Up-regulation Of The Transcription Activity Of β-catenin By EGF-induced Phosphorylation Of α-catenin

Overexpression of epidermal growth factor (EGF) receptor (EGFR) has been reported in many human tumors, including lung, colon, breast, prostate, brain, head and neck, thyroid, ovarian, kidney, and bladder cancers as well as gliomas, and correlates with a poor clinical prognosis in the tumors. Activation of the receptor via EGF promotes migration of tumor cells.β-catenin, a component of cell-cell adhesion structures, interacts with the cytoplasmic domain of E-cadherin and links E-cadherin toα-catenin, which in turn mediates anchorage of the E-cadherin complex to the cortical actin cytoskeleton. In addition to its role in cell-cell adherens junctions,β-catenin is also a key component of the Wnt/Wingless signaling pathway. Wnt signaling plays a central role in development, cell proliferation and differentiation. Therefore,β-catenin is important in these biological functions. Moreover, the transcription activity ofβ-catenin is up-regulated in many kinds of cancer cells. Activation of the Wnt pathway inhibits GSK-3β-dependent phosphorylation ofβ-catenin. Stabilized, hypophosphorylatedβ-catenin translocates to the nucleus and interacts with transcription factors of the TCF/LEF-1 family, leading to the increased expression of oncogenes such as c-myc and cyclin D1. Mutations in APC, AXIN1, or CTNNB1 (which encodesβ-catenin) enhanceβ-catenin stability and subsequent transactivation of TCF/LEF-1, and such transactivation is found in a wide variety of human cancers.However, mutations of Wnt pathway proteins that alter the stability ofβ-catenin are not the only factors that contribute toβ-catenin activation.β-catenin translocates into the nucleus and increases its transactivation without altering its stability and phosphorylation. The underlying mechanism of Wnt-independent regulation still remains unclear until now.Alpha-catenin, which functions as a molecular switch that binds E-cadherin andβ-catenin and regulates actin-filament assembly, has received less attention. Accumulated evidence points toα-catenin as a prognostic factor in cancer progression. Loss or downregulation ofα-catenin has been detected in cell lines derived from leukemia, colon, prostate, and other cancers as well as primary human cancers. The effect ofα-catenin regulation on tumor development occurs at least partially through regulation ofβ-catenin, as evidenced by the fact thatα-catenin overexpression leads to repression ofβ-catenin transcriptional activity, whileα-catenin depletion has the opposite effect. Nevertheless, the mechanism underlying thisα-catenin-dependent regulation ofβ-catenin remains elusive.We have previously shown that EGF treatment induces downregulation of caveolin-1, which promotesβ-catenin transactivation. However,it is not clear whether EGF stimulation affects the stability ofα-catenin andβ-catenin complex. To address this question, we treated EGFR-overexpressing U87E, U373, and A431 cells with EGF. Immunoblotting of immunoprecipitatedα-catenin with aβ-catenin antibody showed that EGF induced disruption of the interaction betweenβ-catenin andα-catenin in all tested cell lines. Immunofluorescence studies revealed that 10 hours of EGF treatment resulted in internalization ofβ-catenin and disruption of colocalization ofβ-catenin andα-catenin in cell-cell contacts. Alpha-catenin overexpression blockedβ-catenin nuclear accumulation induced by EGF stimulation, while depletion ofα-catenin through shRNA expression significantly enhanced TCF/LEF-1 transcriptional activity. These results strongly suggest that EGFR activation results in disruption of theβ-catenin andα-catenin complex, which in turn promotesβ-catenin transactivation.To examine whether the dissociation ofα-catenin fromβ-catenin is due to a possible unknown post-translational modification onα-catenin, we analyzed a tryptic digest of α-catenin immunoprecipitated from EGF-stimulated A431 cells by mass spectrometry. S641 was identified as a phosphorylation site in thisα-catenin peptide. The 641SDFE644 sequence ofα-catenin is closely related to the CK2 phosphorylation motif S/TXXD/E. Then we performed in vitro kinase assays with purified CK2αmixed with purified WT His-α-catenin or His-α-catenin S641A mutant. CK2αoverexpression enhanced the phosphorylation of S641. In converse, depletion of CK2αthrough shRNA expression significantly blocked the phosphorylation.To determine whether CK2α-regulated phosphorylation ofα-catenin affects the complex ofα-catenin andβ-catenin, we performed GST pull-down analyses. We found that CK2α, which phosphorylatesα-catenin, reduced the binding of WTα-catenin, but notα-catenin S641A mutant, toβ-catenin. Immunoblotting of immunoprecipitated FLAG-taggedα-catenin with aβ-catenin antibody showed that expression of CK2α, but not a CK2αK68M kinase-dead mutant, reduced the total amount ofα-catenin binding toβ-catenin. In line with EGF-induced and CK2-dependentα-catenin phosphorylation, EGF treatment reducedα-catenin binding toβ-catenin at endogenous levels, which can be blocked by expression of CK2αshRNA or pretreatment with CK2 inhibitor. Furthermore, a phosphorylation-mimicα-catenin S641D mutant, in which Ser was mutated into Asp, showed a much-reduced binding toβ-catenin in total cell lysate or in the membrane and nuclear fraction in contrast to WTα-catenin orα-catenin S641A mutant. These results indicate that phosphorylation ofα-catenin at S641 by CK2αin response to EGF stimulation reducesα-catenin binding toβ-catenin.To investigate how CK2 is regulated in response to EGF, we investigated whether CK2 is post-translationally modified by EGF-induced activation of protein kinases including EGFR, Src, and ERK. The results of in vitro kinase assay showed that ERK2 could phosphorylated CK2α. Mass spectrometry analysis of ERK2-phosphorylated CK2αprotein revealed T360 and S362 as potential phosphorylation sites. This result was further confirmed by in vitro kinase assay and in vivo 32P-phosphate-metabolic labeling results. MAP kinases bind to their substrates through a docking groove comprised of an acidic common docking (CD) domain and Glu-Asp (ED) pockets. Immunoblotting of immunoprecipitated CK2αwith anti-FLAG-tagged ERK2 antibody showed that mutation of either ERK2 CD domain (D316/319N) or ED pocket (T157/158E) reduced their binding to CK2αin contrast to WT ERK2, and a mutant (T/E-D/N) comprised mutations at both the CD domain and the ED pocket further reduced this binding. These results indicate that ERK2 binds to CK2αthrough its docking groove. Intriguingly, ERK2-mediated CK2αphosphorylation enhanced its activity towards its substrates in vitro. We co-expressed MEK1 Q56P mutant with WT ERK2 or ERK2 K52R kinase-dead mutant and with WTα-catenin or theα-catenin S641A mutant. Immunoblotting with the phospho-α-catenin S641 antibody revealed that activated ERK2, but not its kinase-dead mutant, significantly enhanced phosphorylation of WTα-catenin but not theα-catenin S641A mutant. Consistently, EGF-enhanced phosphorylation ofα-catenin S641 was blocked by pretreatment with MEK inhibitor U0126 or CK2 inhibitor TBB.The TCF/LEF-1 luciferase reporter analysis showed that inhibition of ERK2 or CK2αactivity significantly blockedβ-catenin transactivation. Consistent with the results that phosphorylation ofα-catenin regulatesβ-catenin/α-catenin complex stability, expression ofα-catenin S641D, which has a much reduced binding ability toβ-catenin, significantly lost its inhibitory effect on EGF-inducedβ-catenin transactivation in contrast to WTα-catenin orα-catenin S641A mutant. These results strongly suggest that EGF-induced phosphorylation ofα-catenin S641 greatly abolished its negative regulation onβ-catenin and promotedβ-catenin transactivation.β-catenin transcriptional activity has been closely related to tumor development. To examine the effect of phosphorylation ofα-catenin by CK2 on tumor cell invasion, we performed Matrigel transwell invasion assays. CK2αdepletion by shRNA or stable expression of WTα-catenin orα-catenin S641A mutant in A431 cells or U87E cells greatly reduced EGF-induced cell invasion. In contrast, expression ofα-catenin S641D, which has less effect onβ-catenin transactivation, minimally affected EGF-induced cell invasion.To determine whether our findings have clinical relevance, we examined ERK1/2 activity andα-catenin phosphorylation in serial sections of 46 human primary GBM specimens (WHO grade IV) by immunohistochemical analyses. The data showed that the levels of ERK1/2 activity were correlated with the levels of phosphorylation ofα-catenin at S641. In addition, we comparedα-catenin phosphorylation level in low-grade diffuse astrocytoma (WHO grade II with more than 5 years'median survival time) with high-grade GBM (with less than 1 year of median survival time). Immunohistochemical analyses of 23 human low-grade diffuse astrocytoma specimens showed significantly lower levels of phosphorylatedα-catenin S641 than were present in GBM specimens.We propose a mechanistic model for tumor cell invasion and metastasis that integrates these different components. EGFR activation results in the activation of ERK. ERK2, which interacts directly with CK2αregulated by ERK2 docking groove, phosphorylates CK2α, thereby enhancing CK2αactivity toward phosphorylation ofα-catenin at S641. The phosphorylatedα-catenin loses its binding toβ-catenin, which subsequently activatesβ-catenin-TCF/LEF-1 transcriptional activity. Our studies represent a novel and important mechanism underlying the effects of EGF during tumor development. The demonstration of a mechanistic interplay between EGF and Wnt/Wingless signaling components provides an important insight for further understanding tumor cell invasion and metastasis.