PROJECT SUMMARY The goal of this new proposal is to identify molecular mechanisms involved in controlling expansion of auto- and allo-reactive T cells. The serine/threonine kinase DRAK2 is a negative regulator of T cell receptor (TCR) signaling and has been found to set the threshold for the activation of nave T cells and selected thymocytes. We have determined that Drak2-/- (D2-/-) mice are resistant to experimental autoimmune encephalomyelitis (EAE), an autoimmune demyelinating disease that resembles MS. D2-/- mice are similarly resistant to type I diabetes, an autoimmune disease in which T cells attack insulin producing pancreatic beta cells. D2-/- T cells also possess a defect in rejecting allogeneic transplants. Importantly, D2-/- mice retain functional immunity against a host of viruses. Pharmacologic blockade of DRAK2 signaling may thus afford a unique approach to treat organ specific autoimmune diseases such as MS and may also prolong the survival of allografts. Thus, the need for powerful immune suppressive drugs that are themselves highly problematic when used for long-term control of autoimmune symptoms may be overcome. Although the complete basis for this enigmatic role for DRAK2 remains to be discerned, we have discovered that encephalitogenic D2-/- T cells possess significant survival defects that prevent the elaboration of EAE. In recent studies, we have also determined that DRAK2 contributes to the differentiation of helper T cells (Th) such that in its absence, a greater fraction of tolerogenic CD4+/FoxP3+ regulatory T cells (Treg) are produced both under steady state conditions, and following activation of nave CD4+/CD25--depleted T cells. In studies proposed in this new application, we seek to investigate the means by which DRAK2 contributes to the metabolism of activated effector T cells. We have found that D2-/- T cells are unable to properly coordinate mitochondrial metabolism and die through intrinsic apoptosis, a form of programmed cell death that is instigated by the release of mitochondrial factors such as cytochrome C. Using a combination of protein crosslinking and mass spectrometry, we will characterize the interaction partners of DRAK2 and determine if these interactors are subject to DRAK2 mediated phosphorylation. We will also determine how these proteins form complexes with DRAK2 and will determine if their phosphorylation is required for coordinated mitochondrial metabolism, regulation of the mitochondrial permeability transition pore (mPTP) and the prevention of intrinsic apoptosis. These studies will be highly insightful regarding the unique role(s) that DRAK2 plays in controlling effector T cell metabolism and survival, information that will be vital to the development of therapeutics that target DRAK2 for the treatment of autoimmune diseases such as MS and type-I diabetes, and for the prevention of allograft rejection.