Ly, Tony

Earnshaw, William

al-Rawi, Aymen

Darwin Trust

2023-05-16T12:18:10Z

2023-05-16T12:18:10Z

2023-05-16

The cell cycle is a complex series of events that results in one parental cell dividing into two daughter cells. Division only occurs when cells have successfully replicated their DNA, to create progeny each with a copy of their genome. Protein phosphorylation is one mechanism that regulates the cell cycle. Kinases such as the polo-like family, the Auroras, and the Cyclin dependent kinases (CDKs) along with phosphatases, such as PP2A and PP1, form dynamic regulatory circuits that activate and inactivate substrate proteins through reversible phosphorylation of targeted residues. CDK1 is the main regulator of protein phosphorylation in mitosis, carrying out hundreds of these phosphorylations. Monomeric CDK1, however, is inactive and requires either a Cyclin A or Cyclin B partner to gain activity. Progressive expression and degradation of the two Cyclins determine the activity of CDK1 during the cell cycle. Besides, the Cyclin partner was suggested to mediate CDK1 substrate choice. This role, however, has always been a point of debate. Cks1 is another element in CDK1 complexes with even less understood role in substrate phosphorylation. My project aimed to characterise these substrates based on their dependency on CDK1 activity, its Cyclin partner, and the presence of a Cks1 in its complex, in order to understand how the temporal ordering of their phosphorylation is maintained throughout the cell cycle. To achieve that, I developed an in vitro kinase assay through which I phosphorylated fixed and permeabilized cells using recombinant CDK1 in complex with different subunits and assessed their phospho-proteome using mass spectrometry. Results revealed that both CDK1 activity and its Cyclin partner determine the substrate to be targeted for phosphorylation. The presence of Cks1, on the other hand, increased the number of phosphorylated residues on the targeted substrates. This data also unveiled the ability of CDK1 to phosphorylate sites lacking the +1 Proline in its S/TPXK/R consensus motif in the presence of either Cyclin A2 or Cks1 in its complexes. This non-Proline directed phosphorylation uncovered new details of the mechanism by which CDK1 maintains the temporal ordering of substrates phosphorylation. The data here also reveals CDK1 phosphorylation of mitotic sites for which an upstream kinase has not been reported.

https://hdl.handle.net/1842/40569

http://dx.doi.org/10.7488/era/3334

en

The University of Edinburgh

Cyclin A and Cks1 promote kinase consensus switching to non-proline directed CDK1 phosphorylation Authors: Aymen al-Rawi, Edward Kay, Svitlana Korolchuk, Jane Endicott, Tony Ly Journal: Cell Reports, 2023 DOI: https://doi.org/10.1016/j.celrep.2023.112139

Low Cell Number Proteomic Analysis Using In-Cell Protease Digests Reveals a Robust Signature for Cell Cycle State Classification Authors: Van Kelly, Aymen al-Rawi, David Lewis, Georg Kustatscher, Tony Ly Journal: Molecular Cell Proteomics, 2022 DOI: https://doi.org/10.1016/j.mcpro.2021.100169

Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity Authors: Thomas J Kucharski, Rufus Hards, Sarah E Vandal, Maria Alba Abad, A. Arockia Jeyaprakash, Edward Kaye, Aymen Al-Rawi, Tony Ly, Kristina M Godek, Scott A Gerber, Duane A Compton Journal: The Journal of Cell Biology, 2022 DOI: https://doi.org/10.1083/jcb.202107107

CDK1 phosphorylation

cell cycle substrates

mass spectrometry-based phospho-proteomics

cell cycle

dynamic regulatory circuits

CDK1

protein phosphorylation

mitosis

+1 Proline

S/TPXK/R

non-Proline directed phosphorylation

Biochemical determinants of CDK1 phosphorylation of cell cycle substrates using mass spectrometry-based phospho-proteomics

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy