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Shih. Footnotes Abbreviations used in this paper:CHCclathrin heavy chainMBPmaltose-binding proteinMTmicrotubuleNocnocodazoleWTwild type. et al., 2008). Among its substrates, TACC3 (transforming acidic coiled-coilCcontaining protein 3) has recently emerged as an important player in organizing mitotic spindles (Kinoshita et al., 2005; Pascreau et al., 2005; Peset et al., 2005; Peset and Vernos, 2008). Aurora A phosphorylates TACC3 on S558, which facilitates TACC3 localization to spindles and subsequently ch-TOG recruitment, promoting microtubule (MT) assembly (Brittle and Ohkura, 2005; Barr and Gergely, 2007). Notably, TACC3 depletion causes MT destabilization and chromosome misalignment (Gergely et al., 2003; Schneider et al., 2007), resembling some aberrant mitotic events of cells with aurora A disruption (Marumoto et al., 2003; Sasai et al., 2008). Furthermore, treatment of a selective aurora A inhibitor precluded TACC3 localization to the spindle (LeRoy et al., 2007), correlating with the formation of abnormal mitotic spindles (Hoar et al., 2007). Thus, it is conceivable that the capacity of TACC3 in spindle association is crucial for MT stabilization. Although phosphorylation of TACC3 S558 by aurora A is essential for its spindle localization, the molecular mechanism underlying TACC3 phosphorylation-dependent spindle targeting remains elusive. In addition to being a component of clathrin involved in coating various transport vesicles for protein trafficking (Schmid, 1997), clathrin heavy chain (CHC) is concentrated on the spindle during mitosis and stabilizes the MT fibers (Okamoto et al., 2000; Royle et al., 2005; Yamauchi FAD et al., 2008). CHC GSK-269984A depletion causes destabilized kinetochore fibers, defective chromosome congression, and prolonged mitosis (Royle et al., 2005). Although CHC is also required for mitotic spindle function, the mechanism by which CHC regulates spindle stability is unclear. In this study, we show that CHC mediates phospho-TACC3 interaction and spindle recruitment and also provide a model for CHC stabilization of mitotic spindles. Results and discussion Identification of CHC as a phospho-S558 TACC3Cinteracting protein To identify factors capable of binding to phospho-S558 TACC3, GST-TACC3 522C577 fusion proteins, consisting of TACC3 amino acid GSK-269984A residues 522C577, were phosphorylated by aurora A kinase in vitro and subjected to pull-down assays with nocodazole (Noc)-arrested extracts from HeLa cells. GST-TACC3 522C577 wild type (WT) but not S558A mutant pulled down a distinct band with a molecular mass >170 kD (Fig. 1 A). Mass spectrometry analysis suggested that this band represented CHC. We further substantiated GSK-269984A the specificity of CHC to phospho-S558 TACC3 in the full-length context of TACC3. The GST-S558A mutant showed a marked decrease in both aurora ACmediated phosphorylation and CHC precipitation compared with WT (Fig. 1 B). Under the same binding conditions, the levels of pulled-down ch-TOG and aurora A from mitotic extracts were similar in S558A and WT (Fig. 1 B), which is consistent with data demonstrating that TACC3 binds to both ch-TOG and aurora A via its TACC domain (Lee et al., 2001; unpublished data). These results indicate that the CHCCTACC3 interaction occurs specifically via phospho-S558 of TACC3 and excludes the involvement of any other potential aurora A phosphorylation sites of TACC3 during CHC interaction. Of note, the phosphorylation at S558 by itself was crucial for CHC interaction because phosphorylation-mimic S558D or S558E failed to efficiently pull down CHC protein (Fig. S1 A). Accordingly, TACC3 S558D or S558E was defective in spindle association, similar to GSK-269984A S558A (Fig. S1 B). Open in a separate window Figure 1. CHC associates with phospho-S558 TACC3. (A) The SYPRO ruby gel shows CHC pulled down from Noc-treated HeLa cell extracts by recombinant GST-TACC3 522C577 fusion proteins phosphorylated by recombinant aurora A. CHC peptides detected by mass spectrometry are indicated. (B) Western blotting shows CHC pulled down by recombinant GST-TACC3 proteins phosphorylated by aurora A. Input represents the 5% amount of Noc-treated HeLa cell extracts subjected to the pull-down assays. Coomassie blue staining shows various GST fusion proteins used for each binding reaction. The phosphorylation levels of GST-TACC3 proteins in kinase reactions are shown by autoradiography. (CCE) Western blots show complex formation of endogenous TACC3 and CHC by immunoprecipitation (IP) with the indicated antibodies from mitotic HeLa cells synchronized GSK-269984A by Noc (C), from Noc-synchronized cells with or without additional treatment of 2 M VX-680 (D), or from Noc-synchronized cell lysates with or without treatment of -phosphatase (PPase) before being subjected to immunoprecipitation.