자료유형  

 학위논문

Control Number  

0016931509

International Standard Book Number  

9798379782108

Dewey Decimal Classification Number  

574

Main Entry-Personal Name  

Sexton, Joel.

Publication, Distribution, etc. (Imprint  

[S.l.] : Yale University., 2023

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2023

Physical Description  

1 online resource(138 p.)

General Note  

Source: Dissertations Abstracts International, Volume: 85-01, Section: B.

General Note  

Advisor: Turk, Benjamin E.

Dissertation Note  

Thesis (Ph.D.)--Yale University, 2023.

Restrictions on Access Note  

This item must not be sold to any third party vendors.

Summary, Etc.  

요약Eukaryotic 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.

Subject Added Entry-Topical Term  

Molecular biology.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Cellular biology.

Index Term-Uncontrolled  

Cofilin

Index Term-Uncontrolled  

LIMK1

Index Term-Uncontrolled  

Actin depolymerizing factor

Index Term-Uncontrolled  

Mutagenesis

Index Term-Uncontrolled  

Phosphosite

Index Term-Uncontrolled  

Yeast

Added Entry-Corporate Name  

Yale University Pharmacology

Host Item Entry  

Dissertations Abstracts International. 85-01B.

Host Item Entry  

Dissertation Abstract International

Electronic Location and Access  

로그인을 한후 보실 수 있는 자료입니다.

Control Number  

joongbu:642266