Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Alan K. Howe

Abstract

Focal adhesions (FAs) are dynamic multiprotein complexes that bridge the extracellular matrix (ECM) bound integrins to the actin cytoskeleton. They serve as both structural anchors and signaling hubs, orchestrating various cellular processes, including cell adhesion, cytoskeletal dynamics, and cell migration, in response to mechanical and biochemical cues. One of the key mechanisms through which FAs integrate their structure, remodeling, and signaling is the reversible phosphorylation of tyrosine, serine, and threonine residues (pTyr/pSer/pThr). While pTyr in FAs has been extensively studied, pSer/pThr remains underexplored despite its abundance in these structures.The cAMP-dependent protein kinase (Protein kinase A:PKA) is a serine/threonine kinase that is ubiquitously present, regulating diverse physiological and cellular processes. Despite its ubiquity, PKA and its activity are highly localized within cells by A-kinase anchoring proteins (AKAPs) which bind to PKA and guide it to specific subcellular compartments. PKA plays a crucial role in cell adhesion and cell migration, serving as both an effector and regulator of integrin-mediated adhesions to the ECM. While a number of observations have suggested the presence of a distinct PKA pool within FAs, its presence and activity within these structures have not been definitively demonstrated. Furthermore, the localization and regulatory mechanisms governing PKA within FAs remain unexplored. Here, we conclusively establish the existence of a distinct PKA signaling pool within FAs. We show that PKA is present in isolated FA-cytoskeleton complexes, and we observe real-time PKA activity within FAs in live cells, employing an FA-targeted PKA biosensor generated in this study. In addition, we identify fifty-three high-stringency candidate PKA substrates within FAs and validate tensin3, a bona fide FA protein involved in adhesion maturation, plasticity, and cancer metastasis, as a novel substrate. This demonstration of a discrete FA-associated pool of PKA activity not only opens up a new avenue for localized PKA signaling but also suggests the presence of one or more discrete PKA anchoring proteins within FAs. Through screening of putative FA AKAPs followed by biochemical validation, we identify the archetypal FA protein, talin1, as an AKAP. Talin is a large mechanosensitive scaffold that directly links integrins and actin filaments and is critical for FA assembly by recruiting additional FA components in a force-dependent manner. Our subsequent work reveals that PKA exclusively binds to an amphipathic helix in talin1 that is buried in the native conformation and is exposed when talin is opened by mechanical force. These observations identify the first mechanically-gated AKAP and suggest a novel mechanism for coupling cellular tension and signal transduction. Overall, this work establishes FAs as an important new niche for PKA targets and AKAPs, building up a foundation for further investigation into the role and regulation of PKA within FAs. Additionally, it provides new insights into the role of PKA in cellular mechanotransduction.

Language

en

Number of Pages

174 p.

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Included in

Cell Biology Commons

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