Function and regulation of protein phosphatases in Xenopus early embryonic divisions
The aim of my project is to deepen our understanding of the pathways regulating mitosis in higher eukaryotes. Entry into and exit from mitosis are triggered by changes in the phosphorylation state of multiple cell cycle regulators. The phosphorylation state of these regulators is determined by the counteracting activities of mitotic kinases and phosphatases. In Xenopus laevis oocytes and embryos, the main mitotic kinase cyclin-dependent kinase 1 (Cdk1)/cyclin-B is actively counteracted by protein phosphatase 2A in complex with a regulatory B55 subunit (PP2A-B55). Thus, to facilitate faithful and irreversible entry into mitosis, Cdk1/cyclin-B and PP2A-B55 must be regulated in a manner such that their activities are mutually exclusive. At mitotic entry, Cdk1/cyclin-B-activated Greatwall (Gwl) kinase phosphorylates the small heat stable proteins Arpp19/Ensa which enables them to tightly bind to and inhibit PP2A-B55. While it is well established that both Gwl and Arpp19/Ensa have to be dephosphorylated during mitotic exit to facilitate PP2A-B55 re-activation, the precise underlying mechanism remains elusive. To better analyse mitotic exit, we established a manipulable in vitro Xenopus extract system that allowed us to recapitulate mitotic entry and exit. With this system we could show that protein phosphatase 1 (PP1) plays a key role in the inactivation of Gwl during mitotic exit. However, PP1 critically requires associated regulatory subunits (PIPs) to execute its cellular functions. Thus, in this study we aim to reveal the identity of the PIP necessary for Gwl dephosphorylation and therefore PP2A-B55 reactivation at mitotic exit. To this end we will employ immunoprecipitation and mass spectrometry assays to identify candidate PIPs. These PIPs will then be assayed for their role during mitotic exit in the Xenopus extract system. The identification of the PIP necessary for PP1´s activity against Gwl and its regulation will contribute to our understanding of the regulatory pathways that control mitosis.