Stronach Beth, Lennox Ashley L, Garlena Rebecca A
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219.
Genetics. 2014 Jun;197(2):497-513. doi: 10.1534/genetics.113.160937. Epub 2014 Jan 15.
A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability.
在丝裂原和应激激活的蛋白激酶（MAPK/SAPK）信号通路中，一组高度多样化的蛋白激酶起着早期应答者的作用。例如，人类拥有14种MAPK激酶激酶（MAP3K），它们在下游激活Jun激酶（JNK）信号。一个主要挑战是解读这些上游MAP3K的选择性和冗余功能。利用黑腹果蝇作为模型系统的相对简单性，我们评估了发育和应激反应过程中几种JNK依赖性过程中的MAP3K信号特异性。我们的方法是在两个MAP3K家族成员之间生成分子嵌合体，即混合谱系激酶Slpr和TGF-β激活激酶Tak1，它们在激酶结构域中共有32%的氨基酸同一性，但在序列和结构域结构上有所不同，然后测试各个结构域对蛋白质定位、突变体互补和信号激活的贡献。我们发现野生型激酶的过表达在不同背景下刺激JNK信号，因此细胞能够对两种MAP3K都作出反应，但结果不同。相对于野生型，催化结构域交换的补偿作用微弱或根本没有补偿作用，尽管它们有共同的底物JNK激酶Hep。含有Tak1 C末端结构域的构建体在Tak1信号背景下具有抑制作用，包括肿瘤坏死因子依赖性细胞死亡和先天免疫信号；然而，抑制抗菌基因表达不一定会导致对感染的表型易感性。这些相同的构建体在Slpr依赖性发育信号背景下是中性的，反映了不同的亚细胞蛋白质定位，并由此推断出激活点。总之，我们的研究结果表明，特定MAP3K的选择性部署部分可归因于其固有的序列差异、细胞定位和结合伴侣的可用性。
