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

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization

Structure of a highly conserved domain of rock1 required for shroom-mediated regulation of cell morphology

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. © 2013 Mohan et al

The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice

Shroom3 is an actin-associated regulator of cell morphology that is required for neural tube closure, formation of the lens placode, and gut morphogenesis in mice and has been linked to chronic kidney disease and directional heart looping in humans. Numerous studies have shown that Shroom3 likely regulates these developmental processes by directly binding to Rho-kinase and facilitating the assembly of apically positioned contractile actomyosin networks. We have characterized the molecular basis for the neural tube defects caused by an ENU-induced mutation that results in an arginine-to-cysteine amino acid substitution at position 1838 of mouse Shroom3. We show that this substitution has no effect on Shroom3 expression or localization but ablates Rock binding and renders Shroom3 non-functional for the ability to regulate cell morphology. Our results indicate that Rock is the major downstream effector of Shroom3 in the process of neural tube morphogenesis. Based on sequence conservation and biochemical analysis, we predict that the Shroom-Rock interaction is highly conserved across animal evolution and represents a signaling module that is utilized in a variety of biological processes

Rock-Shrm interaction is required for apical constriction.

<p>(A) Parental or EndoShrm3 expressing T23 MDCK cells were treated with either control or Rock1 and 2 specific siRNAs and stained to detect ZO1. (B) Western blot analysis of Rock1 and Rock 2 knock-down in MDCK cells. (C) T23 MDCK cells expressing EndoShrm3 treated with Rock1/2 siRNA for 2 days, transfected with the indicated hRock1 expression vectors, grown on transwell membranes for 24 hours, and stained to detect ZO1 and hRock1. Z-projections are shown in smaller panels. (D) Quantification of rescue of apical constriction by Rock1 variants. Apical area, as determined by the outline of ZO1 staining, was measured for parental, EndoShrm3 (ES3) expressing, and EndoShrm3 cells treated with Rock1 siRNA (+siRNA). For rescue experiments, apical areas of only those cells that expressed the indicated Rock1 proteins (WT = wildtype, KD = kinase dead; IA = RhoA binding domain mutant, 5A = SBD mutant ⁸⁵⁵LYKTQ⁸⁵⁹ to ⁸⁵⁵AAAAA⁸⁵⁹) were measured. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081075#s3" target="_blank">Results</a> are shown for 15 cells picked at random from a single experiment. The horizontal line indicates the average apical area while ** indicates <i>p</i> = 0.001 relative to the apical area of parental cells.</p

A Central region within the coiled-coil domain interacts with Shrm SD2.

<p>A) Diagram of Rock1 domain structure. Domains and their boundaries within Rock1 are indicated. N- and C-terminal extensions on the Rock1 kinase domain are shown in red. Sequence conservation from a multiple sequence alignment of 22 Rock sequences is shown with sequence positions containing 90% identity indicated in blue. B) Identification of a minimal Shrm SD2 binding domain within Rock1. Purified untagged human Shrm2 SD2 was mixed with beads pre-bound to the indicated his-tagged fragment of Rock1. Complexes were precipitated by spinning down the beads and the resulting samples were resolved on SDS-PAGE. P, pelleted beads; S supernatant. C) Rock fragments were assayed for binding to Shrm SD2. Increasing concentrations of Rock1 (707–946) or (834–913) were added to a reaction mixture containing 50 nM Oregon-Green labeled human Shrm2 SD2 domain in a fluorescence spectrophotometer. The binding isotherm was fit to Equation 1 using a non-linear regression to determine binding affinity (<i>K_d</i>).</p

Rock1 SBD variants show decreased interaction with Shrm.

<p>Fluorescence anisotropy experiments monitored 50-Green labeled human Shrm2 SD2 domain with increasing concentrations of Rock1 (707–946) containing the indicated amino acid substitutions. The change in anisotropy was fit to Equation 1 to determine binding affinities (<i>K_d</i>) as indicated.</p

Crystallographic Data collection and refinement statistics for human Rock1 SBD^SER.

<p>Values in parentheses correspond to those in the outer resolution shell.</p><p>R_merge = (|(ΣI−<i>)|)/(ΣI), where <i> is the average intensity of multiple measurements.</i></i></p><p><i><i>R_work = Σ_hkl∥F_obs(hkl)∥−F_calc (hkl)∥/Σ_hkl|F_obs(hkl)|.</i></i></p><p><i><i>R_free = crossvalidation R factor for 7.3% of the reflections against which the model was not refined.</i></i></p

Opposing Shrm binding sites within the SBD.

<p>A) Surface representation of the Rock SBD colored by the effect of substitutions in cell based and in vitro assays. Included are positions which altered Shrm colocalization (magenta), positions which affected SD2 binding in vitro (red), residues which did not affect SD2 binding in vitro (green), and residues 900–902 which had a subtle affect on SD2 binding (pale green). B) Cutaway view of the Shrm binding region. Ribbon diagram and positions of side chains with a demonstrated affect (sticks) are colored as above. Black represents the Rock surface which has been cut away to reveal the backbone and side chains underneath. A hydrophobic patch comprised of residues Y851, F852, and L855 is indicated for each binding site.</p

The Rock1 SBD is required for localization with Shrm3.

<p>(A–F) Myc-tagged wild type or SBD variants of hRock1 (681–942) were co-expressed with wildtype Shrm3 or a Shrm3 variant lacking the SD2 in Cos7 cells and stained to detect Shrm3 (green) or the myc tag (red). The right-hand panels in A, C–F depict the results of pulldown assays to detect the interaction of the indicated SBD variant and the Shrm3 SD2. Binding of the Rock SBD variants was tested by using immobilized GST-Shrm3 SD2 and lysates from HEK293 cells expressing the indicated SBD protein, followed by western blotting to detect the myc-tagged SBD proteins. Input = total cell lysate, GST = pulldown using GST bound to beads, GST-SD2 = GST-Shrm3-SD2 bound to beads. Arrowhead denotes the myc-tagged Rock protein. (G–I) T23 MDCK epithelial cells were transfected with expression vectors for EndoShrm3 and Rock1 SBD (G), EndoShrm3 and Rock1-SBD ⁸⁵⁵LYKTQ⁸⁵⁹ to ⁸⁵⁵AAAAA⁸⁵⁹ (H) or EndoShrm3ΔSD2 and Rock1-SBD (I), grown on transwell filters overnight to form confluent monolayers, and stained to detect EndoShrm3 (green) and Rock-SBD (red). Dashed lines indicate the position of the Z-projections that are shown in the lower panels. Ap, apical surface; Bsl, basal surface.</p

A conserved region on the SBD surface mediates Shrm binding.

<p>A) Surface view of the SBD dimer colored by sequence conservation. Residues colored light blue are identical in >95% of the Rock sequences in our alignment, while residues that are invariant across all 14 sequences are color darker blue. Residues that were altered in our mutational analysis are labeled. B) Surface of the SBD dimer colored by Surface Triplet Propensity. Scoring is colored as a heat map with lowest scores in dark blue and the highest scores in red. A prominent patch containing residues Y851 and F852 is indicated. C) Residues within the conserved patch contribute to Shrm binding. Human Shrm2 SD2 was mixed with wild-type Rock1 SBD or the indicated mutant and the formation of a Rock-Shrm complex was detected by native gel electrophoresis.</p