Protein kinase CKII regulates the interaction of β-catenin withα -catenin and its protein stability

Stephan Bek 0 1 Rolf Kemler ) 0 0 Department of Molecular Embryology, Max-Planck Institute of Immunobiology , Stuebeweg 51, D-79108 Freiburg , Germany 1 Present address: Stephan Bek, Aventis Pharma Deutschland , Functional Genomics, Industriepark Hoechst, G879/029, D-65926 Frankfurt/Main , Germany - b -Catenin is a multi-functional cellular component and a substrate for several protein kinases. Here we investigated the interaction of protein kinase CKII (casein kinase II) and b -catenin. We show that CKII phosphorylates the Nterminal region of b -catenin and we identified Ser29, Thr102, and Thr112 as substrates for the enzyme. We provide evidence that CKII regulates the cytoplasmic stability of b -catenin and acts synergistically with GSK-3b in the multi-protein complex that controls the degradation of b -catenin. In comparing wild-type and Ser/Thr-mutant b -catenin, a decreased affinity of the mutant protein to a catenin was observed. Moreover, kinase assays in vitro Introduction b -Catenin was originally identified in association with the cell adhesion molecule E-cadherin (Vestweber and Kemler, 1984; Ozawa et al., 1989; Nagafuchi and Takeichi, 1989), but has also recently won increasing interest as an important mediator in the Wnt signaling pathway (for a review, see Polakis, 2000). In the cadherin cell-cell adhesion complex b -catenin plays a central role since it binds to both the cytoplasmic domain of E-cadherin and to a -catenin, which in turn binds directly to Factin and other actin-associated proteins (Ozawa et al., 1990; Stappert and Kemler, 1994; Huber and Weis, 2001). This association with the actin-based cytoskeleton is important for the adhesion function of all classical cadherins, as best demonstrated for the E-cadherin-catenin complex in adherens junctions of epithelial cells (for a review, see Aberle et al., 1996a). Although the respective binding sites between Ecadherin and b -catenin and between a - and b -catenin have been characterized in detail (Stappert and Kemler, 1994; Aberle et al., 1996b; Huber et al., 1997; Pokutta and Weis, 2000; Yang et al., 2001), recent evidence underlines the importance of post-translational modifications in modulating the function of this adhesion complex. Phosphorylation of Ecadherin by casein kinase II (CKII) and glycogen synthese kinase-3b (GSK-3b ) increases the affinity of E-cadherin for b catenin and thus enhances E-cadherin-mediated cell adhesiveness (Huber and Weis, 2001; Lickert et al., 2000). In contrast, reduced cell adhesion has been reported upon tyrosine phosphorylation of b -catenin (Ozawa and Kemler, 1998; Hazan and Norton, 1998; Gaudry et al., 2001) and an association of b -catenin with receptor-tyrosine kinases, EGFdemonstrate a CKII-dependent increase in the binding of wild-type b -catenin with a -catenin. In line with that, cells expressing Ser/Thr-mutant b -catenin exhibit an increased migratory potential, which correlates with an enhanced cytosolic localization and a reduced association with the cytoskeleton of the mutant protein. From these results we conclude that CKII regulates the function of b -catenin in the cadherin adhesion complex as well as its cytoplasmic stability. R, IGF-R and c-Met (Hoschtzky et al., 1994; Hiscox and Jiang, 1999; Playford et al., 2000) as well as with c-src (Kinch et al., 1995), has been shown. In comparison, little is known about the role of serine/threonine (Ser/Thr) phosphorylation of b -catenin in modulating the function of the E-cadherin adhesion complex. It is known that phosphorylation of b catenin by GSK-3b determines its availability as a transducer of Wnt signals. In the absence of Wnt, phosphorylation by GSK-3b targets b -catenin for degradation (Yost et al., 1996; Aberle et al., 1997; Orford et al., 1997). Phosphorylation of b catenin by GSK-3b occurs in a multiprotein complex that includes the tumor suppressor Adenomatous Polyposis Coli (APC) (Munemitsu et al., 1995), Axin (Zeng et al., 1997), or Conductin (Behrens et al., 1998). The composition of this multiprotein complex and the integration of b -catenin depend on the phosphorylation of each component by GSK-3b (reviewed by Seidensticker and Behrens, 2000; Dominguez and Green, 2001). Wnt inhibits GSK-3b -mediated phosphorylation of b -catenin and under these conditions b catenin enters the nucleus and forms complexes with transcription factors of the LEF/Tcf family, resulting in specific transcriptional activation (reviewed by Roose and Clevers, 1999; Hecht and Kemler, 2000). b -catenin is implicated in quite different cellular processes, which requires a fine-tuned regulation of its function, so it is very likely that b -catenin is a substrate for other yet to be identified protein kinases. Indeed, it was recently reported that CKII phosphorylates b -catenin (Song et al., 2000), and that CKII and b -catenin co-immunoprecipitate with Dvl proteins, the mammalian homologues of Drosophila Dishevelled (Dsh). From these results it was concluded that CKII participates in Wnt signaling and may act as a positive regulator in this pathway although the underlying molecular mechanisms are at present poorly understood. CKII exists as a constitutively active tetramer that contains two catalytic (a or a ) and two regulatory (b) subunits (Pinna and Meggio, 1997; Allende and Allende, 1995). Although more than 160 substrates have been identified to date, the regulation of this ubiquitously expressed pleiotrophopic kinase remains unclear. A nuclear shift of CKII-a during G1-phase and in proliferating cells (Seldin and Leder, 1995; Keliher et al., 1996; Landesman-Bollag et al., 1998; McKendrick et al., 1999; Ahmed, 1994) points towards a role of CKII in mitotic control and proliferation. However, due to the broad subcellular distribution, it is generally assumed, that CKII is controlled by different interaction partners and in different subcellular compartiments (for a review, see Faust and Montenarh, 2000). In a search for protein kinases that use b -catenin as substrate we confirmed that CKII also phosphorylates b -catenin. We have now identified amino acid (aa) residues in b -catenin phosphorylated by CKII and performed a mutational analysis to obtain first insights into the biological function of this posttranslational modification. By comparing wild-type (wt) and Ser/Thr-mutated (Ser/Thr-mutant) b -catenin in kinase assays in vitro and in vivo we provide evidence here that CKII regulates the best studied functions of b -catenin (i.e. its central role in the E-cadherin adhesion complex and its tight control of cytoplasmic stability), which is a prerequisite for the canonical Wnt signaling pathway. Materials and Methods Mouse epithelial carcinoma CMT cells (ATTC, CCL-223) and human kidney epithelial 293 cells (ATTC, CRL-1573) were grown at 37C/10% CO2 in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 2 mM L-glutamine. 293 cells stably expressing eithe (...truncated)