Abstract
Cell–extracellular matrix (ECM) detachment is known to decrease extracellular signal–regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell–ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell–ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell–ECM adhesion–induced ERK activation. Furthermore, cell–ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150–206) effectively suppressed the cell–ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell–ECM detachment and links it to decreased ERK signaling.
Highlights
It has been well established that cell–extracellular matrix (ECM) adhesion is crucial for regulation of the extracellular signal–regulated kinase (ERK) signaling pathway, which is central for control of cell behavior including cell proliferation, migration, and survival [1,2,3,4,5,6,7,8,9]
The cell– ECM detachment–induced suppression of mitogen-activated protein kinase kinase (MEK) and ERK activation was abolished in the absence of Ras Suppressor 1 (RSU1) (Fig. 1C, compare lanes 3 and 4), suggesting that RSU1 is essential for cell–ECM detachment–induced suppression of MEK and ERK activation
We have tested the role of RSU1 in cell–ECM adhesion–induced regulation of MEK and ERK signaling in other cell types, in which RSU1 was depleted using the same CRISPR/ Cas9 strategy
Summary
It has been well established that cell–extracellular matrix (ECM) adhesion is crucial for regulation of the extracellular signal–regulated kinase (ERK) signaling pathway, which is central for control of cell behavior including cell proliferation, migration, and survival [1,2,3,4,5,6,7,8,9]. Cell–ECM detachment–induced suppression of MEK and ERK activation was restored in the cells re-expressing RSU1 (Fig. 1D, compare lanes 3 and 4) but not in the control cells (Fig. 1D, compare lanes 1 and 2), confirming an essential role of RSU1 in this process. Re-expression of RSU1 in the RSU1 KO MD231 cells restored the cell–ECM detachment–induced suppression of MEK and ERK activation (Fig. S1).
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