CONTROL OF CALCIUM AND PHOSPHORYLATION-REGULATED SIGNALLING PATHWAYS

进行分子研究以确定钙调素的作用 淋巴因子基因中钙调素依赖性蛋白磷酸酶，钙调神经磷酸酶（CN） 活化及其对免疫抑制剂环孢素A的敏感性 FK-506 使用在细菌中表达的缺失突变体， 催化（A）亚基上约40个残基的结构域 是结合调节（B）亚基所必需和充分的。 这些突变体在人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 免疫抑制剂抗性。 值得注意的是，转染N.克拉萨A 亚基产生与哺乳动物CN-A相同的作用，即， 激活多个转录元件（NF-AT、NF-KB、OAP），以及 与内源性哺乳动物形成药物依赖性复合物 免疫亲和素 因此，真菌和哺乳动物的酶都具有保守性。 独特的催化特异性和亲免素识别;这可能 这意味着生物学上需要由亲免素调节，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。 在 原位杂交和免疫定位研究表明， 钙调素依赖性磷酸二酯酶的限制性表达模式 (PDE)PDE 1B 1亚型，与神经元中的重要作用一致 含有D1多巴胺能受体。 与PDE 1A 2同种型相反， PDE 1B 1似乎不与钙调素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 环AMP降解的模式响应于刺激。 一个潜在 磷脂酰肌醇特异性磷脂酶C酶同系物 描述于细菌和T.布鲁塞。 使用基于PCR的方法，CAPP-36 从牛、鼠和人来源获得CDNAS，并且表达CDNAS。 研究表明这种亚型的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后， 它在哺乳动物细胞中的过度表达。

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.