4.5 Article

Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding

Journal

BIOCHEMICAL JOURNAL
Volume 465, Issue -, Pages 149-161

Publisher

PORTLAND PRESS LTD
DOI: 10.1042/BJ20140793

Keywords

B-cell; covalent small-molecule inhibitor; hydrogen peroxide (H2O2); immunoreceptor tyrosine-based activation motif (ITAM); T-cell

Funding

  1. National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health American Recovery and Reinvest Act [1RC2-AR058947-01]
  2. National Institutes of Health/National Cancer Institute UC Berkeley Cancer Laboratory training grant [5T32 CA 9179-35]
  3. NATIONAL CANCER INSTITUTE [T32CA009179] Funding Source: NIH RePORTER
  4. NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [P01AI091580] Funding Source: NIH RePORTER
  5. NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES [RC2AR058947] Funding Source: NIH RePORTER
  6. NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [P30DK063720] Funding Source: NIH RePORTER

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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 ITAMbinding 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.

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