nuclear TEAD is phosphorylated

The Western blot in Figure 1C also revealed that the band of TEAD derived from the nuclear fraction had a slightly higher molecular weight (51 kDa) than the one derived from the cytosolic fraction (49 kDa). In silico analysis with GPS 3.0 revealed that TEAD has 58 putative phosphorylation sites. To determine whether the higher molecular weight of nuclear TEAD was due to phosphorylation, we next made a Western blot of nuclear extracts treated or not with alkaline phosphatase and observed that the molecular weight of the nuclear band of TEAD was diminished from 51 to 50 kDa (Figure 1D, left panel), suggesting that nuclear TEAD is more strongly phosphorylated than cytoplasmic TEAD. To confirm this observation, we next made a mobility shift detection assay of TEAD. To this end, we ran nuclear extracts of MDCK cells treated or not with alkaline phosphatase on phosphate-affinity SDS–PAGE with a dinuclear manganese complex of acrylamide-pendant Phos-tag, which binds to and retards the migration of phosphorylated proteins. The right panel of Figure 1D shows that the higher-molecular-weight band of TEAD is not present in the nuclear samples treated with alkaline phosphatase, thus confirming that this band corresponds to phosphorylated TEAD.

Inhibition of nPKCδ promotes the nuclear importation of TEAD and ZO-2

Next, we performed an in silico analysis with GPS 3.0, finding that TEAD has 23 and six putative PKC and PKA phosphorylation sites, respectively. Hence, we explored whether the inhibition of different PKC isozymes and PKA altered the nuclear/cytoplasmic distribution of TEAD in cultures of parental and ZO-2 KD cells. Figure 3A reveals that treatment with 27 nM Ro 31-8220, which inhibits cPKCα, γ, and β1 and nPKCε (Young et al., 2005), significantly increases the concentration of TEAD in the nuclei of ZO-2 KD cells but not of parental cells, thus suggesting that in the absence of ZO-2, cPKCα, γ, β1 or nPKCε induces TEAD nuclear exportation. Instead, treatment with 10 nM Gö-6983, which inhibits cPKCα, β, and γ and nPKCδ (Young et al., 2005), exerts no effect on the nuclear concentration of TEAD in ZO-2 KD cells. Thus, it is suggested that the inhibition of nPKCε blocks the nuclear exportation of TEAD in ZO-2 KD cells (Figure 3B).

FIGURE 3:

FIGURE 3:

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ZO-2 modulates the effect exerted by PKC δ on TEAD nuclear accumulation. (A) Confluent monolayers of parental and ZO-2 KD MDCK cells were treated with 10 μM H89 to inhibit PKA, 27 nM Ro 31-8220 to inhibit cPKC α, γ, and β1 and nPKCε, 10 nM Gö-6986 to inhibit cPKC α, β, and γ and nPKCδ, or 0.1% DMSO as vehicle. Cytoplasmic and nuclear fractions were isolated and run in a SDS–PAGE and blotted with antibodies against ZO-2 and TEAD. GAPDH and lamin B1 were employed as loading controls for cytoplasmic and nuclear fractions, respectively. Left, representative images of Western blots from three independent experiments; right, densitometric analysis. Statistics done with one-way ANOVA followed by Fisher’s least significant difference test, *p < 0.05; **p < 0.01; ***p < 0.001. Each bar shows the mean ± SEM. (B) Schematic representation showing that in ZO-2 KD MDCK cells Ro-31822 blocks the nuclear exportation of TEAD mediated by nPKCε. (C) Schematic representation depicting in parental cells ZO-2 entry to the nucleus triggered by the inhibition of PKA with 10 μM H89. (D) Parental MDCK cells were transfected with a nPKCδ construct, and 24 h later nuclear fractions were obtained and processed for Western blot employing antibodies against ZO-2 and TEAD. Lamin B1 was employed as loading control of nuclear fractions. Left, representative image of Western blots from three independent experiments; right, densitometric analysis. Statistics done with Students’ t test, **p < 0.01. Each bar shows the mean ± SEM. (E) MDCK ZO-2 KD cells were transfected with a nPKCδ construct, and 24 h later nuclear fractions were obtained and processed for Western blot employing antibodies against TEAD. Lamin B1 was employed as loading control of nuclear fractions. Left, representative image of Western blots from three independent experiments; right, densitometric analysis. Statistics done with Students’ t test, n.s., nonsignificant. Each bar shows the mean ± SEM. (F) Cartoon showing that nPKCδ blocks ZO-2-TEAD cytoplasmic interaction and nuclear importation.

To further understand the effect of nPKCε inhibition on TEAD function, we performed a luciferase reporter gene assay with the promoter of hCTGF regulated by three TEAD-binding sites (Zhao et al., 2008). Figure 4A confirms that, as we had previously reported (Dominguez-Calderon et al., 2016), hCTGF promoter activity is higher in ZO-2 KD than in parental cells. This has been explained previously in ZO-2 KD cells by the increased entry into the nucleus of YAP that associates to TEAD and functions as a transcriptional activator (Dominguez-Calderon et al., 2016). Figure 4B shows that treatment with permeable nPKCε inhibitor peptide εV1-2 increases hCTGF promoter activity in ZO-2 KD cells and not in parental cells. The increased amount of nuclear TEAD found in ZO-2 KD cells might explain this result as observed after treatment with 27 nM Ro 31-8220 but not with 10 nM Gö-6983 (Figure 3A).

FIGURE 4:

FIGURE 4:

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In ZO-2 KD cells inhibition of nPKCε increases the activity of a promoter regulated by TEAD-binding sites. Reporter gene assays done in parental and ZO-2 KD cells transiently transfected with hCTGF-Luc at a concentration of 100 ng/well (32 mm2). (A) hCTGF promoter activity is higher in ZO-2 KD than in parental cells. hCTGF promoter activity values were normalized to values observed in parental cells. Statistical analysis done with Student’s t test, *p < 0.05. (B) Inhibition of nPKCε increases the activity of hCTGF promoter in ZO-2 KD cells but not in parental cells. hCTGF promoter activity values were normalized to values observed in ZO-2 KD cells without the nPKCε inhibitor. Statistical analysis done with Student’s t test, *p < 0.05. In all cases data were normalized to protein content and transfection efficiency with pGL3-Control. Each bar shows the mean ± SEM from triplicates derived from two independent experiments.

Figure 3A also shows that the cytoplasmic content of TEAD in ZO-2 KD cells increases after treatment with 10 μM H89, which inhibits PKA, or 10 nM Gö-6983, but not with 27 nM Ro 31-8220, thus suggesting that inhibition of PKA and nPKCδ augments the content of TEAD but blocks its nuclear importation. In parental cells, PKA inhibition with H89 has no effect on TEAD in cytoplasmic and nuclear fractions but instead increases the amount of ZO-2 in the nuclei and decreases its content in the cytoplasm, thus suggesting that PKA inhibition induces ZO-2 nuclear accumulation (Figure 3C). To further confirm these results, we next employed two additional doses of H89, observing that in parental cells, the decrease in cytoplasmic ZO-2 was triggered when 1, 10, or 100 μM H89 was employed. The increase in nuclear ZO-2 was not observed when 1 μM H89 was used but was present upon treatment with 10 or 100 μM H89 (Supplemental Figure 1).

Figure 3A also shows that in parental MDCK cells, 10 nM Gö-6983 but not 27 nM Ro 31-8220 augments the nuclear content of TEAD, thus indicating that the inhibition of nPKCδ mediates the nuclear accumulation of this protein. However, this increase in the nuclear content of TEAD is accompanied by an increase in the cytoplasmic amount of TEAD, thus suggesting that inhibition of nPKCδ augments the synthesis or stability of this transcription factor and its nuclear importation. In parental cells, 10 nM Gö-6983 but not 27 nM Ro31-8220 also triggers an increase in nuclear ZO-2 accompanied by a decrease in the cytoplasmic fraction of ZO-2, thus suggesting that inhibition of nPKCδ induces the nuclear importation of ZO-2. To confirm these results, we next transfected parental MDCK cells with a construct for nPKCδ and analyzed by Western blot the content of ZO-2 and TEAD in the nuclear fractions. Figure 3D reveals a diminished amount of both ZO-2 and TEAD in the nuclei of parental cells transfected with nPKCδ. To further show the importance of ZO-2 in this process, we next transfected nPKCδ into ZO-2 KD cells and observed no change in the amount of nuclear TEAD compared with that in nontransfected cells (Figure 3E).

In summary, while in parental cells PKA inhibition induces the nuclear accumulation of ZO-2 but not TEAD, the inhibition of nPKCδ triggers the accumulation at the nucleus of both ZO-2 and TEAD. In cells that lack ZO-2, neither the inhibition nor the transfection of nPKCδ alters the nuclear content of TEAD, thus suggesting that phosphorylation mediated by nPKCδ blocks the interaction between ZO-2 and TEAD that facilitates the entry of TEAD and ZO-2 into the nucleus (Figure 3F).

ZO-2 and TEAD interaction is inhibited by nPKCδ

To test whether ZO-2 and TEAD interact and whether this process is inhibited by nPKCδ, we transfected MDCK cells with Flag-hZO-2, treated them or not with 3 μM röttlerin, which inhibits nPKCδ, and made a proximity ligation assay (PLA) with a mouse antibody against Flag and a rabbit antibody against TEAD (Figure 5A). At time 0 (6th hour after transfection), when as previously demonstrated 74–78% of transfected cells have nuclear ZO-2 (Chamorro et al., 2009; Quiros et al., 2013), positive red fluorescent spots are detected at the nucleus of Flag-hZO-2 transfected cells, identified by their green staining (Figure 5, B, a and a′, and C). The number of red spots was above the background present when the antibody against TEAD was omitted (Figure 5, B, e and e′, and C). The abundance of positive red spots at the nucleus of Flag-hZO-2 transfected cells increases when nPKCδ is inhibited with röttlerin (Figure 5, B, b and b′, and C). However, because röttlerin has also been found to inhibit Ca2+/calmodulin kinase II (CaM-kinase II), MAP kinase–activated protein 5 (PRAK/MK5), and MAP kinase–activated protein 2 (MAPKAP-K2) (Gschwendt et al., 1994; Davies et al., 2000), we next proceeded to test the interaction between TEAD and ZO-2 when instead of using röttlerin, nPKCδ was overexpressed by transfection. Figure 5 shows that the number of red spots decreases when nPKCδ was transfected together with Flag-hZO-2 (Figure 5, B, c and c′, and C) and achieves the highest value when transfected nPKCδ is inhibited with röttlerin. In this case, we also observe the appearance of abundant red spots in the cytoplasm (Figure 5, B, d and d′, and C), which further confirms that nPKCδ inhibition allows the interaction of TEAD and ZO-2 in the cytoplasm. Instead, 24 h posttransfection, when previous observations demonstrated that the percentage of cells with nuclear ZO-2 has diminished to 17% (Chamorro et al., 2009; Quiros et al., 2013), the red spots at the nuclei are barely detectable in all the experimental conditions (Figure 5, B, f, f′–j, j′, and C) and resemble the background signal obtained when no anti-TEAD antibody was added (Figure 5, B, j and j′, and C).

FIGURE 5:

FIGURE 5:

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Inhibition of nPKCδ with röttlerin augments ZO-2 and TEAD interaction at the nucleus. (A) Scheme of the experimental procedure followed. MDCK cells were transfected with Flag-hZO-2 or Flag-hZO-2 plus nPKCδ. Cells were fixed and processed for a PLA done with a mouse antibody against Flag and a rabbit antibody anti-TEAD at 0 and 24 h posttransfection (time 0 corresponds to the initial 6 h required after transfection for proteins to be expressed). Where indicated, 3 μM röttlerin was added 1 h before fixation. (B) Representative immunofluorescence images of PLA. Red fluorescent spots reveal the positive interaction between transfected Flag-hZO-2 and endogenous TEAD. Cells transfected with Flag-hZO-2 were identified with a mouse antibody against Flag, followed by a secondary goat anti-mouse IgG coupled to Alexa Fluor 488. Background corresponds to experiments done without the anti-TEAD antibody. Nuclei detected with DAPI. Transf, transfected. *, nuclei of transfected cells. Images obtained from two independent experiments. (C) Quantitative analysis of PLA done using BlobFinder. At least 25 cells were analyzed per experimental condition. Statistical analysis done with two-way ANOVA followed by Tukey’s multiple comparisons test, ****p < 0.0001. Each bar shows the mean ± SEM.

To further confirm that the interaction of TEAD and ZO-2 is mediated by nPKCδ, we analyzed the presence of TEAD in a ZO-2 immunoprecipitate, finding that inhibition of nPKCδ with röttlerin augments the amount of TEAD associated with ZO-2 in MDCK cells (Figure 6A). In addition, in intestinal epithelial cells IEC-18, we confirmed the presence of TEAD in a ZO-2 immunoprecipitate (Figure 6B). We also showed with a pull-down assay the interaction between TEAD and ZO-2 in the human kidney cell line HEK293T. For this purpose, the cells were transfected with the amino (coding PDZ domains 1, 2, and 3), or AP (coding the acidic and proline-rich regions) constructs of cZO-2, introduced in the pcDNA4/HisMax vector. Then, the corresponding proteins were purified from extracts of HEK293T cell using Ni affinity columns, run in SDS–PAGE, and stained with Coomassie blue. Figure 6C reveals the presence of bands of 62 and 45 kDa that respectively correspond to amino and AP segments of cZO-2. The identity of the amino pulled-down segment was confirmed in a blot with the anti–ZO-2 antibody that recognizes this section of the protein, as indicated by the manufacturer (Figure 6D, top panel). The blot with the antibody against TEAD revealed the presence of this transcription factor in the pull down of the amino and AP segments of ZO-2, thus suggesting that these segments of ZO-2 associate to TEAD (Figure 6D, bottom panel). We also transfected HEK293T cells with the 3PSG (coding PDZ-3, SH3, and GuK domains) segment of ZO-2. However, the identity of this ZO-2 segment could not be confirmed because it generated a very faint band upon staining with Coomassie blue (unpublished data) and cannot be recognized by any commercial anti–ZO-2 antibody.

FIGURE 6:

FIGURE 6:

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The interaction between TEAD and ZO-2 is inhibited by nPKCδ. (A) The amount of TEAD that coimmunoprecipitates with ZO-2 is augmented in cells treated for 1 h with 3 μM röttlerin. Blotting was done with antibodies against ZO-2 and TEAD. Top panel, representative image; bottom panel, densitometric analysis. Statistics done with Students’ t test, **p < 0.01. Each bar shows the mean ± SEM. Co-IP, coimmunoprecipitation; IP, immunoprecipitation. (B) In extracts derived from intestinal IEC-18 cells, TEAD coimmunoprecipitates with ZO-2. PIS, preimmune serum. (C) 6XHis-tagged amino (NH3) and AP segments of cZO-2 were purified with Ni affinity columns from extracts of HEK293T cells, run in SDS–PAGE, and stained with Coomassie blue. (D) Western blot analysis of pull downs of amino and AP segments of cZO-2. Top panel, anti–ZO-2 antibody recognizes only the 3PSG segment of ZO-2. Bottom panel, anti-TEAD antibody gives a positive signal in pull downs of amino and AP segments of cZO-2. (E) Cartoon showing that the inhibition of nPKCδ allows ZO-2-TEAD interaction, which facilitates the nuclear importation of these proteins. At the nucleus nPKCδ also ruptures ZO-2-TEAD association.

Altogether, these results indicate that ZO-2 and TEAD associate and that the inhibition of nPKCδ allows ZO-2 and TEAD to interact in the cytoplasm and enter together into the nucleus (Figure 6E).

Activation by nPKCε of NES in ZO-2 induces the nuclear exportation of ZO-2 and TEAD

Previously, we demonstrated the activation of ZO-2 NES-1 through the phosphorylation of Ser-369 by PKCε (Chamorro et al., 2009). Hence, we next analyzed in islets of cells what happened with TEAD after treatment with the nPKCε permeable activating peptide (εV1-7). In the islets of MDCK cells treated with peptide εV1-7, ZO-2 is no longer present at the nucleus (Figure 7A, b and e) in comparison to control cultures (Figure 7A, a and d), as had been previously reported (Quiros et al., 2013). Similarly, the expression of TEAD at the nucleus is barely detectable after treatment with the nPKCε permeable activating peptide εV1-7 (Figure 7A, h and k) in comparison to the control condition (Figure 7A, g and j). Treatment with εν1-2, a nPKCε permeable inhibitor peptide, concentrated ZO-2 at the nuclei (Figure 7A, c and f) and increased the amount of nuclear TEAD in comparison to control cells (Figure 7A, i and j). The same results were obtained when this experiment was done in intestinal epithelial cells IEC-18 (Figure 7B). Therefore, we conclude that nuclear exportation of ZO-2 triggers the translocation of nuclear TEAD to the cytoplasm, while the nuclear accumulation of ZO-2 facilitates the nuclear concentration of TEAD.

FIGURE 7:

FIGURE 7:

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Nuclear exportation of ZO-2 induced by PKCε triggers TEAD translocation from the nucleus to the cytoplasm. (A) Representative images obtained from three independent experiments of the immunofluorescence of ZO-2 (a–f) and TEAD (g–l) in control sparse cultures of parental MDCK cells (first column), treated for 24 h with 2 μM nPKCε permeable activating peptide (εV1-7) that activates the nuclear ZO-2 exportation (second column), or with 2 μM nPKCε permeable inhibitor peptide (εν1-2) that blocks the nuclear exportation of ZO-2 (third column). Nuclei were stained with DAPI. (B) Representative images obtained from three independent experiments of the immunofluorescence of intestinal epithelial cells IEC-18. ZO-2 (a–f) and TEAD (g–l) in control sparse cultures of IEC-18 cells (first column) treated for 24 h with 2 μM nPKCε permeable activating peptide (εV1-7) that activates the nuclear ZO-2 exportation (second column) or with 2 μM nPKCε permeable inhibitor peptide (εν1-2) that blocks the nuclear exportation of ZO-2 (third column). Nuclei were stained with DAPI. (C) Quantification of nuclear TEAD immunofluorescence per cell in arbitrary units (AU) of the experiment described in A. At least 50 nuclei were analyzed per experimental condition. Statistical analysis done with one-way ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05; ****p < 0.0001. (D) Quantification of nuclear ZO-2 immunofluorescence per cell in arbitrary units (AU) of the experiment described in B. At least 50 nuclei were analyzed per experimental condition. Statistical analysis done with one-way ANOVA followed by Tukey’s multiple comparisons test, **p < 0.01; *** p < 0.001; ****p < 0.0001. (E) Quantification of nuclear TEAD immunofluorescence per cell in arbitrary units (AU), of the experiment described in B. At least 50 nuclei were analyzed per experimental condition. Statistical analysis done with one-way ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05; ***p < 0.001.

We previously showed that newly synthesized ZO-2 translocates to the nucleus in sparse cultures. Then as the culture becomes confluent, it exits the nucleus and reaches the TJ region in the plasma membrane (Chamorro et al., 2009). Therefore, we next tested whether blocking the translocation of nuclear ZO-2 to the cytoplasm as the culture becomes confluent altered the nuclear content of TEAD. For this purpose, we transfected sparse parental cells with HA-cZO-2 or a construct where the four NES of cZO-2 are mutated (HA-cZO-2 MutNES) and analyzed 48 h later the expression of TEAD in the nucleus of the transfected cells. Figure 8 shows that the amount of TEAD at the nucleus is not altered when ZO-2 is kept at the nucleus due to the lack of functional NES.

FIGURE 8:

FIGURE 8:

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Nuclear accumulation of ZO-2 due to NES inactivation has no effect on nuclear concentration of TEAD. (A) Representative immunofluorescence images derived from three independent experiments of sparse parental MDCK cells 48 h after transfection with HA-cZO-2 or a construct where the four NES of cZO-2 were mutated (HA-cZO-2 MutNES 0,1,2,3). First column, HA staining; second column, TEAD staining. Observe the strong HA staining present in the nucleus of a cell transfected with HA-cZO-2 MutNES 0,1,2,3 in comparison to that of cells transfected with HA-ZO-2. Arrows, nuclei of transfected cells. (B) Quantification of nuclear TEAD immunofluorescence in cells transfected with HA-cZO-2 or HA-cZO-2 MutNES 0,1,2,3. AU, arbitrary units. Thirty-five nuclei of transfected cells were analyzed per experimental condition. Statistical analysis done with Students t test, ns, nonsignificant. Each bar shows the mean ± SEM.

Altogether, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD in a process regulated by PKA and nPKC δ and ε (Figure 9).

FIGURE 9:

FIGURE 9:

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Schematic representation of TEAD and ZO-2 movement in and out of the nucleus, regulated by the NLS and NES of ZO-2 and by the activity of PKA and nPKC δ and ε. In epithelial MDCK cells, nPKCδ blocks ZO-2 and TEAD interaction, inhibiting nuclear importation and exportation of the ZO-2/TEAD complex. PKA blocks ZO-2 nuclear importation, while nPKCε activates a NES of ZO-2 and the exportation of TEAD either alone or associated to ZO-2.

DISCUSSION

In epithelial MDCK cells, the subcellular distribution of ZO-2 relies heavily on the degree of cell–cell contact. Thus, in sparse cultures, ZO-2 is found at the TJs present in the developing monolayer and abundant at the nuclei. In contrast, in confluent cultures with mature TJs, ZO-2 is mainly concentrated at the cell borders (Islas et al., 2002). Here, we have studied whether the nuclear concentration of the transcription factor TEAD is regulated by ZO-2 because the absence of ZO-2 increased the transcriptional activity of artificial and natural promoters driven by TEAD-binding sites, while the overexpression of ZO-2 abolished this activity (Dominguez-Calderon et al., 2016).

In sparse cultures of MDCK cells, we observe that both ZO-2 and TEAD accumulate at the nucleus and detect that the amount of nuclear TEAD is diminished in cells where the expression of ZO-2 has been silenced, as well as in sparse parental cells transfected with a ZO-2 construct lacking NLS, indicating that ZO-2 nuclear importation facilitates the entry of TEAD into the nucleus. This observation is reinforced with our finding that in parental cells, the inhibition of nPKCδ triggers the interaction between ZO-2 and TEAD at the cytoplasm, facilitating the entry and accumulation at the nucleus of both interacting proteins. The site phosphorylated by nPKCδ that blocks the interaction between ZO-2 and TEAD remains undefined and could be in either ZO-2 or TEAD, as both proteins display putative nPKCδ phosphorylation sites (ZO-2, 22 sites; TEAD, two sites, according to GPS 3.0), and experimental evidence reveals that TEAD is phosphorylated by several PKC isozymes (Jiang et al., 2001). Moreover, a previous report noted that cytoplasmic translocation of TEAD could be induced by hyperosmotic stress through a process mediated by p38 (Lin et al., 2017). However, TEAD is a poor substrate for p38 phosphorylation, and the mutation of the four putative p38 phosphorylation sites in TEAD did not alter its stress-induced cytoplasmic translocation, suggesting that other kinases different from p38 are involved (Lin et al., 2017).

Nevertheless, the blockade of the nuclear entry of TEAD and ZO-2 by nPKCδ could be explained by the fact that at the carboxyl basic region of the bpNLS-2 of ZO-2, three serine residues present there (S257, S259, and S261) are putative nPKCδ phosphorylation sites. Previously we demonstrated that their phosphomimetic mutation obstructs the nuclear accumulation of ZO-2, likely due to the neutralization of the positive charges present at the carboxyl-terminal segment of bpNLS-2 (Quiros et al., 2013).

Our results also indicate that in MDCK cells, the exportation of TEAD from the nucleus is triggered by the activation of nPKCε. This process can happen in a ZO-2–independent manner as observed in ZO-2 KD cells and in a ZO-2–dependent way in parental cells, which involves the activation of ZO-2 NES by nPKCε. The latter observation further confirms that the interaction with ZO-2 facilitates the movement of TEAD. Nevertheless, it should be mentioned that TEAD also contains a bipartite NLS and a NES, both conserved in numerous species whose functionality has been tested in Drosophila (Magico and Bell, 2011). Therefore, the interaction of ZO-2 with TEAD might only reinforce the movement of the transcription factor.

The role of ZO-2 as a nuclear translocator of proteins was previously observed. Thus, ZO-2 was found to enhance the nuclear localization of YAP-2 through a process that required the presence of the NLS of ZO-2 (Oka et al., 2010). YAP is a transcriptional activator of promoters regulated by the TEAD transcription factor (Zhao et al., 2008). It binds to the first PDZ domain of ZO-2 through its carboxyl-terminal PDZ-binding motif (Oka et al., 2010). Likewise, the movement of ARVCF into the nucleus requires the interaction of the PDZ-binding motif of this protein with the amino PDZ-containing region of ZO-2 and the presence of the NLS of ZO-2 (Kausalya et al., 2004). ARVCF protein at the nucleus binds to splicing factors and contributes to the regulation of alternating splicing (Rappe et al., 2014).

In summary, our observations indicate that ZO-2 acts as a platform that facilitates the nuclear importation and exportation of TEAD.

MATERIAL AND METHODS

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Cell culture

Parental (control) and ZO-2 KD MDCK II cells were kindly provided by Alan Fanning (University of North Carolina, Chapel Hill, NC) and cultured as previously described (Van Itallie et al., 2009). These cells stably express a mixture of three different shRNAs against ZO-2 in the pSuper vector. Parental cells instead express only the empty vector. The stable clonal ZO-2 KD MDCK cell line here employed (IC5) was obtained based on zeocin resistance. Sparse cultures were plated at a density of 2.5 × 104 cells/cm2, whereas confluent cultures were seeded at a density of 5 × 105 cells/cm2.

IEC-18 epithelial cells derived from the rat small intestine and obtained from the American Type Culture Collection (Cat. CRL-1589; ATCC, Manassas, VA) were grown in DMEM F-12 (Cat. 12500-062; Thermo Fisher Scientific, Waltham, MA) supplemented with 5% fetal bovine serum (FBS) and penicillin–streptomycin 10,000 (U/μg/ml) (Cat. A-01; In Vitro, Mexico).

HEK293T epithelial cells derived from the human embryonic kidney (Cat. CRL-3216; ATCC, Manassas, VA) were grown in a high-glucose DMEM (Cat. 11965-118; Thermo Fisher Scientific, Waltham, MA) supplemented with 5% FBS and penicillin–streptomycin 10,000 (U/μg/ml) (Cat. A-01; In Vitro, Mexico).

Transfections

Transfections were done following the manufacturer’s instructions using Lipofectamine 2000 (Cat. 11668-019; Life Technologies, Carlsbad, CA). Because the Lipofectamine manufacturer indicates that 6 h is the optimal time required for the transfected construct to be expressed as protein, time 0 in Figure 5 corresponds to 6 h after transfection.

ZO-2 KD cells were transfected with a human full-length ZO-2 construct with altered shRNA-binding sites (pTRE-hZO-2), generously provided by Alan Fanning (University of North Carolina, Chapel Hill, NC).

To generate hZO-2 ΔU2 in a construct with CMV10 promoter and a tag of 3 × Flag, we liberated with KpnI and XbaI restriction enzymes the hZO-2 ΔNLS construct inserted into a vector containing CMV2 promoter and a tag of 2 × Flag (generously provided by Marius Sudol, National University of Singapore, Singapore). hZO-2 ΔNLS lacks base pairs 313–873 of hZO-2 that code for the U2 segment (amino acids 105–291). Full-length hZO-2 contained within a vector with CMV10 promoter and a 3 × Flag (Flag-hZO-2) (generously provided by Otmar Huber, Jena University, Germany) was cut with KpnI and XbaI restriction enzymes to eliminate full-length hZO-2 from the vector. The hZO-2 ΔNLS construct was then inserted into this empty vector containing CMV10 promoter and a 3 × Flag. Parental cells were transfected with either Flag-hZO-2 or hZO-2 ΔNLS within a vector with CMV10 promoter and three Flag tags (Flag-hZO-2).

In some experiments, parental and ZO-2 KD MDCK cells were transfected with mouse nPKCδ (plasmid 16386; Addgene, Watertown, MA) or Flag-hZO-2 plus nPKCδ.

HEK293T cells were transfected with amino (398–962 nucleotides [nt]), 3PSG (1595–3019 nt), or AP (3029–3923 nt) segments of cZO-2, in the pcDNA4/HisMax vector that had been previously reported (Betanzos et al., 2004).

Immunofluorescence

Immunofluorescence experiments on MDCK cells were done as previously described (Quiros et al., 2013) with the only modification that cells were fixed with 10% (vol/vol) formaldehyde in phosphate-buffered saline. As first antibodies, we employed a rabbit monoclonal against TEAD (Cat. 12292; dilution 1:200; Cell Signaling, Danvers, MA); mouse monoclonal antibodies against Flag (Cat. F3165; dilution 1:1500; Sigma Aldrich, Darmstadt, Germany); and rabbit polyclonal against ZO-2 (Cat. 71-1400; dilution 1:200; Invitrogen, Carlsbad, CA). As secondary antibodies, we employed donkey antibodies against rabbit immunoglobulin G (IgG) coupled to Alexa Fluor 488 (Cat. A21206; dilution 1:1000; Invitrogen, Carlsbad, CA) or Alexa Fluor 594 (Cat. A21207; dilution 1:1000; Invitrogen, Carlsbad, CA) and against mouse IgG coupled to Alexa Fluor 488 (Cat. A11001; Life Technologies, Eugene, OR).

Quantification of TEAD immunofluorescence intensity at the nucleus was done using ImageJ. Nuclei images stained with DAPI (4′,6-diamidino-2-phenylindole) were used to define the region to be subsequently quantitated for TEAD immunofluorescence.

Western blot

Western blot was done following a standard procedure previously described (Quiros et al., 2013), but using a different RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) and a commercial sample buffer (Cat. NP0008; Invitrogen, Carlsbad, CA). The following primary antibodies were employed: a rabbit monoclonal against TEAD (Cat. 12292; dilution 1:2000; Cell Signaling, Danvers, MA); mouse monoclonals anti-GAPDH (Cat. Sc-32233; dilution 1:15,000; Santa Cruz Biotechnology, Santa Cruz, CA) and lamin B1 (Cat. 33-2000; dilution 1:2000; Invitrogen, Carlsbad, CA); and rabbit polyclonal against ZO-2 (Cat. 71-1400; dilution 1:2000; Invitrogen, Carlsbad, CA). As secondary antibodies, we employed goat polyclonals anti-rabbit IgG coupled to peroxidase (Cat. A9169; dilution 1:20,000; Sigma Aldrich, St. Louis, MO) or anti-mouse IgG coupled to peroxidase (Cat. 62-6420; dilution 1:10,000; Invitrogen, Carlsbad, CA). The procedure was followed by Immobilon chemiluminescence detection (Cat. WVKLS 0500; Merck KGaA, Darmstadt, Germany).

For the mobility shift detection assay of phosphorylated TEAD, phosphate affinity SDS–PAGE was done with the acrylamide-pendant Phos-tag ligand (Cat. AAL-107; Wako Pure Chemical Industries, Richmond, VA) following the manufacturer’s instructions. In Phos-tag SDS–PAGE, molecular weight estimations using molecular weight markers are not possible. Because the manufacturer recommends using a dephosphorylated sample of the target protein as a marker, in Figure 1D, right panel, we have placed the indication of 50 kDa in the lower band of TEAD that is still present after alkaline phosphatase treatment.

Cell fractionation assay

Cytoplasmic and nuclear fractions of parental and ZO-2 KD MDCK cells transfected or not with hZO-2 were obtained as previously reported by others (Shaiken and Opekun, 2014). Briefly, cells were lysed in isotonic buffer A (40 mM HEPES, pH 7.4, 120 mM KCl, 2 mM EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), 0.4% glycerol, 10 mM β-glycerophosphate, and 0.2% NP-40) while rotating for 30 min at 4°C. Nuclei were pelleted by centrifugation at 1000 × g for 5 min. The supernatant was centrifuged further at 10,000 × g for 10 min to obtain the cytosolic fraction. The pellet of nuclei was sequentially and gently washed with 0.1% NP-40 and non–detergent containing buffer A and centrifuged at 1000 × g for 5 min. The supernatant was discarded, and the nuclear pellet was resuspended in RIPA buffer.

Alkaline phosphatase treatment

Nuclear pellets obtained from confluent MDCK monolayers were resuspended in NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol, pH 7.9; Cat. B7003S; New England Biolabs, Ipswich, MA). The nuclei were then sonicated and incubated for 60 min at 37°C with 20 U of calf intestine alkaline phosphatase (Cat. M0290S; New England Biolabs, Ipswich, MA). Samples were then processed for Western blot.

Proximity ligation assay

The Duolink in situ proximity ligation assay (PLA) (Cat. DUO92101; Sigma Aldrich, Darmstadt, Germany) was done according to the manufacturer’s instructions. Using a rabbit antibody against TEAD (Cat. 12292; dilution 1:200; Cell Signaling, Danvers, MA) and a mouse antibody anti-Flag (Cat. F3165; dilution 1:1500; Sigma Aldrich, Darmstadt, Germany). After the Duolink reaction was completed, the preparations were washed three times with Duolink B-buffer. The cells were stained with a donkey anti-mouse antibody coupled to Alexa Fluor 488 to detect cells transfected with Flag-hZO-2.

Quantitative analysis of PLAs was done using BlobFinder (Allalou and Wahlby, 2009) developed by the Centre for Image Analysis at Uppsala University.

Immunoprecipitation

Immunoprecipitation of ZO-2 was done using 1 μl of ZO-2 antibody (Cat. 71-1400; Invitrogen, Carlsbad, CA) per 300 μg of protein in the lysate of parental MDCK cells and following a protocol previously described (Raya-Sandino et al., 2017). The radioimmunoprecipitation assay buffer employed contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40 (vol/vol), and the protease inhibitor cocktail Complete (Cat. 11697498001; Roche Diagnostics, Mannheim, Germany). The blot for TEAD in the ZO-2 immunoprecipitate was done using the Tidy blot reagent (Cat. STAR209; BioRad, Hercules, CA) following the manufacturer’s instructions.

Protein purification

HEK293T cells were transfected with previously reported constructs containing the amino (398–962 nt), 3PSG (1595–3019 nt), or AP (3029–3923 nt) segments of cZO-2 in the pcDNA4/HisMax vector (Betanzos et al., 2004). After 24 h the cells were lysed and the extracts were subjected to affinity chromatography with Complete His-Tag Purification Columns (Cat. COHISC-RO; Sigma Aldrich, St. Louis, MO), following the manufacturer’s instructions. The purified fractions were run in a SDS–PAGE, stained with Coomassie blue, and blotted with antibodies against ZO-2 and TEAD.

Reporter gene assays

Parental and ZO-2 KD MDCK cells were plated in 96-well plates at a density of 2.5 × 104 cells/cm2. After 24 h, cells were transfected using Lipofectamine 3000 (Cat. L3000015; Thermo Fisher, Waltham, MA) with 100 ng of a construct of the human CTGF promoter region that contains three putative TEAD-binding sites cloned into the basic luciferase reporter vector pGL3-6xOSE-Luc (Zhao et al., 2008) generously provided by Kun-Liang Guan from the Moors Cancer Center, University of California at San Diego, or with 75 ng of the pGL3-Control (Cat. E1741; Promega, Madison, WI) plasmid with constitutive activity. After 6 h, the transfection medium was removed and replaced with a fresh culture medium or medium with 2 μM nPKCε permeable inhibitor peptide εv1-2. After 20 h of treatment, the cells were harvested and suspended in reporter lysis buffer (Cat. E3971; Promega, Madison, WI). Protein extraction was done by a heat shock lysis cycle of 5 min at 70°C followed by 1 min at 37°C and 3 min in agitation. Finally, luciferase activity was determined using the luciferase assay system (Cat. E1500; Promega, Madison, WI) and the Infinite M200 Pro series (Tecan, Männedorf, Switzerland) equipment. Luciferase activity values were normalized to protein content and expressed relative to those recorded in parental cells or in parental and ZO-2 KD cells without the nPKCε inhibitor. In all cases, transfection efficiency was normalized using pGL3-control as a reference.

Drugs

The nPKCε permeable inhibitor peptide εv1-2 and the nPKCε permeable activating peptide εv1-7 were kindly provided by Daria Mochly-Rosen (Stanford University, Stanford, CA) and dissolved in water as a stock at a concentration of 1 mM and used at a final concentration of 2 μM.

Röttlerin, an nPKCδ inhibitor (Cat. 557370; Calbiochem, Darmstadt, Germany), was prepared as a 12 μM stock in dimethyl sulfoxide (DMSO) and used at a final concentration of 3 μM.

Supplementary Material

Click here for additional data file. (470.2KB, pdf)

Acknowledgments

This work was supported by grants to L.G.-M. from the Miguel Alemán Valdés Foundation 2018, SEP-Cinvestav FIDSC2018/33, and National Council of Science and Technology of Mexico (Conacyt) FORDECYT-PRONACES-140644/2020. H.G.-G. and L.G.-G. were recipients of doctoral fellowships from the Conacyt (282075 and 340209), and H.G.-G. was also the recipient of a scholarship from the Mexiquense Council of Science and Technology (2018AD0009-11).

Abbreviations used:

AP

alkaline phosphatase

AP-1

activator protein 1

AU

arbitrary units

bp

bipartite

CAM-kinase II

Ca 2+/calmodulin kinase II

Co-IP

coimmunoprecipitation

CTGF

connective tissue growth factor

cyt

cytoplasmic

DAPI

4’,6-diamidino-2-phenylindole

DMSO

dimethyl sulfoxide

EGTA

ethylene glycol-bis(2-aminoethylether)- N, N, N’, N’-tetraacetic acid

ICM

inner cell mass

IP

immunoprecipitation

KO

knockout

LATS1/2

large tumor suppressor kinase 1/2

LEF

lymphoid enhancer family

MAGUK

membrane-associated guanylate kinase

MAPKAP2

MAP kinase-activated protein 2

MDCK

Madin-Darby canine kidney

mESC

mouse embryonic stem cells

mTSC

mouse trophoblast cells

NES

nuclear exportation signal

NLS

nuclear localization signal

nPKC

novel protein kinase C

ns

non significant

nt

nucleotide

nuc

nuclear

PIS

preimmune serum

PKA

protein kinase A

PLA

proximity ligation assay

PRAK/MK5

MAP kinase-activated 5

shRNA

short hairpin RNA

TAZ

transcriptional activator with PDZ-binding domain

TCF

T cell factor

TCF4

transcription factor 4

TEAD

transcriptional enhanced associated domain

TJ

tight junction

Transf

transfected

VGLL

vestigial like

WNK4

with no lysine kinase 4

WT

wild type

YAP

Yes-associated protein

ZO-2

zonula occludens.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E20-07-0470) on May 19, 2021.

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