The ROCK1 PH domain interacts with active Rho to transduce cell contraction signals

Abstract

The spatio-temporal regulation of cell contraction is crucial for numerous biological processes. In particular, contraction pulses in the cell cortex contribute to mechanotransduction and tissue rearrangements during embryonic development. We previously identified a signaling network that generates mechanosensitive contraction pulses in adherent mammalian cells via positive and negative feedback regulation of the small GTPase RhoA. Our investigations into the molecular mechanism of this process revealed surprising observations that challenge prevailing models of ROCK1 regulation. In particular, we identified a novel RhoA binding site in the ROCK1 PHC1 tandem domain that is sufficient for dynamic recruitment leading to increased Rho activity within subcellular regions of the cell cortex. AlphaFold-guided mutagenesis supports a direct interaction between these molecules. Functional investigations show that the PHC1 domain is required for efficient recruitment to active Rho, and that it plays a role in the transduction of Rho activity via ROCK1 to Myosin II activation. Based on the newly identified Rho binding site at the C-terminus of ROCK1, we propose a model for ROCK1 activation, which can resolve inconsistencies between previous biochemical and structural studies.