Phosphorylated Tyr142 β‐catenin localizes to centrosomes and is regulated by Syk

β‐catenin is a central component of adherent junctions and a key effector of canonical Wnt signaling, in which dephosphorylated Ser/Thr β‐catenin regulates gene transcription. β‐catenin phosphorylation at Tyr142 (PTyr142 β‐catenin), which is induced by receptor and Src family Tyr kinases, represents a previously described β‐catenin switch from adhesive to migratory roles. In addition to classical β‐catenin roles, phosphorylated Ser/Thr β‐catenin and total β‐catenin were involved in centrosomal functions, including mitotic spindle formation and centrosome separation. Here we find that PTyr142 β‐catenin is present in centrosomes in non‐transformed and glioblastoma cells and that, in contrast to the Ser/Thr phosphorylated β‐catenin, PTyr142 β‐catenin centrosomal levels drop in mitosis. Furthermore, we show that the inhibitor of Spleen Tyrosine Kinase (Syk) piceatannol decreases centrosomal PTyr142 β‐catenin levels, indicating that Syk regulates centrosome PTyr142 β‐catenin. Our findings suggest that PTyr142 β‐catenin and Syk may regulate centrosomal cohesion. This study highlights the contribution of different phosphorylated β‐catenin forms to the cell and centrosome cycles.


INTRODUCTION
Centrosomes are microtubule (MT) organizing centers that control cell polarity, adhesion, motility and cytokinesis. The two centrosomes present at the onset of mitosis determine the proper formation of bipolar MT spindles that control chromosome segregation. Thus, alterations in centrosome functions and number contribute to chromosome instability and are frequent in cancer [Nigg et al., 2002;Gonczy et al., 2015].
A centrosome consists of two centrioles surrounded by pericentriolar matrix (PCM), where γ-tubulin-ring complexes act as MT-nucleating templates. Centrosome structure and number are regulated through the cell cycle. During mitosis, the centrosome at each pole of the mitotic spindle contains a pair of tightly connected centrioles. However, this association is lost at the end of mitosis, leading to the separation of the daughter cells.
Centriole duplication occurs in S phase, with pro-centrioles that elongate and function as a single MT-organizing center until late G2. At G2-M transition, centrosome maturation involves the exchange of PCM components and further recruitment of γtubulin complexes. In response to activated MT-dependent motor proteins, centrosomes finally separate and instruct the formation of spindle poles that ensure that each daughter cell inherits one centrosome. β-catenin is a component of cell adhesion complexes and a key Wnt signalling effector.
In the cell adhesion complex, β-catenin dissociation from cadherin or from α-catenin, in part induced by its Tyr phosphorylation, decreases cell adhesion while increasing cell migration. Furthermore, in the canonical Wnt signalling pathway, β-catenin acts as transcriptional co-regulator together with transcription factors of T-cell 4 Factor/Lymphoid Enhacer Factor families. Briefly, when signalling is off, β-catenin taking part of the destruction complex is phosphorylated by Glycogen Synthase Kinase (GSK)-3β at Ser33/37/Thr47, which is then degraded by the proteasome. In contrast, in the presence of Wnt, β-catenin (dephosphorylated at Ser33/37/Thr47) accumulates and regulates Wnt target expression. Interestingly, β-catenin phosphorylation at Tyr142 (PTyr142 β-catenin) implies a switch from adhesive to transcriptional functions independent of Wnt [Brembeck et al., 2004;Heuberger et al., 2010], which is regulated by Met and Hepatocyte Growth Factor signalling [David et al., 2008;Nager et al., 2015] and other Tyr kinases [Piedra et al., 2003], PTyr142 β-catenin therefore dissociates from the adhesion complex, localizes to the nucleus and promotes cell migration and axon growth in neurons [David et al., 2008;Nager et al., 2015].
Here we analyze the putative presence of β-catenin phosphorylated at Tyr142 in centrosomes and its regulation. We describe for the first time the centrosome localization of PTyr142 β-catenin in astrocytes and glioblastoma (GBM) cells. Using phosphospecific antibodies, we demonstrate the co-localization/co-fractionation of PTyr142 β-catenin with centrosome markers. Cell cycle analysis indicates that centrosomal PTyr142 β-catenin levels fall in mitosis, paralleling a drop of Syk centrosomal levels reported previously [Zyss et al., 2005]. Furthermore, we show that Syk phosphorylates β-catenin at Tyr and that centrosome PTyr142 β-catenin levels decrease upon exposure to the Syk inhibitor piceatannol (Pic). Thus, we identify a PTyr142 β-catenin centrosomal pool regulated by Syk that, different to PSer/Thr βcatenin [Huang et al., 2007], vanishes from the centrosome in mitosis.

Investigation with experimental animals was approved by the Experimental Animal
Ethic Committee of UdL (CEEA 10-02/08, CEEA 11-02/08) and followed the Helsinki declaration. Animals were anesthetized with a lethal dose of inhaled isoflurane and decapitated within the animal facility.

CELL TRANSFECTION
For knockdown using siRNA, RNAimax Lipofectamine was used. Briefly, 25 µl of Optimem containing siRNA sequences (80 nM; Sigma; VPDSIRNA2D for β-catenin and PDSIRNA2D for GAPDH, used as control), together with 0.2 µg of Green Fluorescence Protein DNA plasmid, were mixed with 25 µl of Optimem plus 1 µl of RNAimax Lipofectamine. The mix was incubated for 15 min at room temperature (RT) 7 and added dropwise to cells for 6h. Then media was changed for complete media (MEM including FBS).
Mitosis arrest was carried out on cells plated on PDL-coated coverslips in complete media. Cells were deprived of serum for 20h and then grown in complete media for the 16-20h before methanol fixation.

WESTERN BLOTTING
Proteins were resolved by SDS-PAGE and transferred to Immobilon-P using a semi-dry apparatus. Membranes were blocked with Tris-buffered saline plus Tween-20 (TBS-T) 10 (20 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.05% Tween-20) containing 5% non-fat dry milk (1h, RT), washed with TBS-T and incubated overnight with the primary antibody. Membranes were then incubated with peroxidase-conjugated secondary antibodies before developing, using either Enhanced chemiluminescence (ECL) or Supersignal reagents.

RESULTS
Different cell types, including primary rodent fibroblasts, striatal astrocytes and human GBM (U251MG and U87MG) cells were immunostained against PTyr142 β-catenin and γ-tubulin, a centrosome marker. The co-localization of PTyr142 β-catenin and γtubulin immunostainings suggested the presence of PTyr142 β-catenin in centrosomes in all tested cell types, including cancerous and non-transformed cells ( Figure 1A). This immunostaining was not observed in absence of primary antibody (data not shown) or in β-catenin silenced cells ( Figure 1B). To confirm the centrosomal localization of PTyr142 β-catenin, we performed centrosome subfractionation and immunoprecipitation experiments. Centrosome fractions were isolated from U251MG cells, using a discontinuous sucrose gradient centrifugation method [Hsu et al., 1998].
We then studied whether centrosome PTyr142 β-catenin is regulated in mitosis. To this end, we used U251MG cells upon cell cycle-arrest induced by serum deprivation for 20h followed by a period of 16-20h of release in complete media, in which mitotic figures were captured. Centrosomal PTyr142 β-catenin immunostaining was compared during the different mitotic phases identified by Hoechst (to label the DNA) and α-tubulin (to reveal the MT reorganization) stainings ( Figure 3). While PTyr142 β-catenin appeared centrosomal in interphase cells (arrows; Figure 3), PTyr142 β-catenin decreased abruptly in metaphase centrosomes and remained absent from centrosomes until the end of telophase/cytokinesis, when it reappeared at the centrosomes of the daughter cells (Figure 3). These findings indicate that centrosomal PTyr142 β-catenin is lost in mitosis, suggesting that its presence negatively affects mitotic progression.
Next, we sought to investigate which kinase regulates PTyr142 β-catenin at the centrosome. Syk appeared as a possible candidate because it is associated to interphase centrosomes, is removed from centrosomes in mitosis [Zyss et al., 2005] and phosphorylates cell adhesion proteins (E-cadherin and α-catenin) [Larive et al., 2009].
Immunostaining experiments confirmed that Syk localizes to centrosomes in GBM cells (data not shown), in agreement with subfractionation experiments. Therefore, we performed in vitro kinase assays using recombinant Syk and β-catenin Wild-type (WT) or Y142F. Results indicate that Syk phosphorylates WT β-catenin in Tyr residues, which was inhibited by the prototypical ATP-competitive kinase inhibitor Staurosporine and by the Syk inhibitor Pic [Oliver et al., 1994] ( Figure 4A). In addition, PTyr phosphorylation of Y142F β-catenin was reduced compared to that of WT protein, and its phosphorylation level was similar with or without Pic (close to basal phosphorylation levels detected in absence of ATP; Figure 4A).
Next, we tested whether regulating Syk activity could affect centrosomal PTyr142 βcatenin in GBM cells treated with Pic. Centrosomal PTyr142 β-catenin immunostaining decreased in GBM cells treated with Pic vs. control cells (by 40-50% in U251MG and U87MG cells, respectively, according to fluorescence intensity measurements; Figure   4B and 4C). Centrosomal PTyr142 β-catenin fluorescence intensity was not statistically 13 significant when control cells were compared to cells treated with sodium pervanadate, a Tyr phosphatase inhibitor and Syk activator [Larive et al., 2009] (Figure 4C). In addition, U251MG cells treated with Pic showed an increased % of cells displaying two well separated centrosomes compared to control cells ( Figure 4D), suggesting that PTyr142 β-catenin may be involved in centrosomal cohesion/separation. Together, these results indicate that Syk phosphorylates centrosomal β-catenin in Tyr142.

DISCUSSION
Although β-catenin's best studied roles include the transcriptional regulation of Wnt targets and cell adhesive functions [Valenta et al., 2012], the association of β-catenin with the centrosome has been known for years [Kaplan et al., 2004], when early studies showed its contribution to mitotic spindle formation and centrosome separation [Bahmanyar et al., 2008;Kaplan et al., 2004]. Mitogenic Wnt/β-catenin signalling regulates cell proliferation, and β-catenin and Wnt pathway components located to the centrosomes assist mitosis [Niehrs and Acebron, 2012]. Indeed, in addition to β-catenin, other Wnt pathway components have been localized to the centrosomes [Mbom et al., 2013] and regulate centrosome functions. Axin1 is involved in MT nucleation [Fumoto et al., 2009], whereas Axin2 regulates centrosome cohesion [Hadjihannas et al., 2010].
Moreover, APC and Dishevelled regulate the attachment and orientation of the mitotic spindle in which Wnt receptors Frizzled and LRP6 cooperate [Kikuchi et al., 2010].
PTyr142 β-catenin could associate to the centrosome in the phosphorylated state or be induced by a centrosomal kinase. Looking for putative Tyr kinases that could phosphorylate centrosomal PTyr142 β-catenin, we identified Syk as a putative candidate. Syk is a Tyr kinase localizing to the centrosome [Zyss et al., 2005;Fargier et al., 2013]. Interestingly, Syk is persistent in interphase centrosomes but its levels drop in mitosis due to degradation by the proteasome system [Zyss et al., 2005]. We therefore addressed whether centrosomal Syk could regulate PTyr142 β-catenin by two approaches: overexpression of Syk active or inactive mutants and by treatment with the Syk inhibitor, Pic [Oliver et al., 1994;Larive et al., 2009]. Whereas cells overexpressing Tyr130Glu active Syk showed co-localization between active Syk and PTyr142 βcatenin at centrosomes, cells expressing Lys402Arg inactive Syk still displayed centrosomal PTyr142 β-catenin (results not shown). We reasoned that endogenous Syk could still be phosphorylating β-catenin upon expression of exogenous inactive Syk.
Immunostaining for α-tubulin allowed the visualization of MTs and, together with Hoechst (blue), helped identifying the mitotic stage. Note that interphase and daughter cells (after cytokinesis) show centrosomal PTyr142 β-catenin immunostaining (arrows).