Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

The ROCK‐I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin‐based focal adhesions. ROCK‐I phosphorylates the unconventional G‐protein RhoE on multiple N‐ and C‐terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA‐induced stress fibre assembly. Here, we provide a molecular explanation for multi‐site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK‐I kinase domain. RhoE interacts with the C‐lobe αG helix of ROCK‐I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK‐I catalytic site. Disruption of the ROCK‐I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE‐mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK‐I through protein(s) binding to its effector region. We propose that ROCK‐I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.