Activating and Inhibitory Mutations in Adjacent Tyrosines in the Kinase Domain of ZAP-70 (*)

ZAP-70 is a 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that it is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.

[1]  S. Hubbard,et al.  Expression, Characterization, and Crystallization of the Catalytic Core of the Human Insulin Receptor Protein-tyrosine Kinase Domain (*) , 1995, The Journal of Biological Chemistry.

[2]  H. Kitayama,et al.  Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. , 1995, Blood.

[3]  L. Samelson,et al.  Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists , 1995, Science.

[4]  R. Aebersold,et al.  F(Pmp)-TAM, a Novel Competitive Inhibitor of the Binding of ZAP-70 to the T Cell Antigen Receptor, Blocks Early T Cell Signaling (*) , 1995, The Journal of Biological Chemistry.

[5]  S. Hubbard,et al.  Crystal structure of the tyrosine kinase domain of the human insulin receptor , 1994, Nature.

[6]  R. Aebersold,et al.  Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. , 1994, The Journal of biological chemistry.

[7]  A. Weiss,et al.  ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR zeta in murine thymocytes and lymph node T cells. , 1994, Immunity.

[8]  H. Kung,et al.  Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain , 1994, Molecular and cellular biology.

[9]  J. Fargnoli,et al.  Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement. , 1994, The Journal of biological chemistry.

[10]  S. Courtneidge,et al.  Protein tyrosine kinases, with emphasis on the Src family. , 1994, Seminars in cancer biology.

[11]  T. Hope,et al.  Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. , 1994, Science.

[12]  W. Kuo,et al.  ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. , 1994, Science.

[13]  R. Klausner,et al.  Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif , 1994, Molecular and cellular biology.

[14]  A. Cohen,et al.  Defective T cell receptor signaling and CD8+ thymic selection in humans lacking Zap-70 kinase , 1994, Cell.

[15]  A. Weiss,et al.  Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. , 1994, Science.

[16]  S. Malek,et al.  Tandem SH2 domains of ZAP-70 bind to T cell antigen receptor zeta and CD3 epsilon from activated Jurkat T cells. , 1993, The Journal of biological chemistry.

[17]  B. Seed,et al.  T cell activation by clustered tyrosine kinases , 1993, Cell.

[18]  R. Abraham,et al.  Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. , 1993, The Journal of biological chemistry.

[19]  Arthur Weiss,et al.  ZAP-70: A 70 kd protein-tyrosine kinase that associates with the TCR ζ chain , 1992, Cell.

[20]  J. O’Shea,et al.  Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Low,et al.  Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli , 1992, Protein science : a publication of the Protein Society.

[22]  J. Tavaré,et al.  Analysis of the order of autophosphorylation of human insulin receptor tyrosines 1158, 1162 and 1163. , 1992, Biochemical and biophysical research communications.

[23]  A. Kong,et al.  A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the zeta chain and CD3 components of the activated T cell antigen receptor. , 1992, The Journal of biological chemistry.

[24]  Arthur Weiss,et al.  The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Tavaré,et al.  The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain. An assessment by replacement with neutral and negatively charged amino acids. , 1991, The Journal of biological chemistry.

[26]  N. Abraham,et al.  Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation , 1990, Molecular and cellular biology.

[27]  M. Fournel,et al.  The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation. , 1990, Oncogene.

[28]  R. Klausner,et al.  Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  B. Kemp.  Peptides and Protein Phosphorylation , 1990 .

[30]  I. G. Fantus,et al.  Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. , 1989, Biochemistry.

[31]  J. Bolen,et al.  Alterations in tyrosine protein phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes , 1989, Molecular and cellular biology.

[32]  S. Cheng,et al.  pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527 , 1989, Molecular and cellular biology.

[33]  J. Avruch,et al.  Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells. , 1988, The Journal of biological chemistry.

[34]  K. Arai,et al.  SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat , 1988, Molecular and cellular biology.

[35]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[36]  T. Kmiecik,et al.  Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation , 1987, Cell.

[37]  T. Roberts,et al.  Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src , 1987, Cell.

[38]  W. Rutter,et al.  Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose , 1986, Cell.

[39]  H. Iba,et al.  Activation of the transforming potential of p60c-src by a single amino acid change. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[40]  G K Lewis,et al.  Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product , 1985, Molecular and cellular biology.

[41]  H. Oppermann,et al.  Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src). , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[42]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.