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doi: 10.1038/msb.2013.9.

SH3 interactome conserves general function over specific form

Affiliations

SH3 interactome conserves general function over specific form

Xiaofeng Xin et al. Mol Syst Biol.2013.

Abstract

Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The worm and yeast SH3 domain peptide-binding specificity repertoire is conserved. SH3 domain specificities (36 worm, orange labels; 24 yeast, black labels), visualized as sequence logos, are grouped by similarity in a tree (see Experimental Procedures). Tree branches are colored according to their specificity class: I (red), II (blue), and atypical (black). Underlined labels indicate domains that exhibit multiple specificities. Multiple peptide libraries were used to determine specificity: X6-PXXP-X6 (*), X6-PXXP-X6 and X12 (**), X6-PXXP-X6 and X7-R/K-X7 (***), or X12 (no asterisk), where P is proline, R is arginine, K is lysine, and X is any amino acid (see Experimental Procedures).
Figure 2
Figure 2
SH3 domain binding specificities are conserved between yeast paralogs, but not necessarily between yeast and worm orthologs. Protein domain architecture and SH3 domain binding specificity are shown for the yeast and worm SH3 protein paralogs and orthologs with available peptide phage display data. Yeast and worm SH3 proteins are grouped by green boxes, orthologs are grouped by blue boxes, and paralogs are grouped by red boxes. Lower left corner of boxes shows the sequence identity between the SH3 domains in the box. The domain architecture is defined by SMART (Letunic et al, 2009; Schultz et al, 2000), but is not drawn to scale. ADF, Actin depolymerization factor/cofilin-like domain; FCH, Fes/CIP4 homology domain; MYSc, myosin ATPases; PH, Pleckstrin homology domain; SAM, Sterile alpha motif; VHS, domain present in VPS-27, Hrs and STAM.
Figure 3
Figure 3
Overlap between Y2H and phage display predicted PPIs. Each worm SH3 domain with a phage-derived specificity profile represented as a PWM was used to score and rank all worm proteins for matches to this PWM. The plot shows the fraction of PPIs with a rank higher than the value on thex axis. Full Y2H is red, filtered Y2H (Supplementary Table 3) is green, and the expected results for randomly distributed PPIs is black. The observed enrichments are all highly significant (P<10−6).
Figure 4
Figure 4
Endocytosis is conserved between yeast and worm SH3 interactomes and is connected to species-specific functions. Gene functions significantly enriched in yeast and worm SH3 interactomes are visualized as an enrichment map (Merico et al, 2010). Nodes represent enriched gene functions in yeast (green), worm (blue), and both organisms (red). Edges link gene functions that share genes. Edge thickness is proportional to number of shared genes and edge color is blue for shared worm genes and green for shared yeast genes. Clusters of functionally related nodes were manually circled and labeled.
Figure 5
Figure 5
SH3-mediated PPIs are poorly conserved from yeast to worm. PPIs between worm proteins from our network with yeast orthologs are shown (worm PPIs are blue, yeast PPIs are green, and conserved PPIs are red). Diamonds indicate SH3 containing baits. Names in parenthesis indicate yeast orthologs of worm proteins involved in conserved interactions. Conserved worm proteins that are not involved in interactions with other conserved worm proteins are not shown.
Figure 6
Figure 6
Binding motif distribution is optimized to prevent excessively competitive interactions. (A,B) Number of binding motifs is correlated with the number of interacting SH3 proteins, in the subset of our worm (A) and yeast (B) interactomes with predicted binding sites. (C,D) No correlation is observed between number of SH3 domains in a protein and its number of PPIs in the same worm (C) and yeast (D) SH3 interactomes as in (A) and (B). Circle area is proportional to the number of proteins represented.
Figure 7
Figure 7
Experimental validation of predicted interactions and endocytosis functions in worm. (A) The worm AMPH-1 SH3 domain physically interacts with TBC-2 and not the SDPN-1 SH3 domain or GST controls, as shown by western blot. GST bait proteins, visualized by Ponceau S staining, are shown under the corresponding western blot lanes. (B–D) GFP-TBC-2 localization to endosomes (B) is disrupted in amph-1 (C) and rme-1 (D) mutants. Comparable confocal images show living intestinal epithelial cells expressing GFP-tagged TBC-2 from an integrated low-copy number transgene in wild-type,amph-1(tm1060), andrme-1(b1045) mutant animals. Scale bar, 10 μm. (E) Average fluorescence intensity of GFP-TBC-2 labeled puncta quantifies disruption of TBC-2 endosome localization across multiple experiments. Error bars, standard deviation (n=18 each, six animals of each genotype sampled in three different regions of each intestine). Significant differences in the one-tailed Student’st-test are indicated (***P=0.001).
Figure 8
Figure 8
Experimental validation of predicted endocytosis functions in human. (A–C) Co-localization of SH3D19-GFP (C-terminal tag) with CLTA-TagRFP-TEN-1 in SK-MEL-2 cells. (D–F) Co-localization of GFP-MAP3K9 (N-terminal tag) with CLTA-TagRFP-TEN-1. (G,H) Localization of GFP-CTTNBP2NL and GFP-BZRAP1 to actin stress fibre-like structures. (I) Localization of GFP-FGR to focal adhesion-like structures. Scale bars, 10 μm.
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