The Linker Phosphorylation Site Tyr292 Mediates the Negative Regulatory Effect of Cbl on ZAP-70 in T Cells

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-G306E, 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, p116, 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.

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

“…The interaction between SHP-2 and c-Cbl has been described earlier [44,45], but effects of this interaction for T cell function has not been shown to date. Potential target molecules of SHP-2/Cbl complex in T cells that are located upstream of PKC in the TCR signal transduction cascade are CD3-e, CD3-f, Lck, ZAP-70 or in particular PI3K [52][53][54][55][56][57]. By targeting one or more of these molecules for ubiquitin-mediated degradation, the increased complex formation between SHP-2 and c-Cbl could account for reduced effector function in full-length SHP-2-transduced Th cells.…”

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

“…rIgG 1 13B8.2 drives the gp120-induced phosphorylation pattern toward a Zap70-negative regulation pathway, explaining why anti-CD4 13B8.2 Ab treatment induces inhibition of NF-B activation and transcription inhibition of the viral genome, whereas gp120 binding leads to NF-B activation and productive transcription of the viral genome in the case of HIV infection (23). Of great interest is that Zap70 is required in infected donor cells for efficient cell-tocell HIV transmission to recipients and for formation of the virological synapse (58 (57). Tyr 319 residue is autophosphorylated in vitro and becomes phosphorylated in vivo upon TCR triggering (56), leading to positive regulation of TCR-mediated signaling.…”

Cloning and characterization of human Src-like adaptor protein 2 and a novel splice isoform, SLAP-2-v

“…Recruitment of c-Cbl to the activated TCR leads to its association with the Syk family kinases, SYK and ZAP-70. The association between c-Cbl and the Syk family kinases has been demonstrated to result in a decrease in their activity and protein levels (Lupher et al, 1998;Murphy et al, 1998;Rao et al, 2000), resulting in an overall downregulation of signalling from the TCR. The mechanism by which c-Cbl negatively regulates SYK and ZAP-70 is not fully understood, however, it has been proposed that c-Cbl ubiquitin ligase activity may be involved in this process.…”