Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Pharmacology
First Advisor
Turk, Ben
Abstract
Eukaryotic cells require cofilin, also known as actin depolymerizing factor (ADF), to regulate the severing and rapid reorganization of actin filaments during processes such as cell division and migration. Cofilin severing activity is facilitated by a binding interaction between actin and the cofilin N-terminus. The cofilin N-terminus contains a phosphorylation site that reversibly inhibits actin severing when phosphorylated by members of the LIM kinase (LIMK) family. These two functions of the N-terminus, actin severing and phospho-regulation, converge to promote strict evolutionary conservation of a Ser-Gly-Val/Ile/Met/Thr motif, but how the N-terminal motif conforms and contributes to these two requirements has remained unclear. Here I characterize the distinct sequence constraints for cofilin severing activity and phospho-regulation and how they promote N-terminal sequence conservation. I first detail the structure of cofilin and the mechanism for actin filament severing before describing cofilin regulation and its role within the eukaryotic cell. Next, I present a dual-functionality screen of a cofilin N-terminus mutagenesis library in yeast that identifies cofilin residues important for actin binding and phospho-regulation. This screen harnesses the reliance of yeast growth on cofilin functionality to deplete non-functional mutants from a combinatorial library of 16,000 cofilin variants. The screen also identifies sequences selectively inhibited by inducible exogenous LIMK1 expression. The functionality and phosphorylation of selected cofilin mutants was validated using in vitro actin-binding and radiolabel kinase assays. Taken together, these results demonstrate that the cofilin sequence requirements for severing activity and phosphorylation by LIMK1 are less restrictive than what evolutionary conservation of native cofilin sequences would imply. Unexpectedly, these results identified cofilin N-terminal sequences that remained capable of actin severing despite phosphorylation by LIMK1. Phosphosite motif conservation is primarily thought to maintain residues that promote specific phosphorylation by a regulatory kinase. This work demonstrates that the residues of a phosphosite can also contribute to its capacity for phospho-regulation and drive motif conservation unrelated to kinase-substrate specificity.
Recommended Citation
Sexton, Joel Andrew, "Disentangling Sequence Constraints on the Cofilin N-terminal Phosphorylation Site" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1100.
https://elischolar.library.yale.edu/gsas_dissertations/1100
