Eyes absent proteins: characterization of substrate specificity and phosphatase activity of mutants associated with branchial, otic and renal anomalies.

The eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C-terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low-molecular-weight compounds and synthetic phosphotyrosine (pY)-containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N-terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high-affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio-oto-renal syndrome (BOR), branchio-oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild-type levels.

Monitoring phosphatase reactions of multiple phosphorylated substrates by reversed-phase HPLC.

In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999-2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.

Synthetic strategies to a backbone-side chain cyclic SHP-1 N-SH2 ligand containing N-functionalized alkyl phosphotyrosine.

The cyclic peptide EGLNc Psi [CON((CH(2))(3)NH)pYNleE(NHCH(2)CO)]L-NH(2) (1) was designed and synthesized according to a native interaction partner of tyrosine phosphatase SHP-1. We introduced N-aminopropyl-phosphotyrosine to enable backbone-side chain cyclization with a glutamic acid derivative as counterpart for cyclization. Different approaches have been compared to find a strategy for the generation of backbone and backbone-side chain cyclic phosphopeptides.

Modulation of SHP-1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands.

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high-affinity binding partner for protein tyrosine phosphatase SHP-1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N-SH2 domain. Here, we focused on a set of peptides comprising C-terminally extended linear and conformationally constrained side chain-bridged cyclic N-SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP-1 through binding to both, the N-SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE--NH(2), displayed favorable binding affinity, but reduced ability to stimulate SHP-1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP-1. In summary, results presented in this study suggest that inhibitory N-SH2 ligands of SHP-1 may be obtained by designing bivalent compounds that associate with the N-SH2 domain and simultaneously occupy a specific binding site on the PTP domain.