The multistep propagation of discrete intracellular signals allows cells to respond to cues from the extracellular environment. Among the most ubiquitous and well-studied of these are the phosphorylation cascades that culminate in the activation of mitogen-activated protein kinases (MAPKs). The enzymatic activity of MAPKs is markedly influenced by extracellular events. As a rule, p38 MAPK activity is induced by environmental stress (e.g. osmotic shock, hypoxia, heat shock, ultraviolet radiation) and pro-inflammatory stimuli and cytokines such as LPS, IL-1, TGF-beta, and TNF-alpha. The most membrane-proximal enzyme activated in the classic MAPK cascade is a serine/threonine kinase known as a MAPK kinase kinase, or MAPKKK, and the MAPKKKs that lead p38 activation include TAK1, ASK1, and MTK1 (human)/MEKK4 (mouse). Growth Arrest and DNA Damage inducible 45 (Gadd45a) was initially identified as a stress-responsive gene. Our studies of Gadd45a-deficient mice found that they died at an early age of a lupus-like autoimmune disease. Because of the known ability of Gadd45-family proteins to bind and activate MTK1/MEKK4, we asked how the absence of Gadd45a might affect p38 activation. We found that, rather than being hypoactive, p38 was spontaneously active in T-lineage cells. An in-depth analysis of this initial observation led to the findings that T cells have an alternative mechanism for the activation of p38 in which the T cell receptor (TCR) proximal kinase ZAP-70 phosphorylates p38 on Tyr-323 (Y323). We have characterized this pathway in great detail, and in the past year have made the following discoveries with regard to pancreatic cancer, an inflammatory neoplasm. 1. All patients pancreatic ductal adenocarcinoma (PDAC) have tumor infiltrating T cells (TIL) with alternatively activated p38, as measured by the presence of phosphorylated Y323 (pY323). 2. 20% of these patients have high levels ( 10% of TIL positive for pY323), which was associated with especially aggressive disease. 3. We developed a cell-permeable fragment of Gadd45a tagged with 11 arginines (11R) 71-85 that accumulated in T cells and inhibited the alternative but not the cannonical p38 pathway. 4. Treatment of two mouse models of PDAC (one of which is mutant K-ras oncogene driven, like the human disase) with (11R) 71-85 statistically-significantly slowed tumor progression and lengthed survival. Therefore, TCR-activated p38 plays a critical role in PDAC in mice and, very likely, humans. The data support the importance of the alternative p38 activation pathway as a molecular target. Another series of experiments is aimed at understanding why the alternative p38 pathway is required for the upregulation of NFAT2, a necessary transcription factor upstream of pro-inflammatory cytokines. We have found that the alternative pathway is required because pY323 p38 (1) induces nuclear translocation of NFAT1 via phosphorylation of a novel serine residue that is required for association with the phosphatase calcineurin, and (2) induces the upregulation of the transcription factor c-Fos, which is required along with NFAT1 for the transcriptional upregulation of NFAT2.
