Protein phosphorylation is a post-translational modification controlled by two counteracting enzyme families, protein kinases and phosphatases. Protein phosphatases have been demonstrated to regulate many physiological pathways and exhibit distinct specificity in vivo. However, the factors determining their specificity are not well understood. In this study, the intrinsic specificity of various families of phosphatases has been explored utilizing combinatorial peptide library screening.
The sequence specificity of eight classical-protein tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRD, PTPRO, PTP-PEST, PTP1B, SHP-1, and SHP-2) was determined. While PTPRA showed no selectivity, the other PTPs exhibited similar preferences for peptides containing acidic residues but disfavored basic ones. However, the enzymes differed in their level of selectivity and catalytic activity. Most of the classical-PTPs screened in this study also contains substrate recruiting/regulatory domains; it is likely that the in vivo PTP specificity is enhanced by the recruiting/regulatory domains.
The sequence specificity of atypical dual-specificity phosphatases (DUSPs), which only contain a single catalytic domain, was examined to determine whether they exhibit more stringent specificity than the classical-PTPs. The screening of Vaccinia VH1-related (VHR) DUSP against pY-peptide libraries revealed two distinct classes of substrates. While class I peptide substrates are similar to the pY motifs derived from reported VHR protein substrates, the novel class II peptide substrates of the consensus (V/A)P(I/L/M/V/F)X1-6pY exhibit an alternative-binding mode to VHR, as suggested by site-directed mutagenesis and molecular modeling. ROBO1 and LASP1, which contain the class II consensus motif, were demonstrated to be VHR substrates in vitro.
The haloacid dehydrogenase superfamily (HADSF) phosphatases have been shown to dephosphorylate a wide range of substrates including sequence with pY, phosphoserine (pS), and phosphothreonine (pT). The study utilized pS/pT library screening approach for comprehensive analysis of phosphatase specificity to advance the identification of pS/pT protein substrates. The screening of human small C-terminal domain phosphatase 1 (SCP1) revealed its preference for pS-containing peptides with proline residues at the pS+1 position and hydrophobic residues, especially at the pS-1 to pS-3 positions. The tumor suppressor p53 protein, which contains the consensus sequence obtained from screening, was discovered to be a putative SCP1 substrate. This result was validated by in vitro dephosphorylation of p53-pS33 by SCP1 and modulation the phosphorylation of pS33 level in HEK293 cells.