Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an E. coli expression system

Abstract

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

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

“…One expects that these PTPases may have broad specificity for any pY-containing protein brought into apposition. Our previous studies (Cho et al, 1991(Cho et al, , 1992bLee et al, 1992) on synthetic pY-peptides corresponding to phosphotyrosyl sites in proteins such as tyrosine kinases of the insulin receptor, PDGF receptor, and EGF receptor classes or of the src classes, cell cycle kinases such as CDC2, and from phospholipase C-y revealed a range of k,,,/K, values. The single 41-kDa catalytic domain of PTPO shows distinct specificity from LAR and the T-cell specific CD45, and processes several pY-peptides with low-micromolar K , values.…”

“…Part of the data for LAR-Dl and CD45 (from IR-5 to src527) are reproduced from earlier reports (Cho et al, 1991(Cho et al, , 1992bLee et al, 1992). These include the data obtained with all the IR peptides, EGFR, lck394, lck505, and src527 phosphotyrosyl peptides.…”

“…Except for purification purposes, which used p-nitrophenyl phosphate as a substrate, the enzyme activities were determined by measuring released inorganic phosphate using the malachite green reagent as described (Cho et al, 1991(Cho et al, , 1992b. For the HPTPP assay, the reaction was initiated by addition of HPTPP to a mixture containing pY-substrate, 0.1 M Hepes (pH 7.6), 2 mM EDTA, and 10 mM dithiothreitol (DTT).…”

“…These results are in agreement with the results obtained earlier by Dixon and colleagues, who detected a phosphoenzyme intermediate and identified a cysteine residue as the nucleophile in the active site using 977 rat LAR (Guan & Dixon, 1991;Pot et al, 1991;Pot & Dixon, 1992). We have also utilized synthetic phosphotyrosyl peptides of 9-12 residues corresponding to known pY sites in proteins in signal transduction cascades to characterize these PTPases for specificity and selectivity (Cho et al, 1991(Cho et al, , 1992b. While LAR-Dl and the D1D2 fragment showed a wide range of discrimination, the relative rank ordering of pY peptide recognition, by the kc,,/K,,, catalytic efficiency criterion, was very similar between the pure LAR-Dl and the pure CD45-DID2 catalytic fragments.…”

Protein-tyrosine Phosphatase-mediated Decrease of Epidermal Growth Factor and Platelet-derived Growth Factor Receptor Tyrosine Phosphorylation in High Cell Density Cultures

“…Preliminary analysis of the substrate specificity of PTPs have revealed some sequence specificity. For example, when a dodecapeptide derived from the insulin receptor containing three phosphorylated tyrosine residues was used as substrate, both CD45 as well as leukocyte antigen-related phosphatase preferentially dephosphorylated the tyrosine corresponding to amino acid 1146 of the insulin receptor (25,26). It is therefore possible that the observed density-dependent difference in receptor phosphorylation reflects specific phosphorylation of some sites, rather than complete dephosphorylation of a subset of receptors.…”