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Updated: Jul 17, 2025

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
Published on: March 28, 2008
The focal adhesion protein talin is a mechanically-gated A-kinase anchoring protein (AKAP)
Abstract:
The cAMP-dependent protein kinase (Protein Kinase A; PKA) is a ubiquitous, promiscuous kinase whose activity is focused and specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to the extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), intracellular complexes coupling ECM-bound integrins to the actin cytoskeleton, suggesting the existence of one or more FA AKAPs. Using a combination of a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1-R13. Direct binding assays and nuclear magnetic resonance spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Finally, single-molecule experiments with talin1 and PKA, and experiments in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. These observations identify the first mechanically-gated anchoring protein for PKA, a new force-dependent binding partner for talin1, and thus a new mechanism for coupling cellular tension and signal transduction.
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