The Stress-Activated Protein Kinase Pathway

应激激活蛋白激酶途径

• 批准号: 7050139
• 负责人: John M Kyriakis
• 金额: $ 32.23万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-12-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7050139
• 关键词: CD40 molecule; JUN kinase; RNA interference; acid aminoacid ligase; enzyme activity; enzyme induction /repression; enzyme mechanism; genetically modified animals; immunocytochemistry; laboratory mouse; mitogen activated protein kinase; proteasome; serine threonine protein kinase; ubiquitin

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）： 由丝裂原、应激和炎症激活的信号转导途径是许多临床重要病症的发病机制的核心，包括癌症、糖尿病、缺血性损伤（如心脏病发作和中风）、关节炎、感染性休克、发烧和副作用放射和化疗。细胞外信号调节激酶 (ERK)、Jun-N 末端激酶/应激激活蛋白激酶 (JNK/SAPK) 和 p38 是三种丝裂原激活蛋白激酶 (MAPK) 信号通路，可被多种刺激激活。 MAPK 在很大程度上负责激活蛋白 1 (AP-1) 转录因子复合物的募集，这是细胞外刺激改变基因表达的主要机制。 MAPK 是三层 MAPK-激酶-激酶 (MAP3K) MAPK-激酶 (MKK) MAPK 核心信号模块的各种重叠阵列的远端组件。缺少对近端信号如何与 MAPK 核心通路耦合的理解。这些信息对于新型抗增殖、抗糖尿病和炎症治疗策略的开发以及潜在新药物靶点的识别极其有益。我们正在进行的研究已经开始揭示 ERK、SAPK 和 p38 上游的一些 MAP3K 的调节机制。我们的初步研究表明，生发中心激酶 1 (GCK1) 和肿瘤坏死因子 (TNF) 受体相关因子 (TRAF) 是应激调节 MAP3K MAPK/细胞外信号调节激酶 (ERK) 激酶的潜在上游激活剂-激酶-1 (MEKK1)。 GCK1（但不是 TRAF2）还可以激活 JNK/SAPK 以及可能的 ERK 上游的混合谱系激酶 3 (MLK​​3) aMAP3K。 GCK1 本身似乎部分受到泛素 (Ub) 蛋白酶体依赖性降解的瞬时抑制的调节。我们使用哺乳动物细胞 RNA 干扰 (RNAi) 进行的遗传学研究表明，MLK3 不仅是 JNK/SAPK 丝裂原激活所必需的，也是 ERK 丝裂原激活所必需的。最后，我们发现 gck1 的破坏会损害 JNK/SAPK 的 CD40 激活，并导致缺乏成熟表面免疫球蛋白的 B 淋巴细胞的产生。我们打算扩展这些发现。因此，我们将使用生化方法来确定GCK1如何激活MLK3。我们还将使用 gck1 敲除小鼠和 GCK1 RNAi 来确定 GCK1 是否是体内 MLK3 丝裂原激活所必需的。我们将确定 GCK1 泛素化和/或刺激诱导的 GCK1 稳定是否需要已知的 GCK1 相互作用蛋白（也具有 E3 Ub 连接酶活性（MEKK1、TRAF6））。我们将使用生化和 RNAi 方法来探讨 MLK3 是否是 ERK 特异性 MEK 的主要调节因子，或者 MLK3 是否调节 Raf 家族的 ERK 特异性 MAP3K。最后，我们将使用免疫细胞化学方法来确定 B 细胞 Ig 类别转换是否需要 GCK1。