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

Abstract

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 alphaG 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.

Figure 1

Overview of ROCK-I:RhoE complex. Each subunit of the ROCK-I dimer binds one molecule of RhoE, forming a dyad-symmetrical molecule. The complex dimerises through the dimerisation domain (pink) of ROCK-I. Structural figures were created using PYMOL (www.pymol.org).

Figure 2

RhoE interacts through its α4–β6–α5 module with the C-lobe of the kinase. (A) View of the ROCK-I:RhoE complex, with secondary structural elements at the protein interface coloured cyan. Remainder of ROCK-I in blue and pink (dimerisation domain) and RhoE in orange with switch I and II coloured red. Surface representation of RhoE (B) and ROCK-I (C) rotated in opposite directions to (A) to reveal subunit interface. Colour coding as in (A).

Figure 3

Details of ROCK-I:RhoE interface. (A) View of interface formed by the α4–β6–α5 module of RhoE to the αG and αEF helices, αEF/αF loop and activation segment of ROCK-I. Colour scheme as in Figure 2. (B) Schematic of interface shown in (A). Hydrogen bonds and van der Waals interactions are indicated by grey and red dashed lines, respectively. ROCK-I and RhoE residue labels are in blue and orange. (C) Multiple sequence alignment of RhoE, Rnd1, Rnd2 and RhoA. Residues contacting ROCK-I are indicated by blue and green arrowheads, with blue arrowheads showing mutated residues (Thr173, Val192). Red arrowheads denote residues of the effector region required for RhoE function. Figure was produced using ALSCRIPT (Barton, 1993).

Figure 4

Conformational changes of RhoE and ROCK-I on forming a complex are confined to the ROCK-I:RhoE interface. (A) RhoE with comparison to isolated RhoE (Fiegen et al, 2002; Garavini et al, 2002) and RhoA structures (Ihara et al, 1998) and (B) stereoview of ROCK-I, isolated ROCK-I from Jacobs et al (2006).

Figure 5

RhoE phosphorylation by ROCK-I. (A) View of the ROCK-I:RhoE dimer with a substrate peptide based on GSK3β bound to PKB (magenta) (Yang et al, 2002), modelled into the catalytic cleft of ROCK-I. The distance between the visible N and C termini of RhoE to the phosphoacceptor site in ROCK-I is 20 Å. Colour scheme as in Figure 2. (B) Details of the catalytic site of ROCK-I, F_o–F_c electron density (contoured at 3σ in green) overlapping a GSK3β peptide bound to PKB modelled into the ROCK-I catalytic site.

Figure 6

Disruption of ROCK-I:RhoE interaction and effects on RhoE/ROCK-I responses. (A) The indicated wild-type (WT) and mutant GST–RhoE proteins on glutathione beads were incubated with cell lysates from COS-7 cells transfected with pCAG-myc-ROCK-I¹⁻⁴²⁰ (wild-type and indicated mutants). Proteins that bound to GST–RhoE were resolved by SDS–PAGE and western blotted with anti-myc antibody to identify myc–ROCK-I. The lower panel (Coomassie staining of polyacrylamide gel) shows that equal amounts of each GST–RhoE mutant were used in the binding assay. (B) GST–RhoE was incubated with His-ROCK-I purified from baculovirus-infected insect cells. ROCK-I bound to glutathione agarose immobilised RhoE was identified by western blotting with anti-His antibody. (C) ROCK-I phosphorylation of RhoE in cells. COS-7 cells were co-transfected with the indicated FLAG–RhoE and myc–ROCK-I¹⁻⁴²⁰ expression vectors. After 24 h, cells were lysed and cell lysates were western blotted with anti-FLAG antibody to detect RhoE. ROCK-I-phosphorylated RhoE migrates more slowly. ERK1 panels indicate equivalent loading in each lane.

Figure 7

Effect of RhoE mutants on the actin cytoskeleton. HeLa cells were transfected with the indicated FLAG–RhoE wild-type (WT) and mutant constructs, or with empty vector (control). After 24 h, cells were fixed and stained for actin filaments and with anti-FLAG antibody. (A) Representative images of transfected cells. Left panels of each pair of images show actin filaments (F-actin); cells expressing transfected FLAG epitope-tagged RhoE are indicated with arrows, and were identified by staining with anti-FLAG antibody (right panels of each pair, labelled ‘RhoE'). Note that cells expressing transfected RhoE have much fewer stress fibres than surrounding untransfected cells, except for cells expressing V56Y and F57A mutants. (B) Cells expressing transfected RhoE were scored for loss of stress fibres (compared with surrounding untransfected cells) and for rounded, ‘branching' phenotype; 100 FLAG antibody-stained cells were counted on each of three coverslips; mean % of cells in each category ±s.e.m. is shown. Note that the ‘branching' phenotype is a stronger response to RhoE than loss of stress fibres. (C) Model for RhoE and ROCK-I regulation. RhoE exerts negative feedback regulation of ROCK-I. Stabilised RhoE (through RhoA-activated ROCK-I phosphorylation or RhoE over-expression) antagonises RhoA-stimulation of ROCK-I through either activation of p190 RhoGAP or through regulation of an alternative RhoA effector.