Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed

Specificity Profiling of Protein Phosphatases toward Phosphoseryl and Phosphothreonyl Peptides

A combinatorial library method was developed to systematically profile the substrate specificity of protein phosphatases toward phosphoseryl (pS) and phosphothreonyl (pT) peptides. Application of this method and a previously reported phosphotyrosyl (pY) library screening technique to dual-specificity phosphatase (DUSP) VH1 of vaccinia virus revealed that VH1 is highly active toward both pS/pT and pY peptides. VH1 exhibits different and more stringent sequence specificity toward pS/pT than pY substrates. Unlike previously characterized protein tyrosine phosphatases (PTPs), the activity and specificity of VH1 are primarily determined by the amino acid residues C-terminal to the pS, pT, or pY residue. In contrast, the mammalian VH1-related (VHR) DUSP has intrinsically low catalytic activity toward pS and pT substrates, suggesting that its primary physiological function is to dephosphorylate pY residues in substrate proteins. This method is applicable to other DUSPs and protein-serine/threonine phosphatases, and the substrate specificity data will be useful for identifying the physiological substrates of these enzymes

Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

Cyclic heptapeptide cyclo­(FΦRRRRQ) (cFΦR₄, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR₄ binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR₄ ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR₄ and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR₄ was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR₄ a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape