Introduction

Wnt/β-catenin signaling is associated with the development and progression of multiple cancers [1]. Activation of this pathway is linked to accumulation of β-catenin in the nucleus followed by increased expression of genes, including cyclin D1, vegf, and survivin, all of which are the known contributors to cancer progression [2–5].

Protein kinase CK2 regulates essential cellular processes, such as proliferation and death. This enzyme has been shown to interact with and phosphorylate β-catenin, regulating its stability and transcriptional activity [6, 7]. Similarly, AKT/PKB is known to regulate cell progression and viability [8, 9]; however, recently it has been reported that AKT phosphorylates β-catenin at Ser552 which leads to its dissociation from cell–cell contacts, nuclear import and, consequently, increases β-catenin transcriptional activity [10, 11].

The putative role of AKT in regulating β-catenin has emerged as a relevant issue since CK2 interacts with, phosphorylates and hyperactivates AKT [12, 13]. Indeed, it has been shown that the residue Ser129 in AKT is phosphorylated by CK2, and mutation of this residue to alanine causes a marked decrease in AKT activity in vivo and in vitro and, interestingly, a reduced phosphorylation of Thr308, which is known as a canonical residue linked to its activation [12]. This suggests that phosphorylation of Ser129 by CK2 may lead to enhanced activity of AKT. Nevertheless, whether activation of AKT by CK2 does involve increased β-catenin activity both in the normal and cancer cells is still unsolved.

The data presented here indicate that the CK2α-dependent up-regulation of β-catenin transcriptional activity is a process involving the kinase AKT. When the role of CK2α on β-catenin-Tcf/Lef signaling was analyzed upon co-expression of an AKT mutant deficient in Ser129 phosphorylation by CK2, namely AKT-S129A, a decreased β-catenin transcriptional activity was observed which correlated with a diminished expression of the antiapoptotic protein survivin and, subsequently, decreased survival in HEK-293T cells. Therefore, a functional interaction between CK2 and AKT may be occurring in the Wnt/β-catenin pathway-dependent cancer progression.

Materials and methods

Cell culture and transfection

Cells were cultured at 37°C and 5% CO2 in DMEM (Invitrogen, Paisley, Scotland, UK) supplemented with 10% FBS (HyClone, Logan, Utah, USA) and antibiotics (10,000 U/ml penicillin, 10 μg/ml streptomycin). Superfect® was used for transfections according to the instructions provided by the manufacturer (Qiagen, Valencia, CA, USA).

Plasmids

Plasmid for CK2α has been previously described [14, 15]. Plasmid pEGFP-β-catenin encodes GFP-tagged β-catenin wild-type [16], and pCMV6-HA-myr-AKT1-S129A encodes an AKT mutant deficient in phosphorylation by CK2 in Ser129 [12]. Reporter plasmids pLuc-1710 (containing the survivin promoter with Tcf/Lef binding sites) and pLuc-420-3M (mutated Tcf/Lef binding sites) have already been described [17].

Reporter assay

Cells were transfected with different plasmids (15 μg total DNA) as indicated in each figure. Cells were lysed in 100 mM KH2PO4 (pH 7.9)/1 mM DTT/0.5% Triton X-100 and supernatants used to measure luciferase activity. Values were used for calculating the 1710/420-3M ratios.

Microscopy

Cells were grown on glass coverslips and transfected as indicated. After rinsing with PBS, cells were fixed at 4°C in PBS/4% paraformaldehyde and mounted onto slides with 10% Mowiol/2.5% DABCO. Fluorescence was visualized in an inverted epifluorescence microscope Eclipse E400 (Nikon) equipped with a CCD camera DS-RI1 (Nikon).

Results

CK2-dependent phosphorylation of AKT is necessary to enhance cell viability

We initially evaluated the effect of overexpressing the catalytic CK2α subunit and the AKT mutant deficient in Ser129 phosphorylation by CK2, namely AKT-S129A [12], on viability of HEK-293T cells. As observed in Fig. 1, overexpression of CK2α enhanced a 26% cell viability whereas no effect was observed with the AKT-S129A mutant. Interestingly, when AKT-S129A was co-expressed together with CK2α, this mutant almost completely abolished the CK2α-dependent enhancement in cell viability. These data suggested that CK2-mediated phosphorylation of AKT is important to promote cell viability in HEK-293T cells.

Fig. 1
figure 1

CK2α-dependent enhance of cell viability was impaired by the AKT-S129A mutant. HEK-293T cells were co-transfected with plasmids encoding HA-CK2α and/or HA-AKT-S129A mutant. Eight hours post-transfection, cells were trypsinized and seeded by triplicate in a 96-well plate and grown for 16–18 h. Viability was determined using the MTS® assay according to the instructions provided by the manufacturer (Promega Corp, Madison, WI). Data depicted (mean±SE) were calculated considering the mock-transfected cells as 100%

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CK2-dependent phosphorylation of AKT promotes nuclear accumulation of β-catenin

Wnt/β-catenin activation is linked to nuclear migration followed by an augmented target gene expression. Therefore, overexpression of an AKT form deficient in Ser129 phosphorylation by CK2 can lead to a decreased nuclear accumulation of β-catenin. Thus, we analyzed the effect of AKT-S129A on β-catenin subcellular localization using the fusion protein GFP-β-catenin. We observed a change in GFP localization from a broadly cell-distributed (Fig. 2a) to a more nuclear-restricted in cells co-expressing HA-CK2α (Fig. 2b). Rather than observing a change in subcellular localization, GFP-β-catenin fluorescence seemed to be decreasing in the nucleus of cells overexpressing AKT-S129A (Fig. 2c), whereas nuclear as well as cytoplasmic fluorescence appeared to be diminished in the cells co-expressing CK2α and AKT-S129A (Fig. 2d). Noteworthy, we observed a change in morphology when CK2α and AKT-S129A were co-expressed, which was highly suggestive of apoptosis in a way similar to when CK2 was inhibited by the specific inhibitor DMAT, which correlates to a reduced expression of the antiapoptotic protein survivin [14].

Fig. 2
figure 2

Nuclear localization of β-catenin was modulated by phosphorylation of AKT by CK2. HEK-293T cells were grown on glass coverslips and transfected with plasmids encoding GFP-β-catenin alone (a) or together with HA-CK2α (b), HA-AKT-S129A (c), or HA-CK2α + HA-AKT-S129A (d). Cells were cultured for 16 h, fixed in paraformaldehyde, mounted onto slides with mowiol, and analyzed for GFP by epifluorescence microscopy

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Impairment of AKT phosphorylation by CK2 inhibits both CK2α-dependent up-regulation of survivin and resistance to apoptosis

The change in cell morphology after co-expression of CK2α and AKT-S129A prompted us to evaluate the expression of survivin, and subsequently the resistance to apoptosis. We analyzed the transcription of survivin gene using a specific survivin’s promoter-dependent reporter assay. As expected, reporter activity was significantly reduced when the AKT-S129A mutant was overexpressed alone (Fig. 3a). In addition, AKT-S129A co-expressed together with CK2α led to a complete blockage of the CK2α-dependent up-regulation of survivin, which was reminiscent of results using both TOP/FOP-Flash (data not shown) and microscopy (Fig. 2). Likewise, protein levels of survivin under the same conditions paralleled to data observed on reporter assay (Fig. 3b). Finally, we evaluated the roles of CK2α and AKT-S129A overexpression on resistance to apoptosis of cells grown in the presence of the specific CK2 inhibitor DMAT (50 μM). CK2α overexpression had a minor effect, whereas AKT-S129A promoted a significant increase of apoptosis (Fig. 3c). Notably, when AKT-S129A was co-expressed together with CK2α, this mutant almost completely abolished the CK2α-dependent effect in resistance to apoptosis, correlating with the above-mentioned effects in cell viability (Fig. 1).

Fig. 3
figure 3

CK2α-dependent expression of survivin and resistance to apoptosis were abolished by the AKT-S129A mutant. a HEK-293T cells were co-transfected with combinations of the survivin-specific reporter vector pLuc-1710 (survivin promoter containing wild-type Tcf/Lef-binding sites) or pLuc-420-3 M (mutated Tcf/Lef-binding sites), and plasmids encoding HA-CK2α wild-type and/or HA-AKT-S129A mutant. Values for luciferase activity were used for calculating the 1710/420-3M ratios (mean ± SE). b Cell lysates of conditions as in A were used to determine survivin protein levels by Western blot using a specific polyclonal antibody (R&D Systems, Minneapolis, MN, USA). c Cells transfected as in A were incubated in the presence of 50 μM DMAT for 12–16 h, and apoptosis was measured by the Caspase Glo® assay according to the instructions provided by the manufacturer (Promega Corp, Madison, WI)

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Discussion

Protein kinase CK2 has been suggested to be part of a protein complex that positively regulates the Wnt/β-catenin signaling pathway by interacting and phosphorylating β-catenin, which result in its increased transcriptional activity [6, 7]. In addition, data indicate that phosphorylation of β-catenin at Ser552 by protein kinase AKT/PKB leads to its increased transcriptional activity [10]. Likewise, we have observed that overexpressions of both a constitutively active and a dominant negative form of AKT resemble to those with wild-type and dominant negative forms of CK2α, respectively [18], supporting the role of AKT in promoting the transcriptional activity of β-catenin as well as the role of CK2α in those events.

Interestingly, it has been demonstrated that CK2 hyperactivates AKT by phosphorylation at Ser129 while mutation of this residue to alanine decreases the activity of AKT both in vitro and in vivo [12]. Indeed, overexpression of an AKT deficient in Ser129 phosphorylation by CK2, namely AKT-S129A, had a negative effect on β-catenin-dependent activity in HEK-293T cells, which is reminiscent of that observed with CK2α dominant negative [18]. These data suggested that phosphorylation of AKT by CK2 is important for regulating the transcriptional activity of β-catenin.

In addition, nuclear levels of β-catenin appeared decreased when the AKT-S129A mutant was overexpressed either alone or together with CK2α, indicating that phosphorylation of β-catenin by AKT would bypass the Axin/APC/GSK3β complex and, consequently, increase the nuclear localization and the transcriptional activity of β-catenin.

The morphological changes observed in HEK-293T cells co-transfected with CK2α and AKT-S129A, were indicative of the occurrence of apoptosis. We have already demonstrated that CK2α overexpression or specific inhibitors modulate the β-catenin-dependent expression of the IAP member survivin, leading to changes in proliferation and resistance to apoptosis in HEK-293T cells [14]. Likewise, we observed a marked reduction in mRNA and protein levels of survivin in colon and breast cancer cell lines upon treatment with the CK2-specific inhibitor TBB, which significantly reduced viability, augmented apoptosis, and altered the cell cycle in these cells [14]. In agreement with our results, decreased expressions of survivin and other IAP members have been observed upon treatment with TNFα or TRAIL in prostate cancer cells [19]. As expected in this study, low mRNA and protein levels of survivin were indeed indicative of the decreased nuclear localization and transcriptional activity of β-catenin, when CK2α and AKT-S129A were co-expressed, leading to both decreased viability and increased apoptosis in human embryonic kidney cells.

In summary, our data support a mechanism of CK2α-dependent regulation of β-catenin transcriptional activity by bypassing the complex Axin/APC/GSK3β but running through the CK2-mediated phosphorylation of AKT at Ser129 (see model in Fig. 4). This phosphorylation results in hyperactivation of AKT which subsequently may phosphorylate β-catenin at Ser552, probably promoting the binding of protein(s) to β-catenin and enhancing its nuclear import and transcriptional activity on cancer-related genes [10, 11, 14]. Despite promoting the β-catenin transcriptional activity, phosphorylation of AKT by CK2 does not affect the cytosolic stability of β-catenin [18], but the protein levels of survivin are augmented, which enhances cell viability and resistance to apoptosis. Taken together, these data suggest a novel functional interaction of CK2 with AKT in the Wnt/β-catenin signaling-dependent cancer progression.

Fig. 4
figure 4

Mechanism model for up-regulation of β-catenin activity by CK2. β-Catenin can be found in normal epithelial cells at the plasma membrane (bound to E-cadherin), cytosolic (N-terminal phosphorylated and targeted for proteasomal degradation), and nuclear (transactivating the expression of its target genes). In colon cancer cells, an aberrant activation of this pathway may occur through CK2, by which β-catenin escapes from binding to E-cadherin, and proteasomal degradation no longer occurs. This mechanism takes into consideration of not only the phosphorylation of β-catenin at Thr393 by CK2 [6, 7] but also the phosphorylation of AKT at Ser129 [12, 13], which subsequently would phosphorylate β-catenin at Ser552 [10]. As a consequence, β-catenin migrates to the nucleus, enhancing the expression of cancer-related genes such as survivin [14]. In cancer cells, survivin should promote increased proliferation and resistance to apoptosis, as well as the ability for invading distant tissues

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