Signal transduction pathways activated by mitogens, stress and inflammation are central to the pathogenesis of a number of clinically significant conditions including cancer, diabetes, ischemic injury (as occurs in heart attack and stroke), arthritis, septic shock, fever and the side effects of radiation and chemotherapy. The extracellular signal regulated kinases (ERKs), Jun-N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) and the p38s are three mitogen-activated protein kinase (MAPK) signaling pathways activated by a wide variety of stimuli. The MAPKs are largely responsible for the recruitment of the activator protein-1 (AP-1) transcription factor complex--a major mechanism by which extracellular stimuli alter gene expression. MAPKs are the distal components of variously overlapping arrays of three tiered MAPK-kinase-kinase (MAP3K) MAPK-kinase (MKK) MAPK core signaling modules. Missing is an understanding of how proximal signals couple to MAPK core pathways. Such information would be extremely beneficial to the development of novel anti proliferative, antidiabetic and inflammatory treatment strategies, and in the identification of potential new drug targets. Our ongoing studies have begun to unravel the mechanisms by which some of the MAP3Ks upstream of the ERKs, SAPKs and p38s are regulated. Our preliminary studies show that germinal center kinase-1 (GCK1), and tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are potential upstream activators of the stress-regulated MAP3K MAPK/extracellular signal-regulated kinase (ERK)-kinase-kinase-1 (MEKK1). GCK1 (but not TRAF2) can also activate mixed lineage kinase-3 (MLK3) aMAP3K upstream of the JNKs/SAPKs and, possibly, the ERKs. GCK1 itself appears to be regulated in part by transient inhibition of ubiquitin (Ub)-proteasome-dependent degradation. Our genetic studies using mammalian cell RNA interference (RNAi) indicate that MLK3 is required not only for mitogen activation of JNK/SAPK, but formitogen activation of ERK. Finally, we find that disruption of gck1 impairs CD40 activation of JNK/SAPK and leads to the generation of B lymphocytes deficient in the presence of mature surface immunoglobulins. We intend to expand upon these findings. Accordingly, we will use biochemical methods to determine how GCK1 activates MLK3. We will also use our gck1 knockout mice, and GCK1 RNAi to determine if GCK1 is required for mitogen activation of MLK3 in vivo. We will determine if known GCK1 interacting proteins which also possess E3 Ub ligase activity (MEKK1, TRAF6) are required for GCK1 ubiquitination and/or stimulus-induced GCK1 stabilization. We will use biochemical and RNAi approaches to explore if MLK3 is a major regulator of ERK-specific MEKs, or if MLK3 regulates ERK-specific MAP3Ks of the Raf family. Finally, we will use immunocytochemical methods to determine if GCK1 is required for B cell Ig class switching.