The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells

Navin Rao, Mark L. Lupher, Ota Satoshi, Kris A. Reedquist, Brian J. Druker, Hamid Band

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Abstract

The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR- induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G3015E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p1115, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.

Original languageEnglish (US)
Pages (from-to)4616-4626
Number of pages11
JournalJournal of Immunology
Volume164
Issue number9
DOIs
StatePublished - May 1 2000

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Protein-Tyrosine Kinases
Phosphorylation
Jurkat Cells
T-Lymphocytes
Luciferases
Mutation
COS Cells
Phosphoproteins
Phenylalanine
Binding Sites
Cell Line

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. / Rao, Navin; Lupher, Mark L.; Satoshi, Ota; Reedquist, Kris A.; Druker, Brian J.; Band, Hamid.

In: Journal of Immunology, Vol. 164, No. 9, 01.05.2000, p. 4616-4626.

Research output: Contribution to journalArticle

Rao, Navin ; Lupher, Mark L. ; Satoshi, Ota ; Reedquist, Kris A. ; Druker, Brian J. ; Band, Hamid. / The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. In: Journal of Immunology. 2000 ; Vol. 164, No. 9. pp. 4616-4626.
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