Experimental evidence of phosphorylation-mediated modulation of domain-motif interactions.

<p>*Numbers in parentheses indicate the distance between the motif and the proximal phosphorylation site/s.</p

Phosphorylation events as double switches.

<p>(<b>A</b>) A protein (black horizontal line) includes a segment that matches two sequence patterns: the first is typical for SH3 domain binding (green), and the second typifies SH2 domain binding (red). The non-phosphorylated form binds SH3 and not SH2 (upper), while phosphorylation inverts the binding preferences (lower). (<b>B</b>) Specificity switches within the PDZ domain family. A protein (black horizontal line) includes a segment that may bind distinct PDZ domains (upper). The non-phosphorylated form binds PDZ^a and not PDZ^b, while phosphorylation inverts these binding preferences (lower).</p

Frequency of various phylogenetic traces of motif-phosphorylation coupling.

<p>The stacked-bar graph details the relative frequency of the three possible phylogenetic traces of the interaction-regulation units (for either intra-motif phosphorylation or near-motif phosphorylation sites): (i) co-appearance of the motif and the potentially phosphorylated residue in the same organism (grey), (ii) the motif appeared before the potentially phosphorylated residue (cyan) (iii) the potentially phosphorylated residue appeared before the motif (red). For each domain we tested if the distribution of the various scenarios deviates from random by a χ² test. Asterisks denote statistically significant results (based on <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341.s008" target="_blank">Table S6</a>).</p

Phylogenetic traces of PDZ interaction-regulation unit evolution.

<p>This matrix summarizes the results for units of PDZ binding motifs and near-motif phosphorylation. The eukaryotic evolutionary tree is depicted above and left to the matrix (abbreviations below). The rows indicate the organism in which the motif probably appeared. The columns indicate the organism in which a potentially phosphorylated residue appeared. The order in which the motif and potentially phosphorylated residue appeared can thus be deduced from the matrix cells. For instance, the brown-framed cell represents the three cases in which the motif appeared in <i>D. melanogaster</i> and the potentially phosphorylated residue appeared in chicken. Accordingly, all cells below the diagonal (cyan) represent cases in which the potentially phosphorylated residue appeared after the motif. The diagonal cells represent cases in which the motif and the potentially phosphorylated residue appeared together. The cells above the diagonal represent cases in which the motif appeared after the potentially phosphorylated residue (red). Organism abbreviations: CHIMP- <i>p. troglodytes</i>, MOUSE- <i>m. musculus</i>, RATUS- <i>r. norvegicus</i>, BOVIN- <i>b. taurus</i>, CHICK- <i>g. gallus</i>, XENTR- <i>x. tropicalis</i>, DANRE- <i>d. rerio</i>, CIONA- <i>c. intestinalis</i>, DROME- <i>d. melanogaster</i>, ANOGA- <i>a. gambiae</i>, CAEEL- <i>c. elegans</i>, YEAST- <i>s. cerevisiae</i>, DICDI- <i>d. discoideum</i>, ARATH- <i>a. thaliana</i> and PLAFA- <i>p. falciparum</i>.</p

Dual sequence patterns used for the identification of potential double switches in human proteins.

<p>Column titles include sequence patterns for motifs that bind SH3 or class I WW domains (in red), and row titles include sequence patterns for motifs that bind different types of SH2 domains, upon motif phosphorylation (in blue). Each table cell includes a merged sequence pattern that hints at a dual binding potential of the motif to both SH2 and SH3 (or WW) domains. The columns under class I WW and SH3-1 titles represent the strict analysis scheme. Sequence patterns were extracted from the ELM database <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Gould1" target="_blank">[14]</a>. (i) An example for a dual motif. The PP.Y.N. sequence pattern is composed of the SH2^Grb2 Y.N. and the class I WW PP.Y patterns. (ii) Note that this sequence pattern encompasses seven positions.</p

Step-wise appearance of motifs and potential phosphorylation sites.

<p>(<b>A</b>) The motif is older than the potential phosphorylation site. The human CDK inhibitor 1B (top line) includes an SH3-binding motif (RxxK, highlighted in red) and a proximal tyrosine that may affect the motif's interaction potential upon phosphorylation <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Chu1" target="_blank">[74]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Harkiolaki1" target="_blank">[75]</a> (highlighted in cyan). The sequence pattern is conserved from <i>C. elegans</i> to human, but the tyrosine is conserved only between rat and human. This suggests that an old domain-binding motif has gained phospho-regulation in more recent organisms. Protein accessions are according to the Uniprot or Ensembl databases. (<b>B</b>) Potential phosphorylation site is older than the motif. The human Tau protein includes an SH3-binding motif (PxxP) and a proximal threonine that inhibits the motif's interaction potential upon phosphorylation <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Reynolds1" target="_blank">[76]</a>. This phosphorylation was also shown to induce a conformational change that unlocks the closed form of the protein <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Lin1" target="_blank">[77]</a>. The motif is conserved from <i>X. tropicalis</i> to human, while the potential phosphorylation site may have appeared earlier in evolution (present in <i>D. melanogaster</i>). This suggests that the domain-binding potential was established close to already functional phosphorylation.</p

Mutually exclusive binding of domain pairs to the same protein segment.

<p>Summary of literature-documented double switches. The second column includes protein sequences, where residues vital for SH2 binding and residues vital for SH3/class I WW binding are in bold and underlined, respectively. Rows (1–3) describe experimentally-verified double switches. Rows (4–5) include examples for which there is evidence for the motif binding to each domain, but not for a direct switch. Note that Y534 in growth hormone receptor is phosphorylated according to a high-throughput experiment. Also note that evidence for Fyn-Cbl interaction exists for the Cbl (552–614) fragment (spanning 62 residues), where Y552 is the only tyrosine, suggesting that this tyrosine is bound by the SH2 domain in Fyn.</p

Coupling between phosphorylation events and domain-binding motifs.

<p>For each domain family (SH2, WW, PDZ and SH3), the bars denote the percent of motifs found to be phosphorylated either within or near them. Solid-colored and empty rectangular bars represent intra-motif phosphorylation and near-motif phosphorylation, respectively. All motifs are derived from the high reliability dataset, while phosphorylation events are derived from three data sets: LTP (low throughput evidence only), HTP (phosphorylation events based on evidence from high-throughput resources), and LTP+HTP (any type of evidence). Asterisks represent statistically-significant results (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#s4" target="_blank">Methods</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi-1002341-t001" target="_blank">Table 1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341.s003" target="_blank">Table S1</a>).</p

Environmental cues imposed on steady state growing yeast cells.

<p>Environmental cues imposed on steady state growing yeast cells.</p

Common Runx3-regulated genes in IL-2-activated CD8-TC/NKC and T-bet/p300 bound genes in Th1.

<p>(A) The majority of the 118 common Runx3-regulated genes in IL-2-activated CD8-TC and NKC (R3_CD8_NK_T) harbor overlapping T-bet (R3CD8_Tbet) and p300 (R3_CD8_p300) bound regions in Th1 cells. (B) Transcription factors/regulators that are common Runx3-regulated genes in IL-2-activated CD8-TC and NKC. </p