T cell alternative p38 activation pathway

T细胞替代p38激活途径

• 批准号: 7592923
• 负责人: Jonathan Ashwell
• 金额: $ 68.76万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592923
• 关键词: Affect; Age; Animal Model; Antigen Receptors; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding; Cell Cycle; Cell Lineage; Cells; Clinical Trials; Condition; Cues; DNA Damage; Depth; Disease; Drug Delivery Systems; Environment; Enzymes; Event; Genes; Goals; Growth; Heat-Shock Response; Human; Hypoxia; Immune; Immune Sera; Immunity; Inflammation; Inflammatory; Inflammatory Response; Interferons; Interleukin-1; Knock-in Mouse; Lead; Lupus; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; MAP Kinase Kinase Kinase; MAP2K6 gene; MAP3K7 gene; MAPK14 gene; Mediating; Mediator of activation protein; Membrane; Mitogen-Activated Protein Kinases; Molecular Target; Mus; Names; Osmotic Shocks; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Protein Family; Protein-Serine-Threonine Kinases; Receptor Signaling; Rest; SB 203580; Signal Transduction; Stimulus; Stress; Substrate Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Toxoplasma gondii; Ultraviolet Rays; cytokine; extracellular; human MAPK14 protein; in vivo; inhibitor/antagonist; prevent; response

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-β, and TNF-α. 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 Gadd45α-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 Gadd45α 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: 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 Gadd45α-deficient T cells is spontaneously phosphorylated on Tyr-323. Gadd45α specifically binds to p38 (whether phosphorylated or not) and inhibits the activity of the p-Tyr-323 form. Importantly, Gadd45α binding does not inhibit the activity of p38 phosphorylated by MKK6 (on Thr-180/Tyr-182). In the past year we have extended our understanding of how p38 is activated in the alternative pathway, and have established an animal model that allows us to explore its importance in normal and pathophysiologic conditions. These recent findings include: 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, suggesting that substrate specificity may differ between these two means of activating the kinase. We have 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-γ when immunized with Toxoplasma gondii. Therefore, the alternative p38 activation pathway is important for normal T cell proliferation and immune/inflammatory responses. Our observations establish the alternative p38 pathway as being the major mechanism for the activation of this important kinase in T cells, and support its possible importance as a molecular target

离散细胞内信号的多步繁殖使细胞可以从细胞外环境中响应线索。其中最普遍和研究的是其中最终导致有丝分裂原激活蛋白激酶（MAPK）激活的磷酸化级联反应。 MAPK的酶促活性受细胞外事件的显着影响。通常，p38 MAPK活性是由环境应激（例如渗透性休克，缺氧，热休克，紫外线辐射）和促炎性刺激以及细胞因子（例如LPS，IL-1，TGF-β和TNF-α）引起的。在经典MAPK级联反应中激活的最膜型酶是一种丝氨酸/苏氨酸激酶，称为MAPK激酶激酶或MAPKKK，而引起P38激活的MAPKKK包括TAK1，ASK1，ASK1，ASK1，ASK1和MTK1（MTK1（HUMAN）/MEKKK4）。最初将生长停滞和DNA损伤诱导的45（GADD45A）鉴定为应激响应基因。我们对GADD45α缺陷小鼠的研究发现，它们死于狼疮样的自身免疫性疾病。由于GADD45家庭蛋白结合和激活MTK1/MEKK4的已知能力，我们询问GADD45α的缺失如何影响p38激活。我们发现，p38在T-linege细胞中自发活性，而不是发动不足。对这一初始观察结果的深入分析导致以下发现：抗原受体刺激的正常T细胞的p38，而不是B细胞可牢固地自磷酸化。自磷酸化似乎在两个规范激活残基THR-180和Tyr-182上。 T细胞p38激活需要LCK和ZAP70，但不依赖于LAT。 TRY-323上的TCR近端激酶LCK，FYN和ZAP70磷酸化p38，该磷酸化p38诱导自磷酸化并增强对其他底物的活性。值得注意的是，即使缺乏Tyr-182的p38也会被Tyr-323磷酸化激活。 TYR-323磷酸特异性抗血清检测到T中Tyr-323-磷酸化的p38（P-TYR-323 p38），但没有通过抗原受体激活的B细胞。 在LCK+ ZAP70- JURKAT T细胞中未检测到P-TYR-323 p38，这意味着ZAP70是体内效应子激酶。 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，表明这是自磷酸化的结果。 来自GADD45α缺陷T细胞的p38在Tyr-323上自发地磷酸化。 GADD45α特别结合了p38（无论是否磷酸化），并抑制p-tyr-323形式的活性。重要的是，GADD45α的结合不会抑制MKK6磷酸化的p38活性（在THR-180/TYR-182上）。在过去的一年中，我们扩展了对p38在替代途径中如何激活的理解，并建立了一种动物模型，使我们能够探索其在正常和病理生理条件下的重要性。这些最近的发现包括：在Tyr-323上磷酸化的p38能够在反式中磷酸化；也就是说，一个p38分子结合并磷酸化另一种分子。 自动传输磷酸化仅涉及THR-180，而不仅涉及MAPK级联中发现的典型Tyr-182，这表明底物特异性在激活激酶的两种手段之间可能有所不同。 我们已经产生了P38“敲入”小鼠，其中Tyr-323被PHE取代（P38YF）。证明了替代途径的生理重要性，TCR介导的激活完全无法激活这些小鼠的T细胞中的p38。 通过TCR刺激，来自p38yf敲击蛋白小鼠的T细胞在细胞周期中从G0转到G1的慢速。此外，当用弓形虫弓形虫免疫时，它们的干扰素-γ少得多。因此，替代p38激活途径对于正常的T细胞增殖和免疫/炎症反应很重要。我们的观察结果确定了替代p38途径是T细胞中这种重要激酶激活的主要机制，并支持其作为分子靶标的重要性