title: Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding.
abstract: Zeta-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling respectively. They are recruited, via their tandem-SH2 (Src-homology domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Visperas
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Winger
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Horton
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Neel H
authors.initials: NH
authors.lastname: Shah
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Aum
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Tao
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Tiago
authors.initials: T
authors.lastname: Barros
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: Qingrong
authors.initials: Q
authors.lastname: Yan
authors.affiliation: †Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California, San Francisco, CA 94158, U.S.A.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Wilson
authors.affiliation: †Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California, San Francisco, CA 94158, U.S.A.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Arkin
authors.affiliation: ‡Department of Medicine, Rosalind Russell and Ephrain P. Engleman Rheumatology Research Center for Arthritis and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, U.S.A.
authors.forename: Arthur
authors.initials: A
authors.lastname: Weiss
authors.affiliation: *Department of Molecular and Cell Biology and Department of Chemistry, California Institute of Quantitative Biosciences and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, U.S.A.
authors.forename: John
authors.initials: J
authors.lastname: Kuriyan
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000187:drug effects; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds; D019000:Phosphotyrosine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D003545:Cysteine