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A Novel Role of Phospho-B-Catenin in Microtubule Regrowth at Centrosome

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A Novel Role of Phospho-B-Catenin in Microtubule Regrowth at Centrosome Oncogene (2007) 26, 4357–4371 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE A novel role of phospho-b-catenin in microtubule regrowth at centrosome P Huang, T Senga and M Hamaguchi Department of Oncology, Division of Cancer Biology, Nagoya University Graduate School of Medicine, Showa ku, Nagoya, Japan b-Catenin is a biologically important molecule playing expression (Huelsken and Behrens, 2002). Recent critical roles in both cell adhesion and transcriptional studies, however, have shown that some components regulation in the Wnt pathway. Here, we show that of Wnt pathway regulate microtubule (MT) function phospho-b-catenin (phosphorylatedat Ser33/37/Thr41), (Gundersen and Cook, 1999; Harwood and Braga, which is reported to be degraded immediately after its 2003). GSK3b itself is present along spindle MTs phosphorylation, accumulatedin the centrosome. Whereas (Wakefield et al., 2003) and can regulate the phospho- phospho-mimicking mutant, S33/37/T41E-b-catenin, rylation of some MT-binding proteins such as tau, couldlocalize to the centrosome, S33A- b-catenin that adenomatous polyposis coli (APC), MAP1B to lacks the phosphorylation site lost its localization to the affect the stability of MTs (Lucas et al., 1998). Another centrosome. Phospho-b-catenin localizedmainly to key component of Wnt pathway, APC, binds to the mother centrosome during the interphase and was plus ends of MTs and stabilizes the growing ends recruitedto daughter centrosome in M-phase. Depletion (Zumbrunn et al., 2001). Similarly, Dishevelled-1, an of b-catenin with small interfering RNA or inhibition of its upstream molecule of b-catenin, is involved in the phosphorylation by LiCl treatment causeddisruption of regulation of MT stability (Krylova et al., 2000; Ciani radial microtubule (MT) array and retardation of the MT et al., 2003). b-Trcp1 may also regulate the timely order regrowth during the recovery from nocodazole treatment. of meiotic and mitotic events (Guardavaccaro et al., In addition, these treatments increased the frequency of 2003). mono-astral MT reorganization. Furthermore, overex- Many b-catenin-binding proteins are also involved in pression of the nonphosphorylatable b-catenin, but not the the regulation of the function of MTs. Dynein binds to phospho-mimicking b-catenin, markedly disrupted radial b-catenin and may tether MTs at adherens junctions. MT array andrepressedthe MT regrowth. In contrast, Overexpression of b-catenin disrupts the cellular loca- phospho-mimicking b-catenin localizedto both of the lization of dynein and dramatically perturbs both the duplicated centrosomes with aberrant larger and denser organization of MTs at the centrosome and the radial MTs array formation. In addition, some of the cells tethering of MTs at adherens junctions (Ligon et al., overexpressing phospho-mimicking b-catenin hadmultiple 2001). b-Catenin, APC, KAP3 and KIF3A-KIF3B are centrosomes. Taken together, this study demonstrates a in the same complex in vivo, and both APC and novel role of phospho-b-catenin in MT organization at the b-catenin are transported along MTs assisted by KAP3- centrosomes. KIF3A-KIF3B (Jimbo et al., 2002). Another b-catenin- Oncogene (2007) 26, 4357–4371; doi:10.1038/sj.onc.1210217; binding protein, EB1, is required for the formation and published online 29 January 2007 maintenance of the radial MT array anchored at the centrosome (Askham et al., 2002). A recent paper has Keywords: phospho-b-catenin; microtubule regrowth; provided direct evidence that b-catenin is associated centrosome with the establishment of bipolar mitotic spindle (Kaplan et al., 2004). In addition, b-catenin itself is reported to be associated with the regulation of the cell cycle. The level of b-catenin increases in S phase, reaching maximum accumulation at late G2/M. Over- Introduction expression of a nonphosphorylatable mutant b-catenin in epidermal keratinocyte induces G2 arrest (Olmeda Traditional Wnt/b-catenin signal transduction pathway et al., 2003). All these studies above indicate a possible plays key role in embryonic development and role of b-catenin on the regulation of the MT function cancer progression through b-catenin-regulated gene during the cell cycle. In this report, we show that phospho-b-catenin is localized to mother centrosome during the interphase Correspondence: Professor M Hamaguchi, Division of Cancer and distributed to both mother and daughter centro- Biology, Nagoya University Graduate School of Medicine, 65 somes in M-phase. Furthermore, we found phospho-b- Tsurumaicho, Showa ku, Nagoya, Aichi 466-8550, Japan. E-mail: [email protected] catenin plays a role on the maintenance of radial MT Received 3 October 2005; revised 9 October 2006; accepted 10 November array during the interphase by anchoring MT to the 2006; published online 29 January 2007 centrosome. Phospho-b-catenin and microtubule regrowth P Huang et al 4358 Results proteasome-dependent pathway. However, we found that phospho-b-catenin was localized at microtubule Phospho-b-catenin localizes mainly to mother centrosome organizing center (MTOC) (Figure 1a). Interestingly, during the interphase phospho-b-catenin was localized to only one of the two We first investigated the subcellular localization of centrosomes in cells (Figure 1b). Deconvolution analysis b-catenin and its phosphorylated form in rat fibroblast showed that phospho-b-catenin enveloped the centro- cell line, 3Y1. In normal cells, b-Catenin appears to be some (Figure 1b), which is similar to the localization distributed mainly in the membrane and diffusely of MT anchor proteins such as ninein and CEP100 throughout the cytoplasm (Sadot et al., 2002). b-catenin (Ou et al., 2002). As phospho-b-catenin is localized to only is phosphorylated by GSK-3b at Ser33, 37 and Thr41, one of the two centrosomes (Figure 1b), we checked and ubiquitinated by b-Trcp1 and degraded in a whether it is mother centrosome or not by staining with Figure 1 Localization of phospho-b-catenin in fibroblast cells. (a and b) Rat 3Y1 cells were immunostained with anti-phospho-b- catenin. MTs and centrosomes were visualized with FITC-conjugated anti-a- and anti-g-tubulin antibody, respectively. Phospho-b- catenin (red) localized to centrosomes (arrowhead). Zaxis reconstructions (b, bottom panels) were generated along the bars indicated in the upper panels. Bars, 20 mm. (c) Mother centrosome (arrowhead) was visualized with mother centrosome marker e-tubulin in human foreskin fibroblasts (HFF). Bar, 20 mm. (d) Rat 3Y1 cells were immunostained with anti-phospho-b-catenin and anti-EB1. Insets show higher magnification of the centrosome. Arrowheads point mother centrosome. Bar, 20 mm. Oncogene Phospho-b-catenin and microtubule regrowth P Huang et al 4359 a mother centrosome marker, e-tubulin. We found the detect mutant b-catenin (S33A) whose Ser33 was co-localization of phospho-b-catenin with e-tubulin replaced with alanine (Figure 2a). To confirm phos- (Figure 1c). EB1 localizes to the mother centrosome and phorylation-dependent localization of b-catenin to the C-terminus of EB1 interacts with b-catenin (Askham centrosome, we constructed a phospho-mimicking et al., 2002). We also observed co-localization of EB1 mutant b-catenin whose Ser33, 37 and Thr41 were and phospho-b-catenin (Figure 1d). However, we replaced to glutamic acid (S33/37/T41E) and transfected could not detect direct interaction of phospho-b-catenin it to COS7 cells. Substitution of serine or threonine to with e-tubulin and EB1 by immunoprecipitation (Supple- glutamic acid is known to mimic phosphorylation. mentary Figure S1a). Although S33/37/T41Eb-catenin localized to the centro- The phospho-b-catenin antibody has been reported to some, we could not see any localization of S33Ab- detect specifically phosphorylated b-catenin (Kielhorn catenin to the centrosome (Figure 2b arrowhead). These et al., 2003), and we confirmed that this antibody did not results with mutant b-catenins are consistent with our Figure 2 S33/37/T41E b-catenin localizes to the centrosome. (a) Lysates of COS7 cells expressing b-catenin WT or mutant were subjected to immunoblotting with rabbit anti-phospho-b-catenin antibody. Then the membrane was reprobed with anti-b-catenin or anti-GFP antibody. Line 1, GFP; Line 2, b-catenin-GFP; Line 3, b-catenin-S33A-GFP; Line 4, b-catenin-S33/37/T41E-GFP. (b) COS7 cells transfected with b-catenin-S33/37/T41E-GFP or b-catenin-S33A-GFP were stained with anti-g-tubulin and the localization of b-catenin mutant was visualized with GFP. Bar, 20 mm. (c) COS7 cells transfected with b-catenin-S33/37/T41E-GFP were stained with anti-g-tubulin. Bar, 20 mm. Insets show higher magnification of the centrosomes. Oncogene Phospho-b-catenin and microtubule regrowth P Huang et al 4360 observation that phospho-b-catenin localizes to the onset of M-phase, phospho-b-catenin was distributed to centrosome. In contrast to endogenous wild-type both mother and daughter centrosomes (Figure 3a). phospho-b-catenin localized to the mother centrosome, Consistent with this observation, there was a dramatic S33/37/T41E mutant unexpectedly localized to both of increase of phospho-b-catenin at G2/M-phase, as the duplicated centrosomes visualized with g-tubulin. compared with that at G1-phase (Figure 3b). We also Moreover, we observed that some of the cells over- found that phospho-b-catenin was present at both expressing S33/37/T41Eb-catenin had multiple centro- centrosomes throughout the whole M-phase (Figures 3c somes, every one of which had association of S33/37/ and Supplementary Figure S2a). This distinctive distri- T41Eb-catenin (Figure 2c). bution pattern led us to speculate that it may take a role in daughter centrosome maturation and spindle pole Phospho-b-catenin is recruited to daughter centrosome in formation. As cells progressed to late telophase, phospho- M-phase b-catenin accumulated at mid-body, indicating a possible We next examined the localization of phospho-b-catenin localization of phospho-b-catenin to the plus ends of in various cell-cycle stages.
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