Substrate specificity of the protein tyrosine phosphatases.

Abstract

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR,68..,,, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR, 9ss. (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affmity binding and catalysis. The K. value for EGFR,85_.,, decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.The tyrosine phosphorylation "status" of a cell is maintained by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) (1, 2). The first PTPase to be purified and sequenced was a 35-kDa protein (PTP 1B) from human placenta (3, 4). PTP1B was shown to share amino acid sequence homology with the cytoplasmic domain of the leukocyte cell surface glycoprotein CD45 (5). CD45 was subsequently shown to have tyrosine phosphatase activity (6). This discovery drew attention to the possible biological role of the PTPases in controlling signal transduction within the cell. Isolation and characterization of cDNA by low stringency hybridization and polymerase chain reaction demonstrated that PTPases constitute a large diversified family of catalysts that can be divided into two structurally distinct groups. One group generally has an extracellular domain, transmembrane spanning region, as well as two duplicated cytoplasmic PTPase domains. The other group of PTPases corresponds to the intracellular family of enzymes that have a single PTPase domain (2).Many PTPases have been cloned but limited information is available about their functions, mechanisms of catalysis, or substrate specificities. One of the central questions in protein phosphorylation is how kinases and phosphatases distinguish the diversity of substrates that they encounter in the cell. In the case of protein kinases, the amino acid sequence surrounding the phosphorylation site plays a crucial role in determining substrate specificity (7,8). For example, the cAMP-dependent protein kinase has a strong preference for phosphorylation of Ser residues that are located two or three residues to the COOH-terminal side of basic amino acids (most commonly Arg). Tyr...

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

“…Previous kinetic and structural studies using synthetic Tyr(P)-containing peptides reveal that PTPs display a range of k cat /K m (substrate specificity constant) values (10 2 to 10 7 M Ϫ1 s Ϫ1 ) for relatively short peptide substrates (26). In addition, the k cat /K m values for the peptides are orders of magnitude higher than that of Tyr(P) alone, suggesting that amino acids flanking the Tyr(P) also contribute to catalytic efficiency (27,28). In fact, substrate recognition by PTPs requires the presence of amino acids on both the N-and C-terminal sides of Tyr(P) (29 -31).…”

“…This method allows the acquisition of explicit kinetic data for all library members and enables the assessment of the contribution of individual amino acids to PTP substrate recognition based on the actual enzymatic activity of the PTP against its putative peptide substrates in solution. The Lyp-catalyzed dephosphorylation of each individual Tyr(P) peptide within the library was monitored by the increase in tyrosine fluorescence (18,39) at pH 7.0 and 25°C. The k cat /K m value, a measure of substrate specificity, was directly calculated from the reaction progress curve for each peptide (see "Experimental Procedures").…”

“…From the N to C terminus, the peptides interact with several surface loops in Lyp (Figs. 3B and 4B), including the Tyr(P) recognition loop (residues 58 -63), the Lyp-specific insert (residues [35][36][37][38][39][40][41][42], the phosphate-binding loop (P-loop, residues 226 -235), and the Q-loop (residues 269 -278). In particular, the Lyp substratebinding site contains a cluster of polar residues from the P-loop and additional basic residues (Lys 32 , and Arg 266 ) that provides a complementary positive surface to the highly electronegative pentapeptide core EEpYDD (Figs.…”

“…3D) 39 . These electrostatic interactions probably explain the overwhelming preference for acid residues at both the ϩ1 and ϩ2 positions.…”

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

“…These data suggest that short peptide sequences in full-length phosphotyrosyl-containing protein substrates may confer in vivo substrate distinctions by the PTPases. A complementary approach to determination of PTPase specificity for defined pY peptide sequences has been undertaken by Dixon and colleagues (Zhang et al, 1993), who used an Ala-scan approach on an EGF receptor phosphopeptide, replacing each residue systematically by Ala to assess catalytic efficiency with recombinant Yersinia PTPase and with PTPl .…”