en
Doctoral thesis
English

Investigating p21-mediated dynamic regulation of CDK1 and cyclin B1

ContributorsPoulain, Lina
Number of pages162
Imprimatur date2024
Defense date2024
Abstract

Cell cycle progression is a tightly regulated process that results in cell proliferation, senescence, or apoptosis. Faithful progression through the cell cycle is – among other proteins – controlled by a family of serine/threonine kinases whose activities strictly depend on a regulatory cyclin subunit and are therefore termed cyclin-dependent kinases (CDKs). CDK activity is further regulated through binding to CDK inhibitors (CKIs). CKIs prevent or limit the phosphorylation of substrates and are divided into two families: INK4 and CIP/KIP. The CIP/KIP (CDK-Interacting Protein/Kinase-Inhibitory Protein) family member p21(CIP1) (referred to as p21) is an intrinsically disordered protein that adopts a defined tertiary structure upon binding to target proteins. It has been proposed that p21 post-translational modifications, in particular phosphorylation of key residues, are important for CDK1 binding. Interestingly, a recent study from our group identified a novel phosphate-binding pocket in B-type cyclins that we speculate is important for p21 binding, once the inhibitor is phosphorylated at specific sites. This phospho-dependent regulation of p21-binding to the cyclin B/CDK1 complex would consequently represent a novel layer of regulation of CDKs. Furthermore, all existing structural studies have been conducted in the context of truncated inhibitor versions and it cannot be excluded that full-length proteins provide additional binding sites to promote high-affinity binding. During my thesis, I aimed to broaden our molecular understanding of CDK1 regulation by determining the structure of p21 in complex with the cyclin B1/CDK1/CKS1 complex and it reveals the dynamic binding mode of full-length p21 to cyclin B1/CDK using cryoEM. By using complementary biophysical and biochemical techniques such as Hydrogen/Deuterium exchange Mass Spectrometry (HDX-MS), I unveil the complexity of this dynamic regulation between p21 and the cyclin B/CDK1 complex. In addition, in vitro kinase activity assays show that p21-mediated regulation is organized into two separate but complementary steps during mitosis; a direct inhibition of CDK1 kinase activity and a conformational reorganization, mediated through the cyclin B1 phosphate-binding pocket, of the full complex allowing a slow transition to a fully active cyclin B1/CDK1 complex. Therefore, the results presented in my thesis provide novel insights into the molecular mechanisms that underly an ordered progression through the cell cycle.

eng
Keywords
  • Cell cycle
  • CDK1
  • CyclinB1
  • P21
  • Mitosis
  • Mammalian
  • CryoEM
  • HDX-MS
  • Metaphase
Research group
Citation (ISO format)
POULAIN, Lina. Investigating p21-mediated dynamic regulation of CDK1 and cyclin B1. 2024. doi: 10.13097/archive-ouverte/unige:176991
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Thesis
accessLevelRestrictedaccessLevelPublic 04/01/2025 CC0
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Creation05/13/2024 6:43:19 AM
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