Structure of a Highly Conserved Domain of Rock1 Required for Shroom-Mediated Regulation of Cell Morphology

Abstract

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.

Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity

“…4, D and F) and in all cases were found to behave like wild type, indicating that the indicated substitutions had no effect on overall structure. Together, these data are consistent with our previous biochemical analysis performed in the absence of structural information on the binding interface (21,30,41,44) and pinpoint residues within both Shrm and Rock that are critical for complex formation in vitro.…”

“…Structural data exist for many domains within Shrm and Rock including the Shrm-binding domain (SBD) of human Rock1 (41), which forms a parallel coiled coil, consistent with previous structural data from the RhoA-binding and central regions of Rock (42,43). Biochemical analysis identified residues important for Shrm binding and found them to be positioned on opposing faces of the Rock coiled coil (41).…”

“…We only obtained suitable diffraction data from crystals of the smallest Shrm-Rock module tested, which contained human Shrm2 SD2 (residues 1427-1610) and Rock1 SBD (residues 834 -913). Diffraction data were weak and highly anisotropic as was observed for Rock1 (41). The SD2-SBD structure was refined to 3.57-Å resolution with an R work/free of 27.3/28.7% (see "Experimental Procedures" and Table 1 for a complete description of the structure determination process).…”

“…Structural data exist for many domains within Shrm and Rock including the Shrm-binding domain (SBD) of human Rock1 (41), which forms a parallel coiled coil, consistent with previous structural data from the RhoA-binding and central regions of Rock (42,43). Biochemical analysis identified residues important for Shrm binding and found them to be positioned on opposing faces of the Rock coiled coil (41). Surprisingly, the structure of the SD2 domain from Drosophila Shrm revealed that this conserved domain adopted a three-segmented, antiparallel coiled coil with mutational analysis indicating two Rock-binding sites located ϳ80 Å apart (44).…”

Coiled‐coils: The long and short of it

“…In the actin cortex, mechanical force transducers have been shown to adopt dramatically different conformations under different loads , suggesting that the precise positioning of components in the cortex likely varies as a consequence of cytoskeletal tension. In terms of regulating ROCK positioning, the scaffold protein Shroom, which interacts with a small segment of the coiled‐coil of ROCK (Fig. ), has been shown to be essential for neural tube closure during embryogenesis .…”