Determinants of Substrate Recognition in the Protein-tyrosine Phosphatase, PTP1 (*)

Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr (EGFR). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the α2′ region of PTP1, most likely Ile, was labeled by the Tyr-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis.

[1]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

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

[3]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[4]  D. Mcnamara,et al.  Identification of amino acids in the N-terminal SH2 domain of phospholipase C gamma 1 important in the interaction with epidermal growth factor receptor. , 1994, Biochemistry.

[5]  G. Prestwich,et al.  Benzophenone photophores in biochemistry. , 1994, Biochemistry.

[6]  Zy Zhang.  Kinetic and mechanistic characterization of a mammalian protein- tyrosine phosphatase, PTP1 , 1995, The Journal of Biological Chemistry.

[7]  P. Ruzza,et al.  Specificity of T-cell protein tyrosine phosphatase toward phosphorylated synthetic peptides. , 1993, European journal of biochemistry.

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

[9]  S. Shoelson,et al.  A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding. , 1993, The Journal of biological chemistry.

[10]  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.

[11]  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.

[12]  J. Dixon,et al.  Expression of a protein tyrosine phosphatase in normal and v-src- transformed mouse 3T3 fibroblasts , 1992, The Journal of cell biology.

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

[14]  B. Neel,et al.  The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence , 1992, Cell.

[15]  R. Colman,et al.  Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase. , 1986, Biochemistry.

[16]  J. Dixon,et al.  Cloning and expression of a protein-tyrosine-phosphatase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

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

[19]  C. Walsh,et al.  Activation of the SH2-containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. , 1994, The Journal of biological chemistry.

[20]  J E Dixon,et al.  Protein tyrosine phosphatases. , 1993, Annual review of biochemistry.

[21]  J. Dixon,et al.  Dissecting the catalytic mechanism of protein-tyrosine phosphatases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

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

[23]  N. Tonks,et al.  Purification of the major protein-tyrosine-phosphatases of human placenta. , 1988, The Journal of biological chemistry.

[24]  N. Williams,et al.  Exploring the adenine nucleotide binding sites on mitochondrial F1-ATPase with a new photoaffinity probe, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate. , 1982, The Journal of biological chemistry.

[25]  C. Walsh,et al.  Potent Stimulation of SH-PTP2 Phosphatase Activity by Simultaneous Occupancy of Both SH2 Domains (*) , 1995, The Journal of Biological Chemistry.

[26]  D. Barford,et al.  Crystal structure of human protein tyrosine phosphatase 1B. , 1994, Science.

[27]  W. DeGrado,et al.  p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide. , 1986, The Journal of biological chemistry.

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

[29]  J. Dixon,et al.  Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate. , 1991, The Journal of biological chemistry.