Abstract
The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.
Highlights
The development of T cells in the thymus involves stringent selection processes to ensure the generation of T cells that are not strongly self-reactive [1,2,3]
C-Cbl is a multiadaptor proto-oncogene that possesses E3 ubiquitin ligase activity by virtue of its RING finger domain, which recruits ubiquitin conjugating enzymes (E2s). It is abundantly expressed in the thymus where it functions as a negative regulator of T cell receptors (TCRs) signaling [6, 7]. c-Cbl KO mice have elevated levels of TCR and CD3 on the surface of CD4ϩCD8ϩ double positive (DP) thymocytes, increased levels of Lck and Fyn, and enhanced activity of the ZAP-70 tyrosine kinase (8 –11)
Because Nur77 and Bim are key mediators of negative selection in DP thymocytes (20 –22) we tested whether the greater susceptibility of c-Cbl A/Ϫ thymocytes to TCR-mediated death may be linked to an enhanced induction of these proteins
Summary
Mice—The generation of c-CblϪ/Ϫ, c-Cbl(C379A), and c-Cbl(Y737F) mice have been previously described [5, 8, 30]. To detect intracellular levels of Nur, thymocytes were stained with anti-CD4 and anti-CD8, fixed, and permeabilized in Cytofix/Cytoperm (BD Biosciences) for 20 min at room temperature and washed in FACS buffer. For the detection of Bim, thymocytes were fixed and permeabilized in Cytofix/Cytoperm, washed twice in FACS buffer ϩ 0.03% saponin, and incubated on ice for 45 min with rat anti-Bim (3C5/10B12 from Andreas Strasser) in FACS buffer containing 0.3% saponin. Bim was detected by sequential incubations with biotinylated anti-rat IgG2a (BD Biosciences) followed by streptavidin phycoerythrin (Molecular Probes), both in FACS buffer containing 0.3% saponin and on ice for 45 min. The cells were fixed and permeabilized in Cytofix/Cytoperm (BD Biosciences) and all procedures were carried out in FACS buffer containing 0.1% saponin as previously described [5]. Antibodies to Bim, Akt, p-Akt(S473), p-ERK(Thr-202/Tyr-204), p-c-Cbl(Tyr-731 and Tyr-774), p-LAT(Tyr-191), p-PKD(Ser-916), and PKD were from Cell Signaling and p-PLC␥-1(Tyr-783) antibodies were from BIOSOURCE
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