Author ORCID Identifier

https://orcid.org/0000-0002-5338-7320

Date of Award

Summer 2024

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

James Moseley

Abstract

The mechanisms that govern cell size have long been topics of study in the field of cell biology. In eukaryotic cells this size control is tied to checkpoints, a set threshold of minimum necessary growth linked to cyclin dependent kinase activity regulation. In the fission yeast Schizosaccharomyces pombe, the Cdk1 regulatory network is conserved, and G2/M represents the major size checkpoint. Prior to mitosis, Cdk1 is inhibited by phosphorylation applied by Wee1 during G2 phase. Once S. pombe cells have satisfied the size checkpoint, Cdk1 is activated through dephosphorylation by Cdc25. Wee1 is a dose-dependent regulator of mitotic entry such that reduction in Wee1 activity results in cells entering mitosis early at small sizes, while increased Wee1 activity causes cells to enter mitosis late at elongated sizes. Control of Wee1 activity is mediated during G2 by the SAD kinases Cdr1 and Cdr2. These kinases coordinate Wee1 inhibition and localize to plasma membrane-associated clusters termed “nodes.” Cdr2 is required to form nodes and its activity is important for retaining Wee1 at a node. Our working model posits that longer retention time of Wee1 at a node allows Cdr1 to inhibit Wee1 catalytic activity through a defined molecular mechanism. Work in this thesis shows development of a new PTet-cdr2 overexpression system that we have validated for Wee1 phenotypes and are using to elucidate the molecular mechanism for Cdr2 regulation of Wee1. We used this system to establish that Cdr2 is a dose-dependent inhibitor of Wee1 through sequestration outside the nucleus. Further, we showed that this inhibition depends on Cdr2 activity and clustering ability. From there, we screened Wee1 for regions likely being phosphorylated by Cdr2 in this mechanism. We have determined that clusters of serine residues in the first 150 amino acids of Wee1 contain the Cdr2-sensitive sites. These findings help to define the mechanism of Wee1 inhibition by Cdr2, where retention at a node dilutes Wee1 away from its substrate Cdk1 in the nucleus. This spatial mechanism works in tandem with Cdr1-based catalytic inhibition to promote mitotic entry at the proper cell size.

Original Citation

Chapter 2 was originally published as:

Berg, R. A., & Moseley, J. B. (2023). Regulation of cell size and Wee1 kinase by elevated levels of the cell cycle regulatory protein kinase Cdr2. The Journal of biological chemistry, 299(2), 102831. https://doi.org/10.1016/j.jbc.2022.102831

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