These outcomes implicate the Scc1CSA2 heterodimer as the binding partner of phospho-Sgo1, although the observed interaction might involve insect cell proteins. Phospho-Sgo1CPP2A maintains cohesin-bound sororin in a hypophosphorylated state Sgo1CPP2A had been proposed to protect SA2 from phosphorylation by Plk1, thereby maintaining centromeric cohesion21,26,27. contains PP2A, Pds5, and hypophosphorylated sororin, but lacks Wapl. Expression of non-phosphorylatable sororin bypasses the requirement for Sgo1CPP2A in centromeric cohesion. Thus, mitotic phosphorylation of Sgo1 targets Sgo1CPP2A to cohesin, promotes dephosphorylation of Pds5-bound sororin, and protects centromeric cohesin from Wapl. PP2A-orchestrated, selective removal of a specific subset of phosphorylation from cohesin and its regulators underlies centromeric cohesion protection. Timely establishment and dissolution of sister-chromatid cohesion during the cell cycle are critical for proper chromosome segregation and require cell-cycle-regulated modifications of cohesin and its regulators1C4. Although the cohesin complex consisting of Smc1, Smc3, Scc1, and SA1/2 is already loaded onto chromatin in G1 phase, it remains dynamic. Concomitantly with DNA replication in S phase, MK-5172 a fraction of cohesin is usually converted to the cohesive state. This conversion requires Smc3 acetylation and, in human cells, subsequent binding of Pds5 and sororin to cohesin5C12. Sororin competes with the cohesin inhibitor, Wapl, for Pds5 binding and opposes Wapl-mediated cohesin removal to establish cohesion in S phase11. Cohesion dissolution in mitosis occurs in two actions in human cells13. In prophase, the mitotic kinases Cdk1 and Plk1 phosphorylate sororin and SA2, respectively, to trigger Wapl-dependent removal of cohesin from chromosome arms11,14C17. Phosphorylation of sororin disrupts the Pds5Csororin conversation, allowing Wapl to access Pds5 and remove cohesin from chromosome arms11. The Sgo1CPP2A complex protects centromeric cohesin from the mitotic kinases and Wapl to enable bipolar attachment of sister chromatids to the mitotic spindle15,18C22. After the satisfaction of the spindle checkpoint, the protease separase cleaves centromeric cohesin to allow sister-chromatid separation. The mechanism by which human Sgo1CPP2A protects centromeric cohesion is not understood. How Sgo1 is usually regulated during the cell cycle is also unknown. Here we study the mechanism and regulation MK-5172 of Sgo1 in human cells. Our results establish a requirement for a phosphorylationCdephosphorylation cascade orchestrated by Cdk and Sgo1CPP2A in centromeric cohesion protection. RESULTS Cdk-dependent phosphorylation of human Sgo1 at T346 in early mitosis Human Sgo1 underwent gel mobility shift in mitosis, consistent with it being phosphorylated19. To study whether Sgo1 phosphorylation was functionally important, we set out to systematically map the phosphorylation sites in Sgo1 in mitosis. We constructed doxycycline (Dox)-inducible HeLa cell lines that stably expressed siRNA-resistant Myc-Sgo1. Myc-Sgo1 was functional, as it rescued the premature sister-chromatid separation phenotype of Sgo1 RNAi cells18,23. Furthermore, ectopic expression of Myc-Sgo1 did not alter cell cycle progression or the timing of chromosome segregation23. Mass spectrometric analysis identified several phosphorylation sites in Myc-Sgo1 immunoprecipitated from mitotic cells (Fig. 1a and Supplementary Fig. 1a). We chose to focus on T346, which was conserved in vertebrate Sgo1 proteins (Fig. 1b). The T346A mutation abolished the slower-migrating Myc-Sgo1 bands on SDS-PAGE gels while the phosphorylation-mimicking T346D mutant had a slower mobility (Fig. 1c). Open in a separate window Physique 1 Human Sgo1 undergoes Cdk-dependent phosphorylation at T346 during mitosis(a) Schematic drawing of human Sgo1 showing its domain structures and the phospho-peptides and phosphorylation sites in mitosis identified with mass spectrometry in this study. (b) Alignment of sequences flanking the MK-5172 T346 site in Sgo1 proteins from human (Hs), mouse (Mm), and (Xl). (c) Lysates of HeLa Tet-On cells transfected with plasmids encoding Myc-Sgo1 WT, T346A, or T346D were blotted with -Myc. (d) Lysates of HeLa Tet-On cells transfected with plasmids encoding Myc-Sgo1 WT or T346A were blotted with -Myc (left panel) or -pT346 Sgo1 (right panel). (e) -Sgo1 IP of HeLa Tet-On cells arrested at G1/S by thymidine (Thy), at mitosis by nocodazole (Noc), or transfected with siSgo1 were blotted with -Sgo1 and -pT346 Sgo1. Lanes are spliced together from the same gel. (f) Representative HeLa Tet-On cells stably expressing Myc-Sgo1 at different mitotic stages were stained with DAPI and the indicated antibodies. The arrows in the pT346 channel indicate nonspecific staining of centrosomes. (g) Recombinant GST-Sgo1 WT and T346A were incubated in the absence or presence of recombinant cyclin BCCdk1 with or without ATP. The samples were resolved on SDS-PAGE and blotted with the indicated antibodies. The bottom panel shows Coomassie-blue staining of GST-Sgo1 proteins. (h) HeLa Tet-On cells stably expressing Myc-Sgo1 were blocked in mitosis by Taxol and treated with the Cdk1 inhibitor RO3306 and the Aurora kinase inhibitor ZM447439 without or without TPO MG132 for the indicated occasions. Lysates of these cells were blotted with -Sgo1 and -pT346 Sgo1. We next generated a phosphorylation-specific antibody against this site. This antibody only acknowledged the slower-migrating species of Myc-Sgo1 WT, but.
