Protein Tyrosine Phosphatase Substrate Research Articles

A Combinatorial Approach to Identifying Protein Tyrosine Phosphatase Substrates from a Phosphotyrosine Peptide Library

A Combinatorial Approach to Identifying Protein Tyrosine Phosphatase Substrates from a Phosphotyrosine Peptide Library

Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition.

The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.

Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases.

The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a "substrate trap." Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.

The transmembrane protein tyrosine phosphatase α dephosphorylates the insulin receptor in intact cells

Protein tyrosine phosphatases (PTPs) are key regulators in a variety of signal transduction processes. However, substrates for most PTPs have not been determined. In a previous report, we demonstrated that in a transient expression system the intracellular phosphatases PTPs 1B and TC preferentially dephosphorylated the precursor form of several receptor tyrosine kinases. In this paper we show that the dephosphorylation of kinase precursors is a specific feature of PTPs 1B and TC that is not shared by two other intracellular PTPs, PTPH1 or SHP-1. By contrast, the receptor phosphatase PTPα preferentially dephosphorylated the β-subunit of the insulin receptor localized on the cell surface. The insulin receptor was a better substrate for PTPα than for other receptor type PTPs. We conclude that the intracellular PTPs 1B and TC regulate the autophosphorylation of receptor tyrosine kinases during their posttranslational processing while receptor type PTPs regulate the mature, cell surface localized receptor tyrosine kinases. © 1997 Federation of European Biochemical Societies.

ICA 512, a Receptor Tyrosine Phosphatase-Like Protein, Is Concentrated in Neurosecretory Granule Membranes

Publisher Summary Islet cell autoantigen (ICA) 512 is a specific membrane marker of neurosecretory granules. It is the first member of the receptor protein tyrosine phosphatases (RPTPs) family found to be resident in an intracellular compartment. Interestingly, ICA 512 contains only one protein tyrosine phosphatase (PTP) “core domain”, rather than two, as usually found in receptor PTPs (RPTPs). Moreover, ICA 512 expressed in bacteria do not display PTP activity when tested with several common PTP substrates. Thus, the function of ICA 512 is still unknown. By Northern blot, ICA 512 transcripts have been detected in pancreatic islets, brain, and pituitary, suggesting an enrichment of ICA 512 in neuroendocrine tissues. To begin addressing the structure and function of ICA 512, its tissue distribution and intracellular localization using rabbit antibodies directed against either its recombinant extracellular domain or recombinant cytoplasmic domain. Confocal microscopy on rat tissues demonstrated that ICA 512 is expressed in virtually all neuroendocrine cells, including neurons of the autonomic nervous system; chromaffin cells of the adrenal medulla; α-, β-, and δ-pancreatic islet cells; cells in the anterior and intermediate pituitary, as well as neurons of the autonomic nervous system and of the hypothalamus and the amygdala in the brain. The strongest ICA 512 immunoreactivity has been found in the posterior pituitary, which contains the nerve endings of neurons located in the hypothalamus and the highest concentration of neurosecretory granules in the entire body. Thus ICA 512 and, thus, tyrosine phosphorylation–dephosphorylation plays a role in the life-cycle of neurosecretory granules.

Prevotella intermedia が産生するアルカリホスファターゼのプロテインチロシンホスファターゼ様の性質について

Prevotella intermedia is frequently detected in the microflora of patients with several types of periodontitis, and thought to be one of the most important etiological agents. P. intermedia was found to have high Alkaline phosphatase (ALPase) activity. ALPases are previously known to be one of the virulence factors to periodontal organisms. However, many enzymological properties of ALPase are unknown. In this study ALPase of Prevotella intermedia ATCC25611 was solubilized by extraction with 1% Triton X-114, and the solubilized enzyme was purified 47.4-fold with 3.3% recovery by using ion-exchange chromatography, gel-filtration and affinity column chromatography. The purified enzyme was observed as a single protein band that corresponded to molecular weight of 54, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be about 150, 000 by gel filtration with TSK-gel G3000SW_<XL>. These findings indicated which P. intermedia ALPase is a dimer or a trimer. The purified enzyme dephosphorylated phosphotyrosine-containing human gastrine, a peptide substrate for protein tyrosine phosphatases (PTPases), and was inhibited by sodium orthovanadate, sodium molybdate and Zn^<2+>, which are inhibitors of PTPases activity. The purified enzyme was indicated that the optimal pH for ALPase activity was about 9.5 and the optimal pH for PTPase activity was about 6.0. The PTPase activity was reduced by approximately 10% of maximum at pH7.0. This result suggests that the purified enzyme can have about 90% of the maximum activity even under physiologically relevant conditions. The N-terminal sequence of the purified enzyme showed about 50% homology with the sequence of amino acids sharing an active site signature motif, (I/V) HCXAGXGR (S/T)G, which all of PTPases conserve. Therefore P. intermedia ALPase was found to have protein tyrosine phosphataselike activity and characterization.

The Active Site Specificity of the Yersinia Protein-tyrosine Phosphatase

Yersinia protein-tyrosine phosphatase substrates have been synthesized employing an expedient methodology that incorporates phosphorylated non-amino acid residues into an active site-directed peptide. While the peptidic portion of these compounds serves an enzyme targeting role, the nonpeptidic component provides a critical assessment of the range of functionality that can be accommodated within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed peptides, with the former serving as a 10-fold more efficient substrate than the latter. In addition, this enzyme catalyzes the hydrolysis of a variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing the achiral phosphotyrosine analog, phosphotyramine, is not only the most efficient substrate described in this study, it is also one of the most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of the general structure, (Glu)4-NH-(CH2)n-OPO3(2-) (n = 2-8), are also hydrolyzed, where the most efficient substrate contains seven methylene groups. Finally, a comparison of the substrate efficacy of the peptide-bound species with that of the corresponding non-peptidic analogs, reveals that the peptide component enhances kcat/Km by up to nearly 3 orders of magnitude.

Protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates.

The structural requirements of substrates for two recombinant protein tyrosine phosphatases (PTPases) are probed using various-sized synthetic phosphotyrosine (pY)-containing peptides corresponding to the autophosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due to its favorable kinetic constants. The contribution of individual amino acids on both sides of pY to binding and catalysis was assessed by kinetic analysis using a continuous, spectrophotometric assay. For both Yersinia PTPase and a soluble recombinant mammalian PTPase of 323 amino acid residues (rat PTP1), efficient binding and catalysis required six amino acids including the pY residue, i.e., four residues N-terminal to pY and one residue C-terminal to pY. Thus, PTPase substrate specificity is primarily dictated by residues to the N-terminal side of pY. The pY moiety and the rest of the peptide interact with PTPases in a cooperative manner. The presence of pY in the peptide substrate is necessary but not sufficient for high-affinity binding, since phosphotyrosine and other simple aryl phosphates exhibit weak binding, and dephosphorylated peptides do not bind to PTPases. Two variations on the pY moiety are also examined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the phosphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, whereas the phosphonomethylphenylalanine analog in which the tyrosyl oxygen is replaced by a CH2 group is a competitive and nonhydrolyzable inhibitor, with Ki values of 18.6 and 10.2 microM, respectively, for the Yersinia PTPase and the rat PTP1.

Acid phosphatase purified from Mycoplasma fermentans has protein tyrosine phosphatase-like activity.

Acid phosphatase purified from Mycoplasma fermentans dephosphorylated phosphotyrosine-containing lysozyme and Raytide, a peptide substrate for protein tyrosine phosphatases. The optimum pH for Raytide was about 5.5. Raytide phosphatase activity was inhibited by potassium fluoride, sodium molybdate, and sodium orthovanadate and was found to exist in some mycoplasmas.

A General Peptide Substrate for Protein Tyrosine Phosphatases

The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a C 18 cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecular-weight PTPs exhibited K m values between 0.5 and 1.3 μM, whereas the receptor forms CD45 and RPTPα gave values of 14 and 35 μM, respectively. All PTPs displayed similar properties toward the peptide including a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to 10 ng in presence of protein substrates.

Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases

Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of N alpha-(9-fluorenylmethyloxycarbonyl)-O-phospho-L-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphorylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and 31P nuclear magnetic resonance (31P NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37 degrees C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with Km's of 68 +/- 9 microM and 42 +/- 11 microM and kcat/Km values of 4.9 x 10(5) s-1 M-1 and 6.9 x 10(5) s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)