Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 May 15;90(10):4446–4450. doi: 10.1073/pnas.90.10.4446

Substrate specificity of the protein tyrosine phosphatases.

Z Y Zhang 1, A M Thieme-Sefler 1, D Maclean 1, D J McNamara 1, E M Dobrusin 1, T K Sawyer 1, J E Dixon 1
PMCID: PMC46528  PMID: 7685104

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, EGFR988-998, 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 EGFR988-998 (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-affinity binding and catalysis. The Km value for EGFR988-998 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.

Full text

PDF
4449

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bliska J. B., Clemens J. C., Dixon J. E., Falkow S. The Yersinia tyrosine phosphatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage. J Exp Med. 1992 Dec 1;176(6):1625–1630. doi: 10.1084/jem.176.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Charbonneau H., Tonks N. K., Kumar S., Diltz C. D., Harrylock M., Cool D. E., Krebs E. G., Fischer E. H., Walsh K. A. Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5252–5256. doi: 10.1073/pnas.86.14.5252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Charbonneau H., Tonks N. K., Walsh K. A., Fischer E. H. The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7182–7186. doi: 10.1073/pnas.85.19.7182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cho H. J., Ramer S. E., Itoh M., Winkler D. G., Kitas E., Bannwarth W., Burn P., Saito H., Walsh C. T. Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system. Biochemistry. 1991 Jun 25;30(25):6210–6216. doi: 10.1021/bi00239a019. [DOI] [PubMed] [Google Scholar]
  5. Cho H., Ramer S. E., Itoh M., Kitas E., Bannwarth W., Burn P., Saito H., Walsh C. T. Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism. Biochemistry. 1992 Jan 14;31(1):133–138. doi: 10.1021/bi00116a019. [DOI] [PubMed] [Google Scholar]
  6. Domchek S. M., Auger K. R., Chatterjee S., Burke T. R., Jr, Shoelson S. E. Inhibition of SH2 domain/phosphoprotein association by a nonhydrolyzable phosphonopeptide. Biochemistry. 1992 Oct 20;31(41):9865–9870. doi: 10.1021/bi00156a002. [DOI] [PubMed] [Google Scholar]
  7. Fischer E. H., Charbonneau H., Tonks N. K. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science. 1991 Jul 26;253(5018):401–406. doi: 10.1126/science.1650499. [DOI] [PubMed] [Google Scholar]
  8. Frangioni J. V., Beahm P. H., Shifrin V., Jost C. A., Neel B. G. The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell. 1992 Feb 7;68(3):545–560. doi: 10.1016/0092-8674(92)90190-n. [DOI] [PubMed] [Google Scholar]
  9. Gu M. X., York J. D., Warshawsky I., Majerus P. W. Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5867–5871. doi: 10.1073/pnas.88.13.5867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  11. Guan K. L., Dixon J. E. Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate. J Biol Chem. 1991 Sep 15;266(26):17026–17030. [PubMed] [Google Scholar]
  12. Guan K. L., Dixon J. E. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science. 1990 Aug 3;249(4968):553–556. doi: 10.1126/science.2166336. [DOI] [PubMed] [Google Scholar]
  13. Kemp B. E., Pearson R. B. Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990 Sep;15(9):342–346. doi: 10.1016/0968-0004(90)90073-k. [DOI] [PubMed] [Google Scholar]
  14. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  15. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  16. Madden J. A., Bird M. I., Man Y., Raven T., Myles D. D. Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor. Anal Biochem. 1991 Dec;199(2):210–215. doi: 10.1016/0003-2697(91)90091-7. [DOI] [PubMed] [Google Scholar]
  17. Pallen C. J., Lai D. S., Chia H. P., Boulet I., Tong P. H. Purification and characterization of a higher-molecular-mass form of protein phosphotyrosine phosphatase (PTP 1B) from placental membranes. Biochem J. 1991 Jun 1;276(Pt 2):315–323. doi: 10.1042/bj2760315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M. Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1982 Feb;79(4):973–977. doi: 10.1073/pnas.79.4.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., Ullrich A., Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tonks N. K., Charbonneau H., Diltz C. D., Fischer E. H., Walsh K. A. Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase. Biochemistry. 1988 Nov 29;27(24):8695–8701. doi: 10.1021/bi00424a001. [DOI] [PubMed] [Google Scholar]
  21. Tonks N. K., Diltz C. D., Fischer E. H. Characterization of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988 May 15;263(14):6731–6737. [PubMed] [Google Scholar]
  22. Tonks N. K., Diltz C. D., Fischer E. H. Purification of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988 May 15;263(14):6722–6730. [PubMed] [Google Scholar]
  23. Wood E. R., McDonald O. B., Sahyoun N. Quantitative analysis of SH2 domain binding. Evidence for specificity and competition. J Biol Chem. 1992 Jul 15;267(20):14138–14144. [PubMed] [Google Scholar]
  24. Woodford-Thomas T. A., Rhodes J. D., Dixon J. E. Expression of a protein tyrosine phosphatase in normal and v-src-transformed mouse 3T3 fibroblasts. J Cell Biol. 1992 Apr;117(2):401–414. doi: 10.1083/jcb.117.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yamaoka K., Tanigawara Y., Nakagawa T., Uno T. A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn. 1981 Nov;4(11):879–885. doi: 10.1248/bpb1978.4.879. [DOI] [PubMed] [Google Scholar]
  26. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]
  27. Zhang Z. Y., Clemens J. C., Schubert H. L., Stuckey J. A., Fischer M. W., Hume D. M., Saper M. A., Dixon J. E. Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase. J Biol Chem. 1992 Nov 25;267(33):23759–23766. [PubMed] [Google Scholar]
  28. Zhu G., Decker S. J., Saltiel A. R. Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9559–9563. doi: 10.1073/pnas.89.20.9559. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES