Biology subjects

Howe, A. K.

Publications and source records attributed to Howe, A. K..

5 recordsLinked to original sources

bioRxiv2023

Protein Kinase A is Functional Component of Focal Adhesions

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify fifty-three high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3) - a well-established molecular scaffold, regulator of cell migration, and component of focal and fibrillar adhesions - as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

cell biology↗

bioRxiv2023

The focal adhesion protein talin is a mechanically-gated A-kinase anchoring protein (AKAP)

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-II regulatory (RII) 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 -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.

cell biology↗

bioRxiv2022

Protein Kinase A Activity In The Leading Edge Of Migrating Cells Is Dependent On The Activity Of Focal Adhesion Kinase

Protein Kinase A (PKA) is a pleiotropic serine/threonine kinase whose localized and dynamic activity is required for cellular migration. Several cell types have shown robust PKA activity in the leading edge during migration. This activity is regulated by changes in actomyosin contractility, but the mechanism of the mechanochemical regulation of PKA remains unclear. Our ongoing investigation into this mechanism led us to discover a novel relationship between PKA and the non-receptor tyrosine kinase Focal Adhesion Kinase (FAK). Here, we find that while inhibition of actomyosin contractility leads to a relatively slow decrease in total cellular levels of phosphorylated FAK over tens of minutes, it decreases the focal adhesion-associated pool of both phospho-FAK and phospho-paxillin within two minutes, similar to the timing of the effect of inhibition of contractility on leading edge PKA activity. We then show that pharmacologic inhibition of FAK rapidly decreases leading edge PKA activity. Additionally, leading edge PKA activity is significantly reduced in FAK-null mouse embryonic fibroblasts and essentially eliminated by also silencing expression of the closely-related kinase Pyk2 in these cells. Importantly, leading edge PKA activity in FAK-null cells is rescued by re-expression of wild-type but not kinase-dead FAK. Taken together, these observations show that FAK activity is required for localized regulation of PKA during cell migration and, in so doing, establish a novel and unexpected connection between an adhesion-regulated tyrosine kinase and a serine/threonine kinase canonically downstream of G-protein coupled receptor and second-messenger signaling.

cell biology↗

bioRxiv2018

Protein Kinase A activity is regulated by actomyosin contractility during cell migration and is required for durotaxis

Dynamic subcellular regulation of Protein kinase A (PKA) activity is important for the motile behavior of many cell types, yet the mechanisms governing PKA activity during cell migration remain largely unknown. The motility of SKOV-3 epithelial ovarian cancer (EOC) cells has been shown to be dependent on both localized PKA activity and, more recently, on mechanical reciprocity between cellular tension and extracellular matrix (ECM) rigidity. Here, we investigated the possibility that PKA is regulated by mechanical signaling during migration. We find that localized PKA activity in migrating cells rapidly decreases upon inhibition of actomyosin contractility (specifically, of myosin ATPase, ROCK (Rho kinase), or MLCK (myosin light chain kinase) activity). Moreover, PKA activity is spatially and temporally correlated with cellular traction forces in migrating cells. Additionally, PKA is rapidly and locally activated by mechanical stretch in an actomyosin contractility-dependent manner. Finally, inhibition of PKA activity inhibits mechanically-guided migration, also known as durotaxis. These observations establish PKA as a locally-regulated effector of cellular mechanotransduction and as a regulator of mechanically-guided cell migration.

cell biology↗

bioRxiv2017

The mechanical microenvironment regulates ovarian cancer cell morphology, migration, and spheroid disaggregation

There is growing appreciation of the importance of the mechanical properties of the tumor microenvironment on disease progression. However, the role of extracellular matrix (ECM) stiffness and cellular mechanotransduction in epithelial ovarian cancer (EOC) is largely unknown. Here, we investigated the effect of substrate rigidity on various aspects of SKOV3 human EOC cell morphology and migration. Youngs modulus values of normal mouse peritoneum, a principal target tissue for EOC metastasis, were determined by atomic force microscopy (AFM) and hydrogels were fabricated to mimic these values. We find that cell spreading, focal adhesion formation, myosin light chain phosphorylation, and cellular traction forces all increase on stiffer matrices. Substrate rigidity also positively regulates random cell migration and, importantly, directional increases in matrix tension promote SKOV3 cell durotaxis. Matrix rigidity also promotes nuclear translocation of YAP1, an oncogenic transcription factor associated with aggressive metastatic EOC. Furthermore, disaggregation of multicellular EOC spheroids, a behavior associated with dissemination and metastasis, is enhanced by matrix stiffness through a mechanotransduction pathway involving ROCK, actomyosin contractility, and FAK. Finally, this pattern of mechanosensitivity is maintained in highly metastatic SKOV3ip.1 cells. These results establish that the mechanical properties of the tumor microenvironment may play a role in EOC metastasis.

cell biology↗