Investigating the role of actin-binding protein palladin in actin regulation

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Vattepu, Ravi
Beck, Moriah R.

Palladin, an actin-binding and bundling protein, plays an important role in normal cell adhesion and motility via organizing the actin cytoskeleton. Palladin exists in multiple isoforms in humans and its canonical isoform contains five immunoglobulin (Ig) domains and Ig3 domain is the minimum requirement for actin-binding and bundling, while Ig4 does not bind directly to actin, the tandem Ig3-4 domain binds and bundles actin more efficiently than Ig3 alone. In our quest to understand palladin’s role in the actin cytoskeleton we have explored the following topics in this dissertation: actin–induced dimerization, phospholipid-binding and regulation of function, structural and functional outcomes of a recently identified point mutation of a critical tryptophan residue, and the role of the linker between the Ig3 and Ig4 domains. First, we demonstrated that actin induces dimerization in the actin-binding domain of palladin, which is confirmed by chemical crosslinking. Our results also provide biochemical proof that the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) functions as a moderator of palladin activity. A mutation in the Ig4 domain of palladin has been found in a pancreatic cancer cell line that displays increased cell motility. Our results reveal a severe disruption of Ig4 domain folding, stability, and actin bundling function. To gain insight into role of the linker between the Ig3 and 4 domain, we have generated a series of mutations to shorten the linker, swap domain linkers, and add phosphomimetic modifications that will allow us to study the effects on actinbinding, bundling, and polymerization. In this report we also highlight the development of a novel His-tag based fluorophore, a tool that will be useful in several future studies, and initial studies of a unique actin polymerization mechanism involving actin oligomerization. Our overall results provided conclusive evidence for Ig3-4 actin bundling mechanism and identified key residues involved in lipid-binding.

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Thesis (Ph.D.)-- Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry