The Stress-Activated Protein Kinase Pathway

描述（由申请人提供）： 由有丝分裂原、应激和炎症激活的信号转导途径是许多临床显著病症的发病机制的核心，所述临床显著病症包括癌症、糖尿病、缺血性损伤（如在心脏病发作和中风中发生的）、关节炎、败血性休克、发热以及放疗和化疗的副作用。细胞外信号调节激酶（ERK）、JNK/SAPKs和p38是三条被多种刺激激活的丝裂原活化蛋白激酶（MAPK）信号通路。MAPKs主要负责激活蛋白-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（MAP 3 K）MAPK-激酶（MKK）MAPK核心信号传导模块的各种重叠阵列的远端组分。缺少的是对近端信号如何与MAPK核心通路偶联的理解。这些信息将是非常有益的发展新的抗增殖，抗糖尿病和炎症治疗策略，并在确定潜在的新的药物靶点。我们正在进行的研究已经开始揭示ERK、SAPKs和p38上游的一些MAP 3 Ks的调节机制。我们的初步研究表明，生发中心激酶-1（GCK 1）和肿瘤坏死因子（TNF）受体相关因子（TRAFs）是应激调节的MAPK/细胞外信号调节激酶（ERK）-激酶-激酶-1（MEKK 1）的潜在上游激活剂。GCK 1（而不是TRAF 2）也可以激活JNK/SAPK上游的混合谱系激酶-3（MLK 3）aMAP 3 K，并且可能激活ERK。GCK 1本身似乎部分受泛素（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）的遗传研究表明，MLK 3不仅是JNK/SAPK的有丝分裂原活化所需的，而且是ERK的形成原活化所需的。最后，我们发现，gck 1的破坏损害CD 40激活的JNK/SAPK，并导致B淋巴细胞缺陷的存在下，成熟的表面免疫球蛋白的产生。我们打算在这些发现的基础上加以扩展。因此，我们将使用生物化学方法来确定GCK 1如何激活MLK 3。我们还将使用我们的gck 1基因敲除小鼠和GCK 1 RNAi来确定GCK 1是否是MLK 3体内有丝分裂原激活所必需的。我们将确定是否已知的GCK 1相互作用蛋白，也具有E3 Ub连接酶活性（MEKK 1，TRAF 6）所需的GCK 1泛素化和/或刺激诱导的GCK 1稳定。我们将使用生物化学和RNAi方法来探索MLK 3是否是ERK特异性MEKs的主要调节因子，或者MLK 3是否调节Raf家族的ERK特异性MAP 3 Ks。最后，我们将使用免疫细胞化学方法来确定GCK 1是否是B细胞IG类转换所必需的。

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.