STRUCTURE/FUNCTION OF PIP KINASE IN TNFR1 SIGNALING

描述(改编自《研究人员摘要》)：所有真核细胞 均受磷脂酰肌醇(PI)信号转导通路的调控。在 PI循环，pI被磷酸化成许多pI衍生的秒的前体 信使和直接与调节的多磷肌醇信使 基本的细胞过程。这个实验室在生物化学方面 编码两个新家族的特征和分离的cDNA 磷脂酰肌醇激酶。这些酶合成了两种 磷脂酰肌醇4，5-二磷酸(PI4，5P2)与细胞凋亡抑制 和促有丝分裂的3-聚磷酰肌醇，PI3，4P2和PI3，4，5P3。一种酶， PIPKII-β与p55肿瘤坏死因子(TNF-α)相关 受体(TNFR1)，并由该受体激活。建议进行的研究 将研究这种受体-激酶相互作用的细胞功能 并对这种新的蛋白激酶的结构和功能进行了研究。这项工作将 涉及以下具体目标：(1)调查结构和 PIPKII-β在肌醇磷脂信使生成中的作用研究一下 以表征ATP为目标的保守序列的功能 结合、催化核心、PIP结合部位和要求 低聚物形成的后果。(2)研究TNFR1信号转导途径 通过PIPKII-BETA。确定哪些是由肿瘤坏死因子调节的细胞事件 由PIPKII-beta介导。确定哪些是磷脂酰肌醇信使 体内产生的肿瘤坏死因子-α和PIPKII-β的作用。(3) 确定TNFR1调节PIPKII-β信号的机制。 TNFR1通过与酵母组装一种蛋白质介体复合体发挥作用 双杂交筛选、TNFR1与PIPKII-β的相互作用部位和 PIPKII-β的功能或活性是否由相关的 蛋白质将被测定。(4)与詹姆斯·赫尔利博士合作 和Glenn Prestwich，PIPKII亚型的三维结构，结合 底物(S)将确定。PIPKII-Beta的晶体已经被 长成的，很容易剥离。将使用其他方法来实现增长 可用于高分辨结构的PIPKII-β晶体 用结合的ATP和PI3P和/或PI4P测定。PIP激酶在人类免疫系统中的作用 肿瘤坏死因子-α信号转导和潜在的细胞凋亡调节有许多 信号在一般疾病和增殖性疾病中的意义， 免疫学，特别是癌症。

DESCRIPTION (Adapted from Investigator's Abstract): All eukaryotic cells are regulated by phosphoinositide (PI) signal transduction pathways. In the PI cycle, PI is phosphorylated to precursors of many PI-derived second messengers and directly to polyphosphoinositide messengers that regulate essential cellular processes. This laboratory has biochemically characterized and isolated cDNAs encoding two novel families of phosphoinositide kinases. These enzymes synthesize both phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and the apoptosis inhibiting and mitogenic 3-polyphosphoinositides, PI3,4P2 and PI3,4,5P3. One enzyme, PIPKII-beta, is associated with the P55 tumor necrosis factor (TNF-alpha) receptor (TNFR1) and is activated by this receptor. The proposed studies will investigate the cellular functions of this receptor-kinase interaction and study the structure and function of this novel kinase. This work will involve the following Specific Aims: (1) Investigate the structure and function of PIPKII-beta in phosphoinositide messenger generation. Study the function of conserved sequences with the objective of characterizing ATP binding, the catalytic core, PIP binding sites, and requirements for, and consequences of, oligomer formation. (2) Investigate TNFR1 signaling through PIPKII-beta. Determine which TNF-regulated cellular events are mediated by PIPKII-beta. Determine which phosphoinositide messengers are generated by TNF-alpha stimulation in vivo and PIPKII-beta's role. (3) Determine the mechanism by which TNFR1 modulates PIPKII-beta signaling. TNFR1 functions by assembling a complex of protein mediators; with the yeast two-hybrid screen, the site of TNFR1 interaction with PIPKII-beta and whether the function or activity of PIPKII-beta is mediated by associated proteins will be determined. (4) In collaboration with Drs. James Hurley and Glenn Prestwich, the 3-D structure of PIPKII isoforms with bound substrate(s) will be determined. Crystals of PIPKII-beta have already been grown which defract well. Additional approaches will be used to grow PIPKII-beta crystals which can be used for high resolution structure determination with bound ATP and PI3P and/or PI4P. A role for PIP kinase in TNF-alpha signaling and potentially apoptosis regulation has many implications for signaling in general and proliferative diseases, immunology, and cancer, in specific.