CONTROL OF CALCIUM AND PHOSPHORYLATION-REGULATED SIGNALLING PATHWAYS

钙和磷酸化调节信号通路的控制

• 批准号: 3789555
• 负责人: R L KINCAID
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3789555
• 关键词: Aspergillus; Neurospora; T lymphocyte; biochemical evolution; biological signal transduction; brain; calcineurin; calcium binding protein; calmodulin; cerebellar Purkinje cell; corpus striatum; cyclic AMP; cyclosporines; developmental genetics; dopamine; enzyme mechanism; gene expression; genetic mapping; genetic promoter element; genetic transcription; immunosuppressive; in situ hybridization; interleukin 2; isozymes; kidney; messenger RNA; molecular cloning; northern blottings; phorbols; phosphodiesterases; phosphorylation; polymerase chain reaction; protein structure; recombinant DNA; regulatory gene; sperm; spleen; transfection; western blottings

Molecular studies of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN), have identified 3 mammalian genes for the catalytic (A) subunit and determined their chromosomal localization in humans. Characterization of A subunit genes in many phyla indicate that mammalian forms arose from an ancestral line that includes the filamentous fungi (Neurospora, Aspergillus) and suggests that gene divergence occurred after the avian/reptilian branch point. Two genes for the regulatory (B) subunit were cloned, one of which is co-expressed with an isoform of the A subunit during testicular development; these two subunits may comprise an isoenzyme that is associated with the sperm flagellum. Biochemical studies with recombinant proteins, expressed in bacteria and in insect cells, show that the B subunit isoforms can associate interchangeably with different A subunits and with a fungal catalytic subunit. These data indicate functional conservation of the B subunit interaction domain, which has now been delineated in mapping studies. An important biological role for CN was established in T-cell regulation. In human cells transfected with a constitutively-active mutant of the A subunit, the interleukin (IL)-2 promoter was activated synergistically by phorbol esters; this suggests a role for CN in T-cell receptor-mediated signaling events that regulate IL- 2 gene transcription. These cells also were resistant to the immunosuppressants, FK-506 and cyclosporin A, in agreement with recent findings that CN forms complexes with the cytosolic drug receptors ("immunophilins"); thus, CN is the primary target of these drugs in T- cells. A CaM-regulated phosphodiesterase (PDE) was cloned from murine brain by PCR amplification of mRNA from microdissected cerebellar Purkinje cell layers. In situ hybridization and Northern blot analysis indicate high expression of PDE mRNA in the striatum, suggesting a role in control of dopamine-mediated cyclic AMP pathways. Peptide microsequencing was completed on a novel 36 kDa CaM-binding protein isolated from brian, showing no homology to any mammlaina proteins. Western blot analysis with anti-peptide antibodies showed highest amounts in brain, spleen and kidney.

钙调蛋白 (CaM) 依赖性蛋白磷酸酶的分子研究， 钙调神经磷酸酶 (CN)，已鉴定出 3 个哺乳动物基因用于催化 (A) 亚基并确定了它们在人类中的染色体定位。 许多门中 A 亚基基因的特征表明，哺乳动物 形式起源于包括丝状真菌在内的祖先谱系 （脉孢菌属、曲霉属）并表明基因分歧发生在 鸟类/爬行动物分支点。调节 (B) 亚基的两个基因 被克隆，其中一个与 A 亚基的亚型共表达 睾丸发育期间；这两个亚基可以包含 与精子鞭毛相关的同工酶。生化研究 与在细菌和昆虫细胞中表达的重组蛋白一起显示 B 亚基亚型可以与不同的亚基互换 一个亚基并带有一个真菌催化亚基。这些数据表明 B 亚基相互作用域的功能保守性，现已 已在绘图研究中描绘出来。 CN 的重要生物学作用 建立了 T 细胞调节。在转染了 A 亚基的组成型活性突变体，白细胞介素 (IL)-2 启动子被佛波酯协同激活；这表明 CN 在调节 IL-的 T 细胞受体介导的信号传导事件中的作用 2.基因转录。这些细胞也对 免疫抑制剂、FK-506 和环孢菌素 A，与最近的研究结果一致 CN 与胞质药物受体形成复合物的发现 （“亲免素”）；因此，CN是这些药物在T-中的主要靶点 细胞。从鼠科动物中克隆了 CaM 调节的磷酸二酯酶 (PDE) 通过 PCR 扩增来自显微解剖小脑浦肯野病毒的 mRNA 细胞层。原位杂交和 Northern blot 分析表明 PDE mRNA 在纹状体中高表达，表明其在控制中的作用 多巴胺介导的环 AMP 通路。肽微测序是 完成了从布莱恩中分离出的新型 36 kDa CaM 结合蛋白， 与任何哺乳动物蛋白没有同源性。蛋白质印迹分析 抗肽抗体在脑、脾和 肾。