Made to measure - keeping Rho kinase at a distance

Abstract

The Rho-associated coiled-coil containing kinases (ROCK) were first identified as effectors of the small GTPase RhoA, hence their nomenclature. Since their discovery, two decades ago, scientists have sought to unravel the structure, regulation, and function of these essential kinases. During that time, a consensus model has formed, in which ROCK activity is regulated via both Rho-dependent and independent mechanisms. However, recent findings have raised significant questions regarding this model. In their recent publication in Nature Communications, Truebestein and colleagues present the structure of a full-length Rho kinase for the first time. In contrast to previous reports, the authors could find no evidence for autoinhibition, RhoA binding, or regulation of kinase activity by phosphorylation. Instead, they propose that ROCK functions as a molecular ruler, in which the central coiled-coil bridges the membrane-binding regulatory domains to the kinase domains at a fixed distance from the plasma membrane. Here, we explore the consequences of the new findings, re-examine old data in the context of this model, and emphasize outstanding questions in the field.

Keywords: GTPase; ROCK; RhoA; coiled-coil; cytoskeleton; kinase; membrane anchor; molecular ruler; stress fibers.

Figure 1.

A historical model of ROCK regulation. ROCK is maintained in an inactive state in the cytosol by autoinhibitory interactions between the carboxy terminal regulatory Rho-binding (RBD), PH and C1 domains. A combination of membrane binding by the PH and C1 domains and the binding of activated, GTP-bound RhoA to the RBD relieves autoinhibiton of the kinase domain and promotes downstream substrate phosphorylation. A second mechanism of activation occurs during apoptosis, in which the kinase domains of ROCK are liberated by proteolytic cleavage of the regulatory PH and C1 domains. The split PH-C1 module has been shown to bind phosphoinositide-containing membranes in vitro.

Figure 2.

The molecular ruler model of ROCK function. ROCK2 is an extended homodimer, 120 nm in length in which the kinase and regulatory domains are separated by 107 nm of parallel, semi-rigid coiled-coil. The kinase domains exist in a constitutively competent conformation mediated by the capped helix bundle (CHB) dimerization domain and an ordered, active conformation activation loop in the absence of phosphorylation. The regulatory domains exert no influence on catalytic activity. RhoA does not bind directly to either ROCK1 or ROCK2 in solution. The coiled-coil of ROCK is highly divergent in sequence, but remarkably conserved in length. Truncations in the coiled-coil, while retaining activity, cause the loss of actin stress fibers in cells, indicating the functional significance of its length. As such, the coiled-coil of ROCK bridges the kinase domains to a fixed distance from the plasma membrane. Scaffold proteins, such as Shroom, may bind to the coiled-coil of ROCK, stabilizing the orientation of the coiled-coil and perhaps restricting the positioning of the kinase domains. This leads to a model in which the phosphorylation of substrates by ROCK is governed by the spatial positioning of both kinase and substrate in the actin cytoskeleton. Proteolytic cleavage during apoptosis presumably liberates the active kinase domains, leading to delocalized activity, unregulated actomyosin contraction, and cell fragmentation.

Figure 3.

Membrane binding by the ROCK:RhoA complex – a topological problem. (A) Schematic illustrating the primary domain composition and structure of ROCK. (B) Structural model of ROCK2 bound to RhoA. A model of ROCK2 was constructed by combining the high-resolution structures of the kinase and regulatory domains (PDB IDs: 2F2U, 2ROV, 2ROW) together with a modeled parallel coiled-coil, 107 nm in length. The complex of ROCK1:RhoA was docked onto the coiled-coil at a position corresponding to the location of the RBD in the primary sequence using the region of canonical coiled-coil in the structure (PDB ID: 1S1C) to which RhoA was observed to bind. In this model, RhoA binds to the coiled-coil at a position 90 nm distal to the kinase domains and 17 nm proximal to the membrane-binding domains. (C) RhoA is anchored in the membrane by a geranylgeranyl (C20) lipid anchor covalently attached to its C-terminus. (D) Docking the complex to a membrane such that the coiled-coil of ROCK is oriented perpendicular to the plane of the membrane results in the lipid anchors of RhoA being too far from the membrane. (E) Docking the complex to a membrane such that the coiled-coil of ROCK is parallel to the plane of the membrane results in the C-termini of RhoA (cyan) projecting in opposite directions. In this topology, only one molecule of RhoA would be capable of inserting its lipid anchor in the membrane.