T Cell Alternative p38 Activation Pathway

T 细胞替代 p38 激活途径

• 批准号: 10014436
• 负责人: Jonathan Ashwell
• 金额: $ 44.69万
• 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/10014436
• 关键词: Affect; Affinity; Age; Arginine; Autoimmune Diseases; Autoimmunity; Binding; Cell Lineage; Cells; Clinical Trials; Cues; DNA Damage; Data; Disease; Drug Targeting; Environment; Enzymes; Event; FOS gene; GADD45A gene; Genes; Genetic Transcription; Goals; Growth; Heat-Shock Response; Human; Hypoxia; Immunity; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interleukin-1; K-ras Oncogene; KRAS2 gene; Lead; Length; MAP Kinase Kinase Kinase; MAP3K7 gene; MAPK14 gene; Malignant neoplasm of pancreas; Measures; Membrane; Mitogen-Activated Protein Kinases; Molecular Target; Mus; Names; Neoplasms; Nuclear Translocation; Osmotic Shocks; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Peptides; Periodicity; Permeability; Phosphorylation; Physiological; Play; Protein Family; Protein-Serine-Threonine Kinases; Receptor Signaling; Role; Series; Serine; Signal Transduction; Stimulus; Stress; Structure; T-Cell Receptor; T-Lymphocyte; TNF gene; Transforming Growth Factor beta; Ultraviolet Rays; Up-Regulation; Variant; Water; ZAP-70 Gene; calcineurin phosphatase; cytokine; design; experimental study; extracellular; in vivo; lupus-like; mitogen-activated protein kinase p38; mouse model; mutant; novel; transcription factor; transcription factor USF; tumor; tumor progression

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. We are currently attempting to characterize the interaction between p38 and Gadd45a. To do this, we are attempting to create cyclic variants of the (11R) 71-85 peptide that will be water-soluble and bind with detectable affinity with p38. If such compounds are established, the plan is to perform studies to obtain structural information that will allow us to design compounds with high affinity that might be useful in vivo.

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活性是由环境应力（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以及LPS，IL-1，TGF-beta和TNF-Alpha等细胞因子引起的。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GADD45A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GADD45A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发现T细胞具有激活p38的替代机制，其中T细胞受体（TCR）近端激酶ZAP-70在Tyr-323上磷酸化p38（Y323）。我们已经详细介绍了这一途径，在过去的一年中，关于胰腺癌（一种炎症性肿瘤）的发现。 1。所有患者的胰腺导管腺癌（PDAC）均具有肿瘤浸润的T细胞（TIL），其替代活化的p38，如存在磷酸化的Y323（PY323）所测量的。 2。这些患者中有20％的水平很高（PY323的TIL阳性10％），这与特别侵略性疾病有关。 3。我们开发了一个具有11个精氨酸（11R）71-85标记的GADD45A的细胞片段，该片段积聚在T细胞中，并抑制了替代方案，但不抑制罐头p38途径。 4。用（11R）71-85统计学上较慢的肿瘤进展和生存时间延长的PDAC的两种小鼠模型（其中一种是突变的K-Ras癌基驱动的突变体K-Ras癌细胞）。因此，TCR激活的p38在小鼠的PDAC中起着至关重要的作用，很可能是人类。数据支持替代p38激活途径作为分子靶标的重要性。另一个一系列实验旨在理解为什么需要替代p38途径才能上调NFAT2，这是促炎性细胞因子上游的必要转录因子。我们发现需要替代途径，因为PY323 p38（1）通过磷酸化与磷酸酶钙调蛋白相关所必需的新型丝氨酸残基诱导NFAT1的核转运，并且（2）诱导了转录因子C-FOS的上调，而NFAT1则与NFAT1相关的转录因子C-FOS以及NFAT1 for Trescriptionalscriptiational for the forscriptiational for the forscriptiational for nofection for nofection for forscriptiational for for nfatectunion for for nfat2222。我们目前正在尝试表征p38和GADD45A之间的相互作用。为此，我们正在尝试创建（11R）71-85肽的环状变体，该变体将是水溶性的，并与p38的可检测亲和力结合。如果建立了这样的化合物，则计划将进行研究以获取结构信息，以使我们能够设计具有可能在体内有用的高亲和力的化合物。

项目成果

T cell priming: let there be light.

T 细胞启动：要有光。

• DOI: 10.1038/cr.2010.72
• 发表时间: 2010
• 期刊: Cell research
• 影响因子: 44.1
• 作者: Jirmanova,Ludmila;Ashwell,JonathanD
• 通讯作者: Ashwell,JonathanD

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

生物活性非 ATP 竞争性 p38 MAP 激酶抑制剂的发现和表征。

• DOI: 10.1177/1087057115615518
• 发表时间: 2016
• 期刊: Journal of biomolecular screening
• 作者: Wilson,BriceAP;Alam,MuhammadS;Guszczynski,Tad;Jakob,Michal;Shenoy,ShilpaR;Mitchell,CarterA;Goncharova,EkaterinaI;Evans,JasonR;Wipf,Peter;Liu,Gang;Ashwell,JonathanD;O'Keefe,BarryR
• 通讯作者: O'Keefe,BarryR