Profiling the substrate specificity of protein tyrosine phosphatases by combinatorial library screening.

摘要

Protein tyrosine phosphatases (PTPs) hydrolyze phosphotyrosine (pY) back to tyrosine and inorganic phosphate, functioning in coordinate with protein tyrosine kinases (PTKs) to regulate a broad range of cell signaling processes. Once viewed as passive housekeeping enzymes, it has recently become apparent that PTPs exhibit substrate specificities in vivo. In this work, we have established a robust combinatorial peptide library screening method to determine the substrate specificities of any PTP. A novel high throughput enzyme-coupled assay has been developed, which for the first time enables the rapid screening of PTP activity and substrate specificities. Three major pY-containing peptide libraries were designed and synthesized on-bead to define the substrate specificities of the PTP domain at both N- and C- terminal sides of pY. The substrate specificity profiles of seven PTPs (PTP1B, TC-PTP, SHP1, SHP2, TULA-1, TULA-2, and VHR) were successfully obtained by the established screening method and validated by solution-phase enzyme kinetics. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, but disfavors basic residues at any position. TC-PTP has broader range of specificity. It prefers acidic residue N-terminal to pY, but exhibits tolerance for basic residues at the C-terminal side and N-terminal positions some distance from pY (-4 and -5). By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of pY. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residue on the C-terminal side of pY but no basic residues.;TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of PTPs. Distinct from the classical PTPs, which utilize a conserved cysteine residue to catalyze the dephosphorylation reaction, the TULA family critically depends on a conserved histidine residue as the catalytic nucleophile. The substrate specificity of TULA-1 and 2 was successfully evaluated by screening three different peptide libraries. Although TULA-1 showed no detectable activity toward any of the pY peptides in the library, TULA-2 recognizes two distinct classes of pY substrates. On the N-terminal side of pY, the class I substrates contain a proline at the pY-1 position, a hydrophilic residue at the pY-2 position, and aromatic hydrophobic residues at positions pY-3 and beyond. The class II substrates typically contain two or more acidic residues, especially at pY-1 to pY-3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of pY, TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library, as well as by pY peptides derived from various pY proteins. TULA-2 is highly active toward peptides corresponding to the pY-323 and pY-352 sites of Syk, and the pY-397 site of focal adhesion kinase and has lower activity toward other pY sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.;The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase is a small member of the dual-specificity phosphatases (DSPs) with only 185 amino acids and no apparent targeting domain. Recent studies have shown VHR is involved in cell cycle progression and cervical cancer, thus making it a novel and promising drug target for the treatment of cervical cancer. Unlike other PTPs, a highly conserved motif NH2AP(I/L/M/F) was observed beyond the pY binding site from the screening, suggesting the existence of a second binding site. Surprisingly, a free N-terminus is required for efficient substrate recognition. Two sets of peptides were tested against VHR to obtain the kinetic constants. Peptides that target both the active site and the second site showed at lease 10-fold higher kcat/ KM compared to the corresponding peptides that only target the pY-binding pocket. The best substrate exhibited a 50-fold lower KM value compared to any reported VHR peptide substrate. Our lab is in the progress of solving the peptide-VHR complex structure to reveal the second binding pocket, and we are also synthesizing specific bidentate inhibitors for VHR.