Immunol. the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, AMG 837 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 transmission strength that regulates the engagement of death-promoting signals. C379A) disrupts the interactions of c-Cbl with E2 enzymes and abolishes its E3 ligase activity (12,C15). Mice with this mutation have many characteristics identical to the c-Cbl KO such as increased expression of TCR, CD3, Lck, and Fyn in DP thymocytes (5). Amazingly, however, unlike the c-Cbl knock-out, the RING finger mutation results in a progressive loss of the thymus even though DP thymocytes from both mutants show equivalent increases in TCR-directed activation of ZAP-70, the Ras and Rac pathways, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and calcium mobilization (5). We showed that thymic loss is not caused by a developmental block, a lack of thymic progenitors, or peripheral T cell activation (5). Rather the phenotype correlates with a marked increase in expression of CD5 and CD69 activation markers on DP thymocytes and AMG 837 increased sensitivity to cell death when cultured with plate-bound anti-CD3, a signal that in WT thymocytes is usually insufficient to induce a death response. In addition, expression of a Bcl-2 transgene rescues thymic loss and blocks anti-CD3-mediated thymocyte apoptosis (5). These findings support the hypothesis that thymic deletion in the c-Cbl RING finger mutant mouse is usually caused by an abnormally intense TCR-directed apoptotic transmission. Surprisingly the only signaling event in c-Cbl(C379A) thymocytes that we found to differ from the c-Cbl KO involved the markedly elevated activation of Akt in response to anti-CD3 cross-linking (5). Furthermore, we exhibited that this mutant c-Cbl protein itself is responsible for the enhanced Akt activation through an increased AMG 837 interaction with the p85 regulatory subunit of PI3K (5). This is caused by high levels of TCR, CD3, and Fyn in the mutant thymocytes that results in a greater proportion of c-Cbl phosphorylation on tyrosine 737, the site recognized by the SH2 domains of p85 (16,C19). Thus analysis of this mouse has provided evidence that tyrosine-phosphorylated c-Cbl can play a positive role in directing thymocyte apoptosis through its conversation with p85 and the resultant activation of the PI3K/Akt pathway. Here VEGFA we expand the study of this pathway by examining the regulation of Nur77 and Bim, two important proteins involved in thymocyte unfavorable selection. Our analysis of the c-Cbl RING finger mutant mouse and a c-Cbl knock-in mouse with a phenylalanine substitution of tyrosine 737 has identified a novel apoptotic pathway directed by c-Cbl that activates Akt and promotes enhanced levels of Bim and Nur77. EXPERIMENTAL PROCEDURES Mice The generation of c-Cbl?/?, c-Cbl(C379A), and c-Cbl(Y737F) mice have been previously explained (5, 8, 30). Mice were maintained on a mixed C57BL/6J x129Sv/J background and experiments were performed in compliance with the Animal Ethics Committee at the University or college of Western Australia (approvals 03/100/275 and 07/100/578). Inhibitors Pharmacological inhibitors used and their working concentrations are: Akt inhibitor VIII, Akti-1/2, 10 m (Calbiochem, 124018); histone deacetylase inhibitor trichostatin A, 33 or 400 nm (Sigma, T 8552); PI3K inhibitors wortmannin, 100 nm, and LY 294002, 25 m (Alomone Labs, W-400 and L-300 respectively); PKC inhibitor Ro 31-8220, 200 nm (Alexis Biochemicals, 270-020-M001); PKC/PKD inhibitor Go6976, 400 nm (Alexis Biochemicals, 270-021-MC05); and MEK inhibitor PD98059, 40 m (Calbiochem, 513000). Circulation Cytometry and Antibody Staining Antibody-stained thymocyte, spleen, and lymph node cell suspensions were acquired on a BD Biosciences FACSCanto and the data were analyzed using FlowJo software (Tree Star Inc). Antibodies used were against: TCR (H57-597), CD4 (RM4C5), CD8 (53-6.7), CD5 (53-7.3), CD69 (H1.2F3), and B220 (RA3-6B2) (BD Biosciences). To detect intracellular levels of Nur77, 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. Thymocytes were then incubated with ice-cold methanol for 30 min at 4 C and washed twice in FACS buffer before incubation with mouse anti-Nur77 (BD AMG 837 Biosciences Transduction Labs) for 45 min at 4 C. Following two washes in FACS buffer thymocytes were incubated with fluorescein isothiocyanate-labeled anti-mouse IgG1 (BD Biosciences). For the detection of Bim, thymocytes were fixed and permeabilized in Cytofix/Cytoperm, washed twice in FACS buffer + 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%.