STRUCTURE/FUNCTION OF PIP KINASE IN TNFR1 SIGNALING

描述（改编自研究者摘要）：所有真核细胞 受磷脂酰肌醇（PI）信号转导途径的调节。 在 在PI循环中，PI被磷酸化为许多PI衍生的第二个 信使，并直接与多磷酸肌醇信使， 重要的细胞过程。 这个实验室从生物化学上 特征和分离的cDNA编码两个新的家族， 磷酸肌醇激酶。 这些酶可以合成 磷脂酰肌醇4，5-二磷酸（PI 4，5 P2）与细胞凋亡抑制 以及促有丝分裂的3-聚磷酸肌醇，PI 3，4 P2和PI 3，4，5 P3。 一种酶， PIPKII-β，与P55肿瘤坏死因子（TNF-α）相关 受体（TNFR 1），并被该受体激活。 拟议的研究 将研究这种受体-激酶相互作用的细胞功能 并对这种新型激酶的结构和功能进行了研究。 这项工作将 具体目标如下：（1）研究结构， PIPKII-β在磷酸肌醇信使产生中的功能。 研究 保守序列的功能，目的是表征ATP 结合，催化核心，PIP结合位点，以及 低聚物形成的后果。 (2)研究TNFR 1信号传导 通过PIPKII测试版 确定哪些TNF调节的细胞事件 由PIPKII-β介导。 确定哪些磷酸肌醇信使是 通过TNF-α刺激体内产生和PIPKII-β的作用。 （三） 确定TNFR 1调节PIPKII-β信号传导的机制。 TNFR 1通过组装蛋白质介体复合物发挥作用;酵母 双杂交筛选，TNFR 1与PIPKII-β相互作用的位点， PIPKII-β的功能或活性是否由相关的 蛋白质将被确定。 (4)与James Hurley博士合作 和Glenn Prestwich，PIPKII同种型的3-D结构与结合的 将确定基质。 PIPKII-β的晶体已经被 生长良好， 将使用其他方法来增长 可用于高分辨率结构的PIPKII-β晶体 测定结合ATP和PI 3 P和/或PI 4P。 PIP激酶在 TNF-α信号传导和潜在的凋亡调节具有许多 对一般和增殖性疾病中信号传导的影响， 免疫学和癌症

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.