Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular Biophysics and Biochemistry
First Advisor
Boggon, Titus
Abstract
LIM kinases 1 (LIMK1) and 2 (LIMK2) are major regulators of cytoskeletal dynamics in the cell. LIMK regulates actin dynamics by phosphorylating the actin-depolymerizing factor (ADF)/cofilin family of actin-binding proteins. Cofilin proteins bind preferentially and cooperatively to ADP-bound subunits in F-actin. This binding event changes the helical rotation of actin filaments, promoting actin filament severing. LIMKs promote actin filament stabilization by inactivating cofilin through phosphorylation of Ser3. Phosphorylation of cofilin at Ser3 deactivates cofilin severing activity by inhibition of cofilin binding to actin filaments. This cycling between actin depolymerization and polymerization impacts higher-order cellular processes, including motility, differentiation, and metastasis. In the past 25 years, LIMK and cofilin have been heavily studied, but important questions remain regarding kinase regulation. Current literature proposes a model of regulation in which the N-terminus, which contains two LIM domains and one PDZ domain, acts as a negative regulator of the C-terminal kinase domain. These N-terminal domains, known to mediate protein-protein interactions, remain understudied in the context of LIMK autoregulation. Previous studies have mainly focused on immunoprecipitation and pull-down assays of fragments of the N-terminal domains to the C-terminus kinase domain . However, no structure of the domains LIM and PDZ is published, nor details about the about an autoregulated complex is known. Thus, how these domains modulate the kinase activity of LIMK has yet to be revealed. The information in this dissertation aims to provide the molecular mechanism and structural details underlying the regulation of LIMK1 activity. I hypothesize that the N-terminus of LIMK1 negatively regulates its kinase activity via a direct "head-to-tail” interaction. I will test this hypothesis using biochemical, biophysical, cell-based, and structural biology techniques to understand molecular mechanisms underlying autoregulation. I will accomplish the goals of this dissertation by setting two aims. In Aim 1, I study the N-terminal domains of LIMK. I use biochemical and structural techniques to gain a molecular-level understanding of the PDZ domains. Specifically, I obtain the crystal structure of the hLIMK2 PDZ domain and map the conservation of this domain using both LIMK1 and LIMK2 sequence alignments. I find a surface in this domain that is conserved from mammals to insects. I use homology- and structure-driven mutations to validate structure-defined and functional mechanisms of PDZ domain regulation. To test the effect of these mutations, I reconstructed the human LIMK pathway in S. cerevisiae. Expression of human LIMK1 phosphorylates and inactivates endogenous yeast cofilin; thus, I observe alterations in LIMK activity by measuring yeast growth. Using this assay, I screened for LIMK1 PDZ mutants that may be involved in kinase autoregulation. I have successfully used radiolabel assays to test the impact of these mutations on kinase activity using cofilin as substrate in vitro. This combination of approaches allowed me to understand better the influence of the PDZ domain in kinase autoregulation. In Aim 2, I used biochemical, biophysical, and activity-based assays to elucidate how the N-terminus domains of LIMK are responsible for autoregulatory interactions with the kinase domain, and if LIMK is regulated in cis or trans. I began by directly addressing whether, in addition to the PDZ, other domains in the N-terminus of LIMK are responsible for kinase autoregulation. I found that the LIMK2 LIM2-PDZ domain fragment reduces the kinase activity of LIMK2 catalytic domain (CAT) in vitro. Furthermore, I used SEC-MALS to study the molecular arrangement of the LIM2-PDZ domains in solution. Additionally, I explore the molecular arrangement of full-length LIMK. I purify human full-length LIMK2 protein and use negative staining electron microscopy to observe global conformational changes between the wild-type protein and kinase inactive D451N mutant to differentiate between intra or intermolecular conformations. Negative staining electron microscopy suggests two different conformations where the full length wild-type LIMK2displays an elongated conformation, while the full length catalytically inactive D451N mutant shows a more compact conformation. These discoveries lead me to propose that the N-terminal domains are responsible for the autoregulation of LIMKs and that the mode of regulation is intramolecular. These findings provide a foundation for studying N-terminal autoregulation of LIMK kinase activity. Here, I present studies of autoregulatory interaction in LIMK in purified systems as well as in a eukaryotic system. This work provides the first crystal of the human LIMK2 PDZ domain and an in-depth study of its fold and conservation. Mutagenesis studies of the PDZ domain reported here provide strong evidence for how this domain undergoes autoregulation. Likewise, I provide insight into the molecular arrangement of LIMK N-terminus domains and full-length protein and provide a low-resolution understanding of its oligomeric state using SAXS and negative stain electron microscopy. Together, I propose how LIMK is autoregulated and identifies the domains responsible this autoregulation.
Recommended Citation
Casanova Sepulveda, Gabriela, "Autoregulation mechanism of LIM domain kinases" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1210.
https://elischolar.library.yale.edu/gsas_dissertations/1210
