ZIA BC 010774 (ZIA) | |||
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Title | T Cell Alternative p38 Activation Pathway | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Ashwell, Jonathan | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $461,571 | Project Dates | 01/01/2006 - 00/00/0000 |
Fiscal Year | 2015 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Arthritis (50.0%) Autoimmune Diseases (100.0%) Cancer (100.0%) Digestive Diseases (50.0%) Interferon (25.0%) |
Pancreas (50.0%) | ||
Research Type | |||
Normal Functioning Endogenous Factors in the Origin and Cause of Cancer |
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Abstract | |||
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 following findings by our laboratory: - p38 from antigen receptor-stimulated normal T cells but not B cells robustly autophosphorylates. The autophosphorylation appeared to be on the two canonical activating residues, Thr-180 and Tyr-182. - T cell p38 activation requires Lck and Zap70 but is LAT-independent. - The TCR proximal kinases Lck, Fyn, and Zap70 phosphorylate p38 on Tyr-323, which induces autophosphorylation and enhanced activity toward other substrates. Notably, even p38 lacking Tyr-182 is activated by Tyr-323 phosphorylation. - A Tyr-323 phospho-specific antiserum detects Tyr-323-phosphorylated p38 (p-Tyr-323 p38) in T but not B cells activated via the antigen receptor. - p-Tyr-323 p38 is not detected in Lck+ Zap70- Jurkat T cells, implicating Zap70 as the effector kinase in vivo. - The alternative pathway appeared to be a major mechanism of p38 activation in T cells, because (1) in Jurkat T cells, p38 containing a Y323F substitution was poorly activated in response to anti-TCR compared to wild type (WT), and (2) dual (Thr-180/Tyr-182) phosphorylation of p38 in TCR-stimulated normal resting T cells was almost completely prevented by the p38 inhibitor SB203580, indicating that it is a consequence of autophosphorylation. - p38 from Gadd45a-deficient T cells is spontaneously phosphorylated on Tyr-323. - Gadd45a specifically binds to p38 (whether phosphorylated or not) and inhibits the activity of the p-Tyr-323 form. Importantly, Gadd45a binding does not inhibit the activity of p38 phosphorylated by MKK6 (on Thr-180/Tyr-182). - p38 phosphorylated on Tyr-323 is able to phosphorylate itself in trans; that is, one p38 molecule binds and phosphorylates another. - Auto-trans-phosphorylation involves just Thr-180 and not the canonical Tyr-182 found in the MAPK cascade. - The substrate specificity of mono-phosphorylated p38 is different from the di-phosphorylated form. This may explain why this alternative pathway has been evolutionarily conserved in T cells, because the biological effects of these two phosphorylated species would be expected to differ in vivo. - We generated p38 ""knock-in"" mice in which Tyr-323 is replaced with a Phe (p38YF). Proving the physiologic importance of the alternative pathway, TCR-mediated activation is completely incapable of activating p38 in T cells from these mice. - T cells from p38YF knockin mice are slow to transit from G0 to G1 in the cell cycle upon stimulation via the TCR. Moreover, they make much less interferon-gamma when immunized with Toxoplasma gondii. Therefore, the alternative p38 activation pathway is important for normal T cell proliferation and immune/infla |