A Peptide Library Approach Identifies a Specific Inhibitor for the ZAP-70 Protein Tyrosine Kinase
| Autor: | Michael B. Yaffe, Kiyotaka Nishikawa, Jack Lai, Steven J. Burakoff, Sansana Sawasdikosol, David A. Fruman, Zhou Songyang, Lewis C. Cantley |
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| Rok vydání: | 2000 |
| Předmět: |
T-Lymphocytes
Molecular Sequence Data Syk chemical and pharmacologic phenomena Peptide Biology Lymphocyte Activation Transfection Jurkat Cells Genes Reporter Peptide Library Humans Amino Acid Sequence Enzyme Inhibitors Kinase activity Protein kinase A Peptide library Molecular Biology chemistry.chemical_classification ZAP-70 Protein-Tyrosine Kinase Phospholipase C gamma Kinase hemic and immune systems Cell Biology Protein-Tyrosine Kinases Molecular biology Amino acid Isoenzymes Kinetics chemistry Biochemistry Type C Phospholipases Interleukin-2 Phosphorylation Peptides |
| Zdroj: | Molecular Cell. 6:969-974 |
| ISSN: | 1097-2765 |
| Popis: | Summary cally blocked signaling downstream of ZAP-70. Our studies extend the range of experimental approaches We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase in- for probing ZAP-70 function in vivo and introduce a technique for developing protein kinase inhibitors that volved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue could be useful for a wide range of protein kinases. for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted Results and Discussion version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates In order to identify high-affinity inhibitors of the ZAP-70 (KI of 2 mM). The related protein Tyr kinases, Lck and protein Tyr kinase, we screened a Tyr-oriented peptide Syk, were not significantly inhibited by the peptide. library by affinity purification rather than by catalytic When introduced into intact T cells, the peptide conversion. The peptide library used contained the seblocked signaling downstream of ZAP-70, including quence Met-Ala-X-X-X-X-Tyr-X-X-X-X-Ala-Lys-Lys-Lys ZAP-70-dependent gene induction, without affecting where X indicates all amino acids except Trp, Cys, or upstream Tyr phosphorylation. Thus, screening Tyr- Tyr. The predicted degeneracy of this library is 178 < oriented peptide libraries can identify selective pep- 6.9 billion. Screening was performed in the presence of tide inhibitors of protein Tyr kinases. 100 mM ATP but in the absence of Mg 21 to prevent |
| Databáze: | OpenAIRE |
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