Mechanical Stressing of Integrin Receptors Induces Enhanced Tyrosine Phosphorylation of Cytoskeletally Anchored Proteins

Abstract

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the ␤1 and the ␣2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.

Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

“…In addition, retrograde flux of FAK may have a profound effect on the state of phosphorylation at focal adhesions and actin filaments, which in turn affects the maturation and turnover of focal adhesions (Webb et al, 2004). Our results further suggest that the flux of focal adhesion proteins requires tyrosine phosphorylation, which, in conjunction with the small GTPases Rac and Rho, represents the primary molecular switches in response to integrin-ECM anchorage and mechanical signals (Burridge et al, 1992;Schmidt et al, 1998). Tyrosine phosphorylation regulates the dynamic turnover of focal adhesion proteins through a number of substrates including paxillin, p130Cas, and FAK (Carragher and Frame, 2004).…”

“…Given the response of tyrosine phosphorylation to mechanical signals (Pelham and Wang, 1997;Schmidt et al, 1998), we asked whether tyrosine phosphorylation might be involved in regulating focal adhesion fluxes. Immunofluorescence localization showed that phosphotyrosine was concentrated only at focal adhesions irrespective of the presence of zyxin tails ( Figure 8A).…”

“…Such responses are often accompanied by the induction or enhancement of centripetal cortical movements, which may manifest as flow of actin arcs in lamella or retrograde movements of integrin-associated beads (Felsenfeld et al, 1996). Chemical signals involved in these processes include the activation of small GTPases, including Rho and Rac (Chrzanowska-Wodnicka and Burridge, 1996), and tyrosine kinases, including Src family kinases (Suter and Forscher, 2001) and focal adhesion kinase (FAK; Burridge et al, 1992;Schmidt et al, 1998), as well as the entry of Ca 2ϩ through stretch-activated ion channels (Guharay and Sachs 1984;Lee et al, 1999;Munevar et al, 2004).…”

“…Such responses are often accompanied by the induction or enhancement of centripetal cortical movements, which may manifest as flow of actin arcs in lamella or retrograde movements of integrin-associated beads (Felsenfeld et al, 1996). Chemical signals involved in these processes include the activation of small GTPases, including Rho and Rac (Chrzanowska-Wodnicka and Burridge, 1996), and tyrosine kinases, including Src family kinases (Suter and Forscher, 2001) and focal adhesion kinase (FAK; Burridge et al, 1992;Schmidt et al, 1998), as well as the entry of Ca 2ϩ through stretch-activated ion channels (Guharay and Sachs 1984;Lee et al, 1999;Munevar et al, 2004).A more intriguing response is the redistribution of some focal adhesion proteins upon mechanical stimulations. Several proteins, including zyxin and vasodilator-stimulated phosphoprotein (VASP), are known to localize to a variable extent at focal adhesions (Crawford et al, 1992;Rottner et al, 2001), or to shuttle between focal adhesions and the nucleus (Nix and Beckerle, 1997;Aplin and Juliano, 2001).…”

Contribution of Bone Tissue Modulus to Breast Cancer Metastasis to Bone

“…RPTP-α can activate Src family kinases (SFKs) within about 300 ms after application of a force on fibronectin beads [65][66][67]. Phosphorylation of mitogen-activated protein kinase (MAPK) has also been reported to increase in response to mechanical stress, suggesting that integrins sense forces regulating gene expression by activation of the MAPK pathway [68], which is known to be downstream of TGF-β signaling [69].…”