The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. common fragile sites for example. Correct DNA Zanosar duplication involves the strictly ordered assembly of various protein complexes onto thousands of genomic sites that will be destined to serve as replication origins1, 2. The origin recognition complex (ORC) first binds the replication origins. This complex promotes the binding of Cdc6 and Cdt1, two proteins that will subsequently facilitate the binding of the MCM proteins to form the pre-replication complex (pre-RC). Pre-RC formation process starts in late M phase and continues during early G1 when cyclin-dependent kinase (Cdk) activity is low. The subsequent initiation of DNA replication involves the activation of the MCM complex via the recruitment of the replication proteins Cdc45 and GINS that occurs at G1/S transition when interphase Cdk activity increases3. It is known that Cdks globally orchestrate transition at origin-bound complexes regulating licensing and initiation events to ensure that each origin is fired only once per cell cycle. During S, G2 and M phases origin licensing is prevented by high levels of Cdk activity that phosphorylate and inactivate multiple pre-RC components. One of these components, Cdt1, is inactivated during S phase by SCF-Skp2-dependent degradation as a consequence of Cdk-dependent phosphorylation4, 5. Another replication factor, Cdc6, is also phosphorylated by Cdk during DNA replication and this phosphorylation downregulates its licensing activity by promoting nuclear exclusion6C8. Finally, ORC1 phosphorylation by Cdk during S phase reduces its chromatin affinity9 and permits its export to the cytoplasm preventing the formation of new pre-RC10. Unlike its negative effect on origin licensing, Cdk activity SHGC-10760 positively regulates origin firing at G1/S transition. In humans, Cdk phosphorylates Treslin, the orthologue of yeast … Nuclear localisation of Ensa in S phase is compatible with a role of this protein in DNA replication. We thus further investigated if Ensa was phosphorylated and activated during S phase by Gwl. To that, we checked Ensa phosphorylation in Ensa immunoprecipitates of HeLa cells during the Zanosar different phases of the cell cycle by using a phospho-antibody that recognises the conserved phosphorylated site of Gwl in both human Arpp19 (S62) and human Ensa (S67). Phosphorylation of Ensa by Gwl significantly increased in S phase compared to G1/S but to a lower extent than in mitosis (Fig.?6b). We next checked the levels of active Gwl on the chromatin during S phase. Hence, thymidine-blocked HeLa cells were lysed to recover cytoplasm and nuclei at 2, 4, 6 and 8?h after release. Chromatin-associated proteins were then extracted by using a buffer containing 300?mM (N3) or 600?mM of NaCl (N6). As shown in Fig.?6c, Gwl was present and bound to the chromatin throughout S phase although to a lesser extent than in cytoplasmic fraction. Moreover, phosphorylation and activation of Gwl was observed in nuclear but not in Zanosar cytoplasmic fractions of thymidine-blocked cells. Gwl phosphorylation in nuclear fractions was maintained 2 and 4?h after release, decreased at 6?h and disappeared at 8?h when S phase is completed. These results indicate that Gwl and Ensa are present and active on the chromatin during S phase. Ensa KD lowers Treslin protein levels It is known that the Gwl/Ensa pathway regulates mitotic entry and progression by promoting the inhibition of PP2A-B55, the phosphatase responsible of cyclin-Cdk substrate dephosphoryaltion19, 24. We investigated the effect of Ensa KD on the global phosphorylation of Cdk substrates by using an antibody directed against the phosphorylated serine in the Cdk consensus motif. As Zanosar shown in Fig.?7a, b, we observed a significant decrease of the phosphorylation of cyclin-Cdk substrates in Ensa KD cells. We hypothesised that the Gwl/Ensa pathway might be activated in S phase to permit the Zanosar phosphorylation of key Cdk substrates responsible for the correct unfolding of S phase. One major Cdk substrate whose phosphorylation controls S-phase length is the TopBP1-interacting protein, Treslin14. We therefore checked whether Treslin was dephosphorylated upon Ensa KD. To our surprise, the main phenotype that we observed after siEnsa treatment was a dramatic drop of this protein in.