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

TY - JOUR

T1 - Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

AU - Komander, David

AU - Garg, Ritu

AU - Wan, Paul T. C.

AU - Ridley, Anne J.

AU - Barford, David

PY - 2008/12/3

Y1 - 2008/12/3

N2 - 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 alpha G helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N- and C-terminal 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.

AB - 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 alpha G helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N- and C-terminal 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.

U2 - 10.1038/emboj.2008.226

DO - 10.1038/emboj.2008.226

M3 - Article

VL - 27

SP - 3175

EP - 3185

JO - EMBO Journal

JF - EMBO Journal

IS - 23

ER -