Significantly, non-MT-nucleating, abnormal -tubulin puncta that have no pericentrin have been described in parental HCT116 cells and correlated with polo-like kinase (Plk) 4 levels (Kuriyama et al., 2009), but the role of mutant -catenin was not tested. All five HCT116 cell lines had a low percentage of abnormal -tubulin puncta that did not contain pericentrin (Fig. of centrosome proteins. HCT116 human colon cancer cell lines, from which the mutant -catenin allele has been deleted, have reduced numbers of cells with abnormal centrosome structures and S-phase-arrested, amplified centrosomes. RNAi-mediated depletion of -catenin from centrosomes inhibits S-phase-arrested amplification of centrosomes. These results indicate that -catenin is required for centrosome amplification, and mutations in -catenin might contribute to the formation of abnormal centrosomes observed in cancers. Keywords:-Catenin, Centrosomes, -Tubulin == Introduction == Mutations in -catenin are found in many cancers and are an early event in colon cancer (Polakis, 1999;Sparks et al., 1998). -Catenin has essential roles in cellcell adhesion, and in the Wnt pathway in which a -cateninT-cell factor (TCF) complex transcriptionally regulates gene expression (Nelson and Nusse, 2004). -Catenin levels are tightly controlled by a destruction complex of Adenomatous polyposis coli (APC) and Axin, which facilitates the phosphorylation of -catenin by casein kinase I (CKI) and glycogen synthase kinase 3 (GSK3), leading to -catenin ubiquitylation and DPN degradation by the proteasome (Easwaran et al., 1999;Hart et al., 1999;Kishida et al., 1998;Kitagawa et al., 1999;Nakamura et al., 1998;Rubinfeld et al., 1996). Mutations in the CKI and GSK3 phosphorylation sites of -catenin (Fig. 1A) are found in human tumors and cancer cell lines and result in stabilization of -catenin (Polakis, 1999). It is thought that the primary consequence of -catenin accumulation is usually unregulated -catenin-mediated transcription of genes controlling cell proliferation (Morin et al., 1997;Polakis, 1999). However, RFC37 expression of stabilized mutant -catenin DPN with increased transcriptional activity in a non-transformed cell line resulted in only a very low, albeit significant, percentage of transformed cells, as measured by anchorage-independent growth in soft agar (Barth et al., 1999). Thus, additional defects caused by stabilization and accumulation of -catenin might be important in cell transformation. == Fig. 1. == Increased levels of -catenin induce formation of extra -tubulin puncta.(A) Schematic representation of -catenin protein. N-terminal (NT) and C-terminal (CT) domains and the 12 central armadillo repeats are indicated (ARM). Mutations in the CK1 and GSK3 phosphorylation sites to generate stabilized -cat* were made: S33A, S37A, T41A and S45A. A stretch of unrepeated amino acids in repeat 10 (gray box) is shown. The CK1 and GSK3 phosphorylation sites (NT), Adenomatous polyposis coli (APC), E-cadherin, and T-cell factor (TCF) binding sites (ARM), transcriptional cofactor binding region (CT) and the KT3 tag used in stable cell lines are shown. (B) Immunoblot of whole-cell lysate from parental MDCK and -cat*-expressing cells. (C) Parental and -cat*-expressing cells immunostained for -tubulin (arrows). -cat*-expressing cells have three or more -tubulin puncta. Level bar: 10 m. (D) Percentage of cells with three or more -tubulin puncta in MDCK cells transiently transfected with indicated constructs: pEGFP-C1 (GFP), GFP-S33A/S37A/T41A/S45A–cat* in pEGFP-C1 (GFP-b-cat*), GFP–catenin in pEGFP-C1, DPN GFP–catenin without the ARM domain name (GFP-ARM;Elul et al., 2003), -cateninengrailed chimera (ENG;Montross et al., 2000). Bars show s.e.m. of three experiments. Mean value is usually shown for experiments performed twice. (E) TCF reporter assays (TOP-Flash) and control reporter assays (FOP-Flash) were performed on lysates from MDCK cells transiently transfected with the indicated constructs. Error bars symbolize s.e.m. A newly identified target for -catenin function is the centrosome. We reported recently that -catenin is usually a component of centrosomes and interacts with centrosomal proteins to regulate centrosome separation (Bahmanyar et al., 2008). The number, business and function of centrosomes is important in chromosome segregation. Human tumors are characterized by numerical and structural chromosomal aberrations that are probably due to abnormal chromosome segregation (Lengauer et al., 1997;Lengauer et al., 1998). Increased centrosome figures and centrosome defects have been observed in aggressive and low-grade tumors, and precancerous lesions (D’Assoro et al., 2002;Nigg, DPN 2002;Pihan et al., 1998;Pihan et al., 2001;Pihan et al., 2003;Salisbury et al., 2004), and chromosomal instability is usually common in colon cancers with stabilized -catenin (Hadjihannas and DPN Behrens, 2006;Hadjihannas et al., 2006). Centrosomes comprise a pair of centrioles that must be duplicated once per cell cycle (Stearns, 2001;Tsou and Stearns, 2006), so that at the onset of mitosis, a cell has two centrosomes that separate to form the poles of the bipolar mitotic spindle (Doxsey et al., 2005;Stearns, 2001;Tsou and Stearns, 2006). Genetic studies inC. elegansand later work in vertebrates showed that SAS-4 (CPAP/CENPJ), SAS-6 and ZYG-1 (Plk4/SAK) are core proteins required.