Author ORCID Identifier

https://orcid.org/0000-0002-9545-1087

Date of Award

Fall 2025

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Arminja Kettenbach

Abstract

Cell division is controlled in large part by reversible protein phosphorylation. The phosphorylation state of proteins during mitosis and other phases of the cell cycle represents a careful balance of activity between kinases and opposing phosphatases. The phosphoprotein phosphatase (PPP) family is responsible for the majority of serine and threonine dephosphorylation. Members of the PPP family are composed of a catalytic subunit paired with a regulatory subunit that targets them towards specific substrates.

The most abundant PPP, PPP2A, paired with its regulatory B55 subunit, is thought to be the phosphatase responsible for the dephosphorylation of substrates during mitotic exit. Despite this important role, few tools exist to specifically examine its function in live cells. To address this need, we developed an auxin-inducible degron system to selectively deplete B55 from live cells. We demonstrated that this resulted in an acceleration of mitotic entry and a delay in mitotic exit. We used phosphoproteomics to identify substrates targeted by PP2A-B55 during mitotic exit. We also showed evidence that the abundance of the competitive inhibitor-substrate ARPP19 that blocks PP2A-B55 activity during mitotic entry is regulated in a PP2A-B55-dependent manner. Our results suggest that degradation of unbound, phosphorylated ARPP19 limits the pool of functional PP2A-B55 inhibitors, ensuring rapid PP2A-B55 reactivation at anaphase onset.

The mechanism by which PP2A-B55 identifies its substrates is another active topic of investigation in phosphatase biology. Several PPPs have been shown to bind their substrates through short linear motifs (SLiMs). A SLiM was proposed for PP2A-B55 but was only validated for a limited number of substrates. To further refine the SLiM definition and to identify more examples of functional SLiMs, we designed a peptide array containing PP2A-B55 SLiMs mined from the human proteome and measured purified PP2A-B55 binding to them. We identified a preference for motifs that include an aromatic residue, as well as a preference for motifs with a basic charge. Our results also support findings that PP2A-B55 prefers to bind folded short helical motifs (SHelMs). Finally, we discovered a novel B55-binding motif on c-Jun and showed that it is both sufficient and necessary to recruit PP2A-B55 in cells.

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