Micro-environmental clues are critical to cell behavior. One of the key elements of migration is the generation and response to forces. Up to now there is no definitive concept on how the generation and responses to cellular forces influence cell behavior. Here, we show that phosphorylation of paxillin is a crucial event in the response to exogenous forces. Application of force induced growth of adhesion sites and this phenomenon was accompanied by a downregulation of Src family kinase activity, which in turn led to a decrease in the phosphorylation of paxillin at the tyrosine residues Y31 and Y118. The force-dependent growth of adhesion sites is mediated by a decrease in the turnover-rate of paxillin in focal contacts. This turnover critically depended on the phosphorylation state of paxillin at Y31/118. Paxillin is an important regulator in the control of the aggregate state of the whole adhesion site since the turnover of other adhesion site proteins such as vinculin is influenced by the phosphorylation state of paxillin as well. Taken together these data suggest that SFK dependent phosphorylation of paxillin is a crucial event in the regulation of adhesion site function in response to force.
Cancer cells normally grow on soft surfaces due to impaired mechanosensing of the extracellular matrix rigidity. Upon restoration of proper mechanosensing, cancer cells undergo apoptosis on soft surfaces (anoikis) like most normal cells. However, the link between mechanosensing and activation of anoikis is not clear. Here we show that death associated protein kinase 1 (DAPK1), a tumor suppressor that activates cell death, is directly linked to anoikis activation through rigidity sensing. We find that when rigidity sensing is decreased through inhibition of DAPK1 activity, cells are transformed for growth on soft matrices. Further, DAPK1 catalyzes matrix adhesion assembly and is part of adhesions on rigid surfaces. This pathway involves DAPK1 phosphorylation of tropomyosin1.1, the talin1 head domain, and tyrosine phosphorylation of DAPK1 by Src. On soft surfaces, DAPK1 rapidly dissociates from the adhesion complexes and activates apoptosis as catalyzed by PTPN12 activity and talin1 head. Thus, DAPK1 is important for adhesion assembly on rigid surfaces and the activation of anoikis on soft surfaces through its binding to rigidity-sensing modules.
Classical soft agar assays indicate that cancer cells do not properly sense the rigidity of their environment, as non-cancer cells typically undergo anoikis under such conditions. However, it is unclear how rigidity sensing activates anoikis. Here, we show that deathassociated protein kinase (DAPK1), which is linked to anoikis and correlates with tumor suppression, catalyzes matrix adhesion assembly and rigidity sensing via its phosphorylation of tropomyosin2.1 (Tpm2.1). Assembly of DAPK1 in adhesions depends upon the talin1 head domain, and occurs early upon formation of nascent adhesions and rigidity sensors. However, on soft matrices, DAPK1 rapidly dissociates from the adhesion complexes and activates apoptosis. When rigidity sensing is decreased through inhibition of DAPK1 activity, cells are transformed for growth on soft matrices and do not activate anoikis. DAPK1 phosphorylation of Tpm2.1 is important for adhesion formation but facilitates anoikis on soft substrates. DAPK1 assembly in adhesions also depends on Y490/Y491 phosphorylation by Src and DAPK1 Y-to-F mutations catalyze apoptosis as does PTPN12 activity.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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