Dsh directly binds the ‘Hook’ domain of Dlg.

<p>(<b>A</b>) Domain architectures of Dsh and Dlg are shown. Dsh (<i>top</i>) consists of an N-terminal DIX (<u>Di</u>shevelled and A<u>x</u>in) domain, a central PDZ (<i>yellow</i>; <u>P</u>ostsynaptic density-95, <u>D</u>iscs large, and <u>Z</u>O-1) domain, and a C-terminal DEP (<u>D</u>ishevelled, <u>E</u>gl-10, and <u>P</u>leckstrin) domain. Dlg (<i>bottom</i>) consists of tandem N-terminal PDZ domains followed by a C-terminal array of PDZ, SH3 (<i>blue</i>; <u>S</u>rc <u>h</u>omology-3), and GK (<i>red</i>; <u>G</u>uanylate <u>K</u>inase) domains. The sequence connecting the SH3 and GK domains has been termed the ‘Hook’ domain (<i>green</i>), which varies in length among species (∼90 amino acids in <i>Drosophila</i>) and undergoes alternative splicing, yielding the specific ‘I3-insert’-containing isoform investigated herein. (<b>B</b>) Structural representation of the SH3-Hook-GK cassette from <i>Drosophila</i> Dlg demonstrates the close association of the SH3 and GK domains. The Hook domain is mostly absent, likely due to conformational flexibility within protein crystals. The SH3-Hook domains act as repressors of GK domain-mediated protein interactions through a poorly understood allosteric mechanism. Image rendered from PDB id: 3TVT with bound ligand removed for clarity. (<b>C</b>) GST pulldown experiments demonstrate a Hook-dependent direct interaction between Dsh and Dlg. GST alone (control) or fused to the PDZ-DEP domains of Dsh (GST:Dsh) were coupled to glutathione agarose and subsequently incubated with soluble 6x-His-tagged Dlg proteins spanning the entire SH3-Hook-GK domains (Dlg) or an SH3-GK tandem with the Hook domain removed (DlgΔHook). Samples were resolved by SDS-PAGE and analyzed by Ponceau red staining (<i>top</i>; GST proteins) or α-His western blots (<i>bottom</i>; Dlg proteins). Purified Dlg proteins input to respective reactions are shown to the <i>right</i>. (<b>D</b>) Dlg binding occurs exclusively through the PDZ domain of Dsh. GST or GST:Dlg-Hook were incubated in the presence of various Dsh domains; <i>left lanes</i> – PDZ alone (indicated by *), <i>middle lanes</i> – both PDZ and DEP domains together (indicated by **), <i>right lanes</i> – DEP domain alone (indicated by ***). Dlg-Hook-specific interactions were detected for PDZ and PDZ-DEP constructs but not for the isolated DEP domain. The top image shows the Ponceau stained membrane depicting comparable input of GST constructs across all conditions; the bottom image is the α-His western blot used to detect bound His-tagged Dsh proteins (an ‘input’ lane of each is shown for reference).</p

Dsh expression activates Dlg-mediated spindle orientation through the GK interacting protein GukH.

<p>(<b>A</b>) <i>Drosophila</i> S2 cells were transfected with the SH3-Hook-GK domains of Dlg fused to the cell adhesion protein Ed (Ed:Dlg) alone or in combination with full-length myc-tagged Dsh (wild-type or the D316A mutant). In additional experiments, transfected cells were treated with RNAi directed against GukH. ‘Induced polarization’ of cortical Ed:Dlg was achieved through Ed-mediated cell contacts, and spindle orientation angles of mitotic cells were measured relative to the center of the induced Ed:Dlg crescent (<i>white dashed lines in Merge images</i>). Shown below cell images are western blots of S2 lysates (20 µg total protein loaded) transfected with either wild-type or D316A Dsh (as pMT:myc fusion constructs), which demonstrate equal protein expression (α-myc). An antibody against β-actin was used as a loading control. (<b>B</b>) Cumulative percentage plots reflect fraction of cells with spindles oriented at or below a given angle for all acquired images (n≥30 for all conditions). Ed alone (<i>black; circles</i>) and Ed fused to the entire SH3-Hook-GK (<i>orange; squares</i>) measurements populate a diagonal expected for completely random orientations between 0-90°. Deletion of the Hook domain (ΔHook; <i>blue; upward triangles</i>) or co-expression of Dsh (<i>green; downward triangles</i>) results in a leftward shift expected for cell populations with increased percent of lower angle measurements. Co-expression of the non-binding Dsh-D316A mutant (<i>red; diamonds</i>) did not improve spindle orientation. *, p <0.05 compared to Ed alone, One-way ANOVA with <i>Dunnett</i>'<i>s post-hoc</i> test. (<b>C</b>) GukH RNAi treatment blocks Dlg-mediated spindle orientation induced by Hook truncation (<i>yellow; squares</i>) or Dsh expression (<i>cyan; upward triangles</i>) to a level statistically equivalent to Ed alone (<i>black; circles</i>). (<b>D</b>) Three additional RNAi sequences were generated against distinct sequence elements of GukH and examined for confirmation of specificity. Each alternative (‘Alt #1-3’) target produced a similar loss-of-function phenotype as that in panel C. (<b>E</b>) Schematic representation of the GukH transcript and location of each RNAi target (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114235#s2" target="_blank">Methods</a> for primer sequence information). Diverse targets include 5′ and 3′ untranslated regions along with two distinct targets within the coding region. All targets were against sequences shared universally among GukH isoforms. The average spindle orientation angle for each condition is shown in parentheses (compare to 26.3° for Ed:Dlg+Dsh in panel B).</p

The Dsh PDZ domain binds a conserved, internal K-K-x-x-x-φ ligand motif within the Dlg I3-insert.

<p>(<b>A</b>) Multiple sequence alignment of Dlg-Hook domains reveals considerable sequence conservation across metazoan evolution. The <i>Drosophila</i> (‘Fly’) Hook domain contains a C-terminal extended sequence (amino acids 726–764) not seen in most other species. The splice isoform of Dlg studied herein includes the I3-insert sequence (<i>solid black box</i>), which contains two segments of high sequence homology across species: a polybasic region (<i>dashed cyan box</i>) followed by stretch containing several hydrophobic amino acids (<i>dashed green box</i>). The highly conserved K-K-x-x-x-φ motif identified herein is also indicated (<i>dashed red box</i>). Amino acid mutations are indicated below with numberings corresponding to fly sequence (* denotes residues chosen for ‘STOP codon’ mutations as Hook domain truncations). (<b>B</b>) The Dlg Hook domain was fused to GST and iteratively truncated from the C-terminus using site-directed mutagenesis of selected amino acids to termination codons. Immobilized GST-Dlg proteins were incubated with the purified Dsh-PDZ domain, and resolved reactions were analyzed by Ponceau red staining. The bound Dsh-PDZ protein band is indicated by an arrow; the asterisk (*) indicates a nonspecific impurity sometimes seen in the GST alone control preparation, which resolves at a slightly higher molecular weight than Dsh-PDZ. Truncation up to amino acid 712 does not affect Dsh-PDZ binding, although further Hook truncation (amino acid 693) results in a complete loss of Dsh/Dlg interaction. (<b>C</b>) Alanine mutation of conserved residues within the I3-insert were analyzed for Dsh-PDZ binding. <i>Left panel</i>: Dlg-Hook domain residues 679-712 fused to GST (either wild-type or indicated mutant) were incubated with Dsh-PDZ and bound proteins were detected using Ponceau red staining of a membrane containing transferred proteins. The K700A mutant failed to bind Dsh, whereas the S705A mutant retained full binding capacity. The F708A mutant also showed significantly reduced binding. <i>Right panel</i>: Identical experiment but with bound Dsh-PDZ detected using an α-His western blot. Results are consistent with Ponceau red analysis. (<b>D</b>) Structural representation of the human Dvl2-PDZ domain (<i>light cyan</i>) bound to two optimized phage-display peptides (<i>orange</i> and <i>yellow</i>) demonstrates the molecular basis of ligand interaction (PDB ids: 3CBX and 3CBY). Both peptides contain N-terminal lysine [Lys(-5)] and C-terminal hydrophobic [Phe(0) or Ile(0)] residues (<i>green</i> and <i>blue</i>) that make contact with conserved PDZ domain residues (<i>red</i>) within the canonical ligand binding groove. Below is shown a multiple sequence alignment of the peptides (‘C1’ and ‘N1’), together with three natural Dsh-PDZ binding partners (Fz, Idax, and Dlg), demonstrating the conservation of the (0; hydrophobic) and (-5; lysine) positions. (<b>E</b>) Alanine mutation of Dlg K699 and F704 within the <b><u>K</u></b>-K-x-x-x-<b><u>φ</u></b> motif reduce binding to Dsh-PDZ. Resolved proteins were analyzed by α-His western blot analysis. (<b>F</b>) Alanine mutation of Dsh-PDZ revealed that both D319A and L325A mutants reduce GST-Hook interaction, whereas the V322A retained full binding capacity.</p