Fig 1.
(A) Coomassie blue staining of a SDS-PAGE gel showing the recombinant DUSP proteins examined. (B) Scanned images of DUSP treated Tyr(P) peptide microarrays. The human Tyr(P) peptides (>6000) were microarrayed in three identical subarrays on each slide. The microarrays were incubated with individual DUSPs and remaining Tyr(P) content was measured using anti- Tyr(P) monoclonal antibody and an Alexa-635 secondary (anti-mouse IgG) antibody. The control reference slide was treated with buffer only. The images were obtained from the same area of each slide. Each spot represents one peptide.
Table 1.
Dual specificity phosphatases examined.
Fig 2.
Distribution of dephosphorylation data.
(A) Representative results of Tyr(P) dephosphorylation of the peptide microarray library by VH1, the poxvirus DUSP. The scatter plot shows the relative florescence units (RFU) of the reference peptide microarray (red dots) ranked from high to low, and the RFUs for corresponding VH1-treated peptides (black dots). Each dot represents the average of triplicate values for one peptide in the library. (B) Untreated reference peptide microarray (red dots) compared with an overlay of quantile normalized results for each DUSP showing Tyr(P) peptide dephosphorylation (black dots). Peptides in each data set were sorted from high to low based on RFU.
Fig 3.
Substrate sequence motifs for each DUSP.
(A) Results (pLogo) were derived using the 500 most dephosphorylated peptides as foreground (n = 500) and all other peptides in peptide library as background set (n = 5532) peptides sequences for each DUSP protein. Over-represented amino acid residues are above and under-represented amino acid residues are below the x-axis. The height of each single letter represents the statistical significance of the amino acid at that position. The horizontal red lines above and below the x axis correspond to Bonferroni-corrected statistical significance values (p ≤ 0.05). Hydrophobic amino acids (A, I, L, V and M), black; acidic amino acids (D and E), red; basic amino acids (R, H and K), blue; neutral amino acids (Q and N), brown; aromatic amino acids (F, W and Y), gray; and polar amino acids (T and S), light blue. Special amino acids G and P are colored in green and C are colored in dark Khaki. Zero position in the center of the peptide sequence represents the Tyr(P) residue in all motif logos. (B) Statistically significant residues that were over-represented at each position are listed for each DUSP.
Fig 4.
Cell signaling pathways represented by highly dephosphorylated peptides.
A total of 29 signaling pathways were identified in the subset of significant peptides recognized by a group of 10 phosphatases. Each phosphatase, as well as its corresponding edges, is color coded. Pathway node size corresponds to number of peptides belonging to that pathway cluster, while edge weight corresponds to the number of peptides in the pathway cluster recognized by the phosphatase. Pathway nodes that are over-represented when compared to a background list encompassing all peptides on the microarray (p<0.001515) are shown in white, while all other pathway nodes are grey.
Fig 5.
Relationship between catalytic recognition of Tyr(P)- peptides and DUSP phylogeny.
(A) Hierarchical clustering of DUSPs was performed based on percent dephosphorylation of microarrayed Tyr(P) peptide substrates. (B) A cladogram representation of phylogenetic relationship of different DUSPs based on the multiple alignment of the catalytic domain amino acid sequences (Jotun Hein method). (C) A conserved region of 15 residues surrounding the active site of each DUSP was used to construct a dendrogram representative of similarity within phosphatase sequences involved in substrate recognition. A maximum likelihood tree is depicted with bootstrap values (out of 1000 replicates) shown in red.
Fig 6.
Structural comparison of DUSP3, DUSP14, DUSP22 and Cdc25B catalytic sites.
(A) Superimposed ribbon representation of structures for DUSP3 (green), DUSP14 (magenta), DUSP22 (cyan) and Cdc25B (yellow). The catalytic sites are centered on the co-crystallized phosphate or 2-(N-morpholino) ethanesulfonic acid (MES) atom. (B) Electrostatic potential surface representation of the catalytic site of DUSP3 (PDB: 1VHR), DUSP14 (PDB: 2WGP), DUSP22 (PDB: 1WRM) and Cdc25B (PDB: 1QB0). Red and blue colored regions denote negative and positive charges, respectively.