The role of multisite phosphorylation in establishing temporal order during mitosis
Cell Biology
Final Report Abstract
Entry into and exit from mitosis are accompanied by dramatic morphological and biochemical changes. These changes are thought to be driven by changes in the activity of the cyclin-dependent kinase 1 (Cdk1), which during mitosis mediates the phosphorylation of hundreds of proteins at thousands of sites. Although Cdk1 activity rises gradually during interphase due to cyclin A and B synthesis, Cdk1 substrates remain largely unphosphorylated. Substrates only become phosphorylated, when Cdk1 activity further increases at the time of mitotic entry due to the toggling of a bistable molecular switch mediated by a doublenegative feedback loop, between the Wee1 kinase and Cdk1, and a positive feedback loop, between the Cdc25 phosphatase and Cdk1. Additionally, substrate phosphorylation is controlled by the antagonistic regulation between Cdk1 and its counteracting phosphatases, PP1 and PP2A-B55, which are both inhibited by Cdk1 activity during mitosis. Exploiting the natural synchronicity of the early embryonic cell cycles in Xenopus laevis egg extracts, I investigated the intricate regulation between Cdk1, PP1 and PP2A-B55, and the phosphorylation of mitotic substrates. I showed that even when bistability in Cdk1 activity is suppressed, mitotic substrate phosphorylation still exhibits a bistable response and sharp phosphorylation thresholds. The bistability arises from an additional double-negative feedback in the regulation of the Cdk1- counteracting phosphatase PP2A-B55 during mitotic transitions. I further found that Cdk1 regulates PP2A-B55 in a biphasic manner; intermediate, sub-mitotic levels of Cdk1 activity stimulate PP2A-B55 activity up to ~4 fold, while mitotic levels of Cdk1 activity inhibit PP2A-B55 to undetectably low levels. As a consequence of this incoherent feedforward regulation, substrate phosphorylation is initially suppressed while PP2A-B55 activity increases concurrently with Cdk1 activity. Only once Cdk1 activity is high enough, PP2A-B55 activity is downregulated and mitotic Cdk1 substrates become rapidly phosphorylated. Similarly, during mitotic exit PP2A-B55 spikes to trigger rapid substrate dephosphorylation and then falls back to its intermediate interphase activity. The work highlights the regulation of PP2A-B55 activity as the determining factor regulating mitotic entry and exit.
