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Molecular and Cellular Biology, October 1999, p. 6652-6664, Vol. 19, No. 10
Laboratory of Cellular and Molecular Biology,
National Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20892
Received 4 February 1999/Returned for modification 23 March
1999/Accepted 25 June 1999
T-cell receptor (TCR) engagement results in the activation of Src
family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to
tyrosine phosphorylation of multiple cellular substrates including the
complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated
upon engagement of growth factor receptors, cytokine receptors, and
immunoreceptors and functions as a negative regulator of tyrosine
kinase signalling pathways. Cbl associates via its phosphotyrosine
binding (PTB) domain to the ZAP-70 pY292 negative regulatory
phosphotyrosine. We recently demonstrated that the oncogenic Cbl
mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in
unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but
not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT
activation. Moreover, 70Z Cbl does not upregulate NFAT in the
ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck,
and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as
mutation of all tyrosines in, or deletion of, the C-terminal region of
70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine
phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further,
70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT,
indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling
pathways. Finally, the upregulation of NFAT observed upon ZAP-70
overexpression is blocked by Cbl in a PTB domain-dependent manner. We
conclude that oncogenic 70Z Cbl acts as a dominant negative to block
the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of
transcription factors.
0270-7306/99/$04.00+0
The Oncogenic 70Z Cbl Mutation Blocks the
Phosphotyrosine Binding Domain-Dependent Negative Regulation of ZAP-70
by c-Cbl in Jurkat T Cells
*
Corresponding author. Mailing address: Laboratory of
Cellular and Molecular Biology, National Cancer Institute, National
Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-5216. Fax: (301) 496-8479. E-mail:
vanleeuj{at}box-v.nih.gov.
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