However, signaling proteins downstream of FasL, TRAIL and NDG-1 like FADD and caspase-8 are not required for negative selection 18, 19. Nur77 and Nor-1 can also act through a non-transcriptional manner to initiate apoptosis. We have previously shown that during the early phase of thymocyte apoptosis, Nur77 and Nor-1 translocate from the nucleus to the mitochondria where they bind Bcl-2 20. Their association with Bcl-2

exposes the BH3 domain within Bcl-2, converting the protein into a potential killer molecule similar to those found in cancer cells 21, 22. However, the upstream signals regulating Nur77′s translocation in thymocytes have not been defined. As Nur77 is heavily phosphorylated, it seems plausible that phosphorylation regulates the protein’s subcellular localization, which has been shown in some cell lines. In prostate and lung cancer cell lines, for example, Nur77′s mitochondrial targeting is dependent on both induction of the JNK kinase GS-1101 mouse and inhibition of the Akt kinase 23. In DO11.10 T-cell hybridomas, expression of a constitutively active Akt protein inhibited Nur77′s transcriptional activities, possibly by stimulating its association with 14–3–3 for nuclear exclusion 24, 25. Also in DO11.10 cells, RSK, a kinase downstream of the ERK1/2 pathway was shown recently to be responsible for phosphorylation of Nur77 required for mitochondria translocation 26. The signals mediating GSK3 inhibitor Nur77′s localization to

mitochondria in primary cells like thymocytes, however, remain unclear. TCR stimulation during negative selection results in activation of several downstream cascades, involving protein tyrosine

until kinases, PKC and MAPK 3. Activation of the protein tyrosine kinases and signaling through the MAP kinase pathway causes activation of ERK1/2, JNK, p38 and ERK5. JNK, p38 and ERK5 have been established as key molecules during negative selection 4 while ERK1/2 are required for positive selection 27. PKC proteins have also been implicated in negative selection 28. The PKC family of serine/threonine kinases consists of multiple isozymes involved in a myriad of signal transduction pathways. PKC isozymes are classified into calcium-independent or classical cPKC (α, β and γ), novel nPKC (δ, ε, η and θ) and atypical aPKC (μ and ζ) 29, 30. In T lymphocytes, PKC isoforms play important roles in facilitating cell survival, activation, differentiation and the induction of cell death 31–33. PKCθ is a nPKC selectively expressed in T cells and muscle and plays a particularly important role in TCR/CD28 signaling pathways 33. In mature T cells, PKCθ functions to activate the JNK/AP-1 pathways and participate in IL-2 induction and activation of NF-κB. However, in thymocytes, the induction of NF-κB is independent of PKCθ signaling, as PKCθ −/− thymocytes treated with anti-CD3 and anti-CD4 or TNF show normal activation of NF-κB 34. Other PKC proteins regulate apoptosis in thymocytes.