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Title

Destruction of Cdc25A Phosphatase during Interphase and in Response to Genotoxic Stress

Date of Award

Spring 5-15-2010

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The Cdc25A protein phosphatase drives cell cycle transitions by activating cyclindependent protein kinases. Failure to regulate Cdc25A leads to deregulated cell cycle progression, bypass of cell cycle checkpoints and genome instability. Overproduction of Cdc25A is frequently observed in human cancers and is due to post-transcriptional mechanisms in many instances. Ubiquitin-mediated proteolysis plays an important role in balancing Cdc25A levels during a normal cell division cycle and when cells are exposed to replication- or genotoxic-stress. During interphase, human Cdc25A is ubiquitinated and targeted for proteolysis by the β-TrCP E3 ligase. Cdc25A contains a regulatory region within its amino terminus that mediates interactions with β-TrCP. My thesis studies focused on identifying phosphorylation sites within the amino terminus of human Cdc25A that are required for its association with and ubiquitination by β-TrCP; determining phosphorylation dependencies within this regulatory region and finally, identifying the protein kinases responsible for phosphorylating Cdc25A within this regulatory region. I used phospho-specific antibodies to demonstrate that human Cdc25A - iii - is phosphorylated on serine 76 (S76), S79, T80, S82 and S88. I then demonstrated that S76 phosphorylation primes Cdc25A for phosphorylation on S79, which then primes Cdc25A for phosphorylation on S82. Phosphorylation of Cdc25A on S76, S79 and S82 was then shown to be required for β-TrCP binding. I demonstrated that Plk3 phosphorylates Cdc25A on T80 and in a collaborative study showed that T80 phosphorylation primes Cdc25A for subsequent phosphorylation on S76 by GSK-3β. The Plk3/GSK-3β pathway regulates S76 phosphorylation in G1- and early S- phase whereas Chk1 takes over in the S- and G2-phases of the cell cycle. Finally, I demonstrated that CK1 phosphorylates Cdc25A on both S79 and S82 in a S76-dependent manner. The priming of Cdc25A by at least three kinases (Chk1, GSK-3β, CK1), some of which also require priming, ensures diverse extra- and intra-cellular signals interface with Cdc25A to precisely control cell division.

Language

English (en)

Chair and Committee

Helen Piwnica-Worms

Committee Members

David Piwnica-Worms, Raphael Kopan, Andrey Shaw, Sheila Stewart, Jason Weber

Comments

Permanent URL: https://doi.org/10.7936/K73B5X31

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