Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB070

Poster Communications

Mechanical stretch induces phosphorylation of keratin intermediate filaments 8 and 18

A. M. Lutz1, T. Felder1, P. Dietl1, E. Felder1

1. Institute of General Physiology, Ulm University, Ulm, Baden-Württemberg, Germany.

Keratin 8 (K8) and 18 (K18) form the intermediate filament (IF) network in cells of simple epithelia. The most widely proposed function of keratin filaments (KFs) is the mechanical stabilization of cells but several studies suggest that KFs are also involved in signaling and other functions. Phosphorylation is the most important post-translational modification of K and it was shown that K8 and K18 are phosphorylated at different serine residues in their head and tail domains. Various kinds of cell stress can induce K phosphorylation. K phosphorylation fulfills different functions in the cell. It changes the distribution of keratins within the cell, alters its polymerization behavior and is associated with keratin granule formation. Previous work of our group showed that K8 serine 431 (ser431) phosphorylation can reduce the tensile force on the desmosome upon stretch and that dephosphorylation of K18 serine 52 (ser52) alters the mechanical properties of KF. Since mechanical stress - in particular cell stretch - can pose a serious threat to epithelia by disrupting cell-cell contacts and destroying the integrity of an epithelial cell layer, we hypothesize that K phosphorylation acts as a mechanoprotective mechanism. Besides confirming this novel role of K phosphorylation, we also investigate how mechanical stimulation induces K phosphorylation, which includes the identification of the involved signaling pathways as well as the primary mechanosensor. In our experiments, we exposed A549 lung carcinoma cells to biaxial stretch for different time durations. A549 cells were grown on elastic PDMS membranes, which were stretched to a maximum of 50 % longitudinal distension. Subsequently, the phosphorylation levels of two potential phosphorylation sites in each K8 and K18 were determined by In-Cell ELISAs and Western blots. We observed an increase in phosphorylation on keratin 8 serine 73 (ser73) and serine 431 and keratin 18 serine 33 (ser33) and serine 52 upon stretch. The increase in phosphorylation was dependent on stretch duration. To elucidate the signaling pathway(s) responsible for phosphorylation of the keratin residues, A549 cells were incubated with various kinase inhibitors prior and during the stretch. Thereby we identified ERK1/2 kinases to be in part responsible for the stretch induced phosphorylation of K8ser73 and K8ser431 and K18ser52, as their inhibition with the potent and selective ERK1/2 inhibitor FR180204 reduced phosphorylation levels significantly. These results confirm that stretch as a form of mechanical cell stress leads to increased phosphorylation of KIF.

Where applicable, experiments conform with Society ethical requirements