Breast cancer is a serious and sometimes life-threatening disease

isassembly factor. The dramatic relocalization of the CPC as cells enter anaphase is related to changes in the activity of cyclin-dependent kinases and their opposing phosphatases: INCENP/Sli15 is phosphorylated by cyclin-dependent kinase 1 and this inhibits its interaction with the spindle, while removal of Cdk1-dependent phosphorylation is a key element driving interaction of the yeast CPC with the spindle mid-zone in anaphase. Relocalization of the CPC to the spindle mid-zone is also important for preventing re-engagement of the spindle assembly checkpoint during anaphase when tension on microtubule-kinetochore attachments is reduced following loss of sister chromatid cohesion. INCENP/Sli15 consists of three domains: the N-terminal region that mediates association with Bir1 and Nbl1, the central domain that binds microtubules and the C-terminal domain or IN-box that is involved in binding to and activating Aurora B/Ipl1 protein kinase. Sli15 is itself a substrate for phosphorylation by Ipl1, becoming phosphorylated by the kinase during in vitro protein kinase assays, while in higher eukaryotes phosphorylation of INCENP by Aurora B on a C-terminal Thr-Ser-Ser motif contributes to full activation of Aurora B. To examine whether Ipl1-dependent phosphorylation of Sli15 is relevant for CPC function or localization, we set out to identify the Ipl1-dependent phosphorylation sites and to examine the potential role that phosphorylation might play. A recent study in which predicted Ipl1 phosphorylation sites in Sli15 were changed to nonphosphorylatable alanine showed that in addition to Cdk1dependent phosphorylation, Ipl1-dependent phosphorylation of Sli15 is also likely to regulate CPC interaction with the spindle and that in cooperation with Cdk1, Ipl1 phosphorylation of Sli15 helps to ensure appropriate microtubule dynamics at different stages in the cell cycle. Here we report the identification of 14 sites in Sli15 that are phosphorylated directly by Ipl1 and confirm that Ipl1-dependent phosphorylation of Sli15 regulates CPC association with spindle microtubules. By mutating SLI15 to encode a protein in which constitutive phosphorylation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 of Sli15 is mimicked, we demonstrate that phosphorylation in vivo is likely to limit the interaction of the CPC with the spindle, and show that phosphorylation of Sli15 or the acidic substitutions that mimic constitutive phosphorylation block the direct binding of Sli15 to microtubules in a novel in vitro binding assay. Furthermore, we find that mimicking constitutive phosphorylation of Sli15 on its Ipl1 phosphorylation sites 169939-93-9 interferes with the cell’s ability to mount a spindle assembly checkpoint response specifically to reduced tension on sister kinetochores. Thus in addition to affecting the spindle association of the CPC, Ipl1dependent phosphorylation of Sli15 may act as a PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19638506 negative regulator of the tension checkpoint response. summarized in sli15 mutant alleles For generating strains with mutant copies of SLI15, a 2.55 kb BamHI-XhoI fragment carrying SLI15 together with 300 bp of upstream and downstream flanking sequence was first excised from pCJ145 and inserted into pRS303. The sli15-20A plasmid was created from pRS303-SLI15 by sequential site-directed mutagenesis using different primers on pRS303-SLI15 with the QuikChange Site-Directed Mutagenesis Kit according to the manufacturer’s instructions. The mutated plasmid was verified by DNA sequencing, linearized with BsiWI and integrated in single copy