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: 3767589
• 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/3767589
• 关键词: Aspergillus; Neurospora; biological signal transduction; calcineurin; calcium binding protein; calmodulin dependent protein kinase; cyclosporines; gene deletion mutation; gene expression; genetic promoter element; immunosuppressive; in situ hybridization; interleukin 2; isozymes; messenger RNA; phosphodiesterases; phosphorylation; polymerase chain reaction; protein sequence; protein structure; regulatory gene

Molecular studies were conducted to define the role of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN), in lymphokine gene activation and its sensitivity to the immunosuppressants, Cyclosporin A and FK-506. Using deletion mutants expressed in bacteria, a minimal domain of approximately 40 residues on the catalytic (A) subunit was shown to be necessary and sufficient for binding the regulatory (B) subunit. Expression of these mutants in human T cells showed that the dimeric A:B complex is needed to activate the interleukin (IL-2 promoter and to confer immunosuppressant resistance. Remarkably, transfection of N. crassa A subunit produces the same effects seen with mammalian CN-A, i.e., activation of multiple transcriptional elements (NF-AT, NF-KB, OAP) and formation of drug-dependent complexes with endogenous mammalian immunophilins. Thus, the fungal and mammalian enzymes have conserved both a distinctive catalytic specificity and immunophilin recognition; this may imply a biological need for regulation by immunophilins, perhaps via naturally occurring ligands. The pleiotropic effect of CN on trans-acting factors affecting IL-2 induction suggests that their activation is indirect, probably occurring at a site upstream of these components. In situ hybridization and immunolocalization studies in mouse brain showed a restricted pattern of expression for the CaM-dependent phosphodiesterase (PDE) isoform, PDE1B1, consistent with an important role in neurons containing D1 dopaminergic receptors. In contrast to the PDE1A2 isoform, it appears the PDE1B1 does not co-localize with CaM-sensitive adenylate cyclase, suggesting that the two enzymes participate in different temporal patterns of cyclic AMP degradation in response to stimuli. A potential homolog of phosphatidyl-inositol specific phospholipase C enzymes described in bacteria and T. brucei. Using PCR-based methods, CAPP-36 CDNAS were obtained from bovine, murine and human sources, and expression studies indicate that MRNA for this isoform is enriched in the cerebellum. Future studies will examine the biological activity of this protein after its overexpression in mammalian cells.

进行分子研究以确定钙调蛋白的作用 淋巴因子基因中的 (CaM) 依赖性蛋白磷酸酶、钙调神经磷酸酶 (CN) 激活及其对免疫抑制剂环孢菌素 A 的敏感性 和FK-506。 使用细菌中表达的缺失突变体，最小 显示了催化 (A) 亚基上大约 40 个残基的结构域 对于结合调节（B）亚基是必要且充分的。 这些突变体在人类 T 细胞中的表达表明，二聚体 A:B 需要复合物来激活白细胞介素（IL-2启动子）并赋予 免疫抑制剂耐药性。 值得注意的是，粗糙猪笼草 A 的转染 亚基产生与哺乳动物 CN-A 相同的效果，即 多个转录元件（NF-AT、NF-KB、OAP）的激活和 与内源性哺乳动物形成药物依赖性复合物 亲免素。 因此，真菌和哺乳动物的酶都保守 独特的催化特异性和亲免素识别；这可能 暗示生物需要通过亲免素进行调节，也许通过 天然存在的配体。 CN 对反式作用的多效性 影响 IL-2 诱导的因素表明它们的激活是 间接的，可能发生在这些组件的上游站点。 在 小鼠大脑的原位杂交和免疫定位研究表明 CaM 依赖性磷酸二酯酶的限制表达模式 (PDE) 同种型，PDE1B1，与神经元中的重要作用一致 含有D1多巴胺能受体。 与 PDE1A2 同工型相比， 看来 PDE1B1 不与 CaM 敏感的腺苷酸共定位 环化酶，表明这两种酶参与不同的时间 响应刺激的环 AMP 降解模式。 有潜力 磷脂酰肌醇特异性磷脂酶 C 酶的同系物 描述于细菌和 T. brucei 中。 使用基于 PCR 的方法，CAPP-36 CDNAS 是从牛、鼠和人类来源获得的，并且表达 研究表明，该异构体的 mRNA 在小脑中富集。 未来的研究将检查这种蛋白质的生物活性 它在哺乳动物细胞中过度表达。