Interaction of Sam68 PxxP peptide mutants with SH3 domains.

<p>W  =  wildtype, M1 to M7  =  mutants, n/a  =  not applicable.</p><p>Compilation of phage-ELISA results indicating changes in binding of the SH3 domains to the PxxP-peptide-mutants compared to the wildtype peptide (++), classified as affinity reduced (+) or interaction abolished (0).</p><p>Core residues of the PxxP motifs are underlined; introduction of alanine point mutations is highlighted by a black background.</p

Analysis of Sam68-binding SH3 domains <i>in vivo</i> by FRET-analysis.

<p>Expression constructs for CFP-tagged Sam68 and YFP-tagged SH3 domains were used to co-transfect 293T cells as indicated. 48 h post transfection, cells were harvested for flow cytometric analysis. Direct protein interaction <i>in vivo</i> was assayed by determining FRET from CFP to YFP by exciting CFP at 405 nm and measuring fluorescence with filters 450/50 (CFP only) vs. 585/42 (CFP + YFP-FRET-signal). (A) Representative diagrams showing the shift of cell populations as a result of FRET. Based on the negative control (CFP, or CFP-Sam68, and YFP on separate plasmids) and the positive control (CFP-YFP-fusion protein on one plasmid) two gates were defined, enclosing cells that do not exhibit FRET (R2, red), or that do exhibit FRET (R3, green), which is manifest by a shift to the left (i.e. lower CFP emission) and simultaneously to the top (i.e. higher YFP-emission). The degree of this shift depends on the FRET-efficiency. (B) FRET signals for all domains assayed. Results are shown as mean ± standard deviation from three independent experiments.</p

Identification of Sam68-PxxP-motifs responsible for SH3-domain-binding.

<p>Peptides corresponding to the seven PxxP-motifs (P0 to P6) of Sam68 were purified as GST-fusions and analyzed for interaction with the indicated SH3 domains by phage-ELISA. Results are expressed semi-quantitatively as half-maximal binding occuring at <10¹¹ (+++), 10¹¹−10¹² (++), >10¹² cfu/ml (+).</p

Interaction of Sam68-PxxP-mutants with SH3 domains.

<p>Expression constructs for CFP-tagged Sam68-mutants defective in either any one of the SH3-interacting PxxP-motifs (Sam68ΔP0, −ΔP3, −ΔP4, −ΔP5), or defective in all (Sam68ΔP0345), were cotransfected with YFP-tagged SH3 domains from Yes, Fyn, p85α, or OSF, or wildtype-Sam68 into 293T-cells and analyzed for direct interaction <i>in vivo</i> by performing FRET-analysis as in Fig. 2. Results are shown as mean ± standard deviation from three independent experiments. Significant reduction (p<0.05 in Student’s T-test) of the signal as compared to wildtype is marked with an asterisk.</p

Secondary structure prediction of wildtype and mutant Sam68.

<p>(A) Schematic representation of Sam68 domains and positions of proline-rich motifs. The three motifs not binding to SH3 domains are enclosed in brackets. RG  =  arginine glycine rich region, NK  =  N-terminal of KH domain, KH  =  hnRNP K homology domain, CK  =  C-terminal of KH domain, YY  =  tyrosine rich region, NLS  =  nuclear localization sequence (B) Prediction of secondary structure by JPred, white bars: helical regions, black bars: extended regions. (C) Prediction of intrinsically disordered regions by IUpred.</p

Sam68-binding SH3 domains as identified by bio-panning of recombinant Sam68 against the human SH3-proteome phage-display library.

a<p>Numbers refer to supplementary table from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540-Krkkinen1" target="_blank">[31]</a>.</p>b<p>SH3 domains already reported as Sam68-binders, compare suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540.s004" target="_blank">Table S2</a>.</p>c<p>For a complete listing see suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540.s005" target="_blank">Table S3</a>.</p

Cytosolic tails of ADAM-metalloproteases and their candidate SH3 binding motifs.

<p><b>A.</b> Schematic presentation of ADAM cytosolic tails with the location of the candidate SH3-binding proline clusters indicated by circles with roman numerals counting from the transmembrane region to the carboxy terminus. The scale bar indicates distance in amino acid residues. <b>B.</b> Potential SH3 binding sequences within the proline clusters shown in A. Established SH3 target motifs (+ΦPxxP, PxΦPx+, PxxDY, where + is K or R and Φ is a hydrophobic residue) occurring individually or in clusters where they partly overlap each other are indicated in bold. <b>C.</b> Western blotting analysis of the ADAM tails expressed as biotinylated fusion proteins in human 293T cells for use as affinity baits in SH3 domain library screening.</p

Outline of phage library screening.

<p>A sequential strategy was used for screening of complete as well as subsequent rationally customized SH3 display libraries in order to exhaustively assess potential SH3 partners of the indicated ADAMs.</p

ADAM8 interacts with TOCA1 and SNX33 in human cells.

<p>SNX33 and TOCA1 were expressed as EGFP-fusion proteins by transiently transfected 293T cells alone or with HA-tagged ADAM8 as indicated on top of the figure. The presence of ADAM8 itself (top panel) or TOCA1 or SNX33 that was associated with it (second panel from top) in anti-HA immunoprecipitates (IP) from the lysates of these cells was examined by Western blotting. Similar total levels of ADAM8, TOCA1, and SNX33 between the transfected cells were confirmed by a Western blotting analysis of the unselected lysates (Lysate). Blotting for the endogenous α-tubulin in these lysates was included as an additional loading control.</p

Summary of the peptide array data for identification of functional SH3 binding sites in the ADAM tails.

<p>The indicated peptides corresponding to the proline-cluster numbering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121301#pone.0121301.g001" target="_blank">Fig 1A</a> were printed in triplicate on arrays slides, and probed with the indicated SH3 domains. Signals of the triplicate spots were averaged, and these values normalized to the average of the peptide giving the strongest signal in each SH3 probing. Shown is a heat map based on these normalized values where white indicates background level binding, and black the strongest peptide binding for each SH3 domain.</p