Affinity selection from peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.

[1]  Hong Sun,et al.  MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo , 1993, Cell.

[2]  J. L. Bailey,et al.  Techniques in protein chemistry , 1989 .

[3]  L. Cantley,et al.  Recognition of Unique Carboxyl-Terminal Motifs by Distinct PDZ Domains , 1997, Science.

[4]  J. Dixon,et al.  A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides. , 1993, Analytical biochemistry.

[5]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

[6]  J Barsoum,et al.  Tat-mediated delivery of heterologous proteins into cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Saha,et al.  CD45 protein tyrosine phosphatase: Determination of minimal peptide length for substrate recognition and synthesis of some tyrosine-based electrophiles as potential active-site directed irreversible inhibitors , 1995 .

[8]  Zhou Songyang,et al.  Use of an oriented peptide library to determine the optimal substrates of protein kinases , 1994, Current Biology.

[9]  F. Jirik,et al.  Characterization of protein tyrosine phosphatase SH-PTP2. Study of phosphopeptide substrates and possible regulatory role of SH2 domains. , 1994, The Journal of biological chemistry.

[10]  Hong Sun,et al.  The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. , 1994, Trends in biochemical sciences.

[11]  Jerry L. Adams,et al.  A protein kinase involved in the regulation of inflammatory cytokine biosynthesis , 1994, Nature.

[12]  A. Ullrich,et al.  Both SH2 Domains Are Involved in Interaction of SHP-1 with the Epidermal Growth Factor Receptor but Cannot Confer Receptor-directed Activity to SHP-1/SHP-2 Chimera* , 1997, The Journal of Biological Chemistry.

[13]  Philip R. Cohen,et al.  PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo(*) , 1995, The Journal of Biological Chemistry.

[14]  M. Gresser,et al.  Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate* , 1997, The Journal of Biological Chemistry.

[15]  D. Barford,et al.  Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. , 1995, Science.

[16]  N. Tonks,et al.  Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST , 1996, Molecular and cellular biology.

[17]  M. Bernier,et al.  A Peptide-based Protein-tyrosine Phosphatase Inhibitor Specifically Enhances Insulin Receptor Function in Intact Cells* , 1996, The Journal of Biological Chemistry.

[18]  D. Solas,et al.  An Efficient Synthesis of N-α-Fmoc-4-(Phosphonodifluoromethyl)-l- phenylalanine , 1996 .

[19]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[20]  M. Bernier,et al.  A Synthetic Peptide Derived from a COOH-terminal Domain of the Insulin Receptor Specifically Enhances Insulin Receptor Signaling* , 1996, The Journal of Biological Chemistry.

[21]  B. Neel,et al.  From Form to Function: Signaling by Protein Tyrosine Phosphatases , 1996, Cell.

[22]  S. Shoelson,et al.  Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking , 1997, Molecular and cellular biology.

[23]  A. Levitzki,et al.  Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor , 1996, Nature.

[24]  R. Aebersold,et al.  Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates. , 1995, European journal of biochemistry.

[25]  Charis Eng,et al.  Catalytic specificity of protein-tyrosine kinases is critical for selective signalling , 1995, Nature.

[26]  P. Roller,et al.  Potent inhibition of insulin receptor dephosphorylation by a hexamer peptide containing the phosphotyrosyl mimetic F2Pmp. , 1994, Biochemical and biophysical research communications.

[27]  T. Hunter,et al.  Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling , 1995, Cell.

[28]  L. Wennogle,et al.  Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection. , 1996, Biochemistry.

[29]  J. Dixon,et al.  Protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates. , 1994, Biochemistry.

[30]  J. Dixon,et al.  Substrate specificity of the protein tyrosine phosphatases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  C. Walsh,et al.  Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors , 1993, Protein science : a publication of the Protein Society.

[32]  B. Jena,et al.  Acidic residues are involved in substrate recognition by two soluble protein tyrosine phosphatases, PTP-5 and rrbPTP-1. , 1993, Biochemistry.

[33]  N. Tonks.  Protein Tyrosine Phosphatases and the Control of Cellular Signaling Responses , 1996 .

[34]  J. Bishop,et al.  Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation. , 1994, The Journal of pharmacology and experimental therapeutics.

[35]  L. Wu,et al.  Why is phosphonodifluoromethyl phenylalanine a more potent inhibitory moiety than phosphonomethyl phenylalanine toward protein-tyrosine phosphatases? , 1995, Biochemical and biophysical research communications.

[36]  T Pawson,et al.  Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav , 1994, Molecular and cellular biology.