HORMONAL REGULATION OF CELLULAR METABOLISM

蛋白质的磷酸化-去磷酸化是最重要的 调节细胞功能的重要机制。蛋白 激酶C（PKC）已成为一个关键的调控元件， 调节许多细胞功能。多个PKC亚型已被 然而，通过分子克隆鉴定， 这些酶基本上是未知的。这些酶被发现具有 不同的组织、细胞和亚细胞分布， 在发育过程中差异表达。由于PKC的激活， 导致靶蛋白磷酸化，我们分离出 几种PKC底物从大鼠脑中的分析， 生理功能。其中，78个氨基酸的CNS特异性 钙调素（CaM）结合蛋白，神经颗粒蛋白，已经被表征为 特异性PKC底物。这种蛋白质在没有Ca 2+的情况下结合CaM 在与磷酸化位点相邻的区域，从而成为一个差的 PKC底物。PKC磷酸化神经颗粒蛋白促进其 从CaM解离。神经颗粒素/钙调素复合物在大鼠脑内的形成 在大鼠脑匀浆中也观察到Ca2+的缺乏，表明 神经颗粒蛋白是一种特异的钙离子非依赖性钙调素结合蛋白。一 合成肽对应的磷酸化和CaM- 神经颗粒蛋白的结合域被PKC磷酸化， 而不是其他蛋白激酶。色氨酸的取代 苯丙氨酸使该肽能够在 磷酸化，一种可用于荧光分光光度测定的特征 的PKC。CA 2+非依赖性B族PKC δ和PKC β已经被证实是一种重要的蛋白质。 分别从大鼠脑颗粒和可溶性级分分离。 这些酶不受Ca2+非依赖性基团的污染 蛋白激酶C α，β，和γ。两种形式的PKC β，PS/DAG- 依赖ε 1和独立ε 2，已经被分离出来， 在Mono Q柱上进行柱层析。PKC ε 1和ε 2的转化 通过将前者与Ca 2+和PS/DAG孵育来实现，尽管 PKC β中固有的激酶活性不依赖于Ca 2+。 因此，受体介导的磷酸肌醇水解可以 触发所有PKC的激活并促进PKC的转化 进入效应子非依赖性酶，以持续激活 当刺激信号减弱时。CNS的基因组结构- 分析了特异性PKC γ和神经颗粒蛋白， 定义这些基因的组织特异性和发育调节表达 两种蛋白质蛋白激酶C γ的多种核蛋白结合元件 基因已经通过足迹和凝胶迁移率变动分析鉴定。一 潜在的负调控元件结合蛋白，其在 与PKC γ的表达呈负相关， 胎鼠脑核提取物。的结构特征 neurogranin基因目前正在研究中。

Phosphorylation-dephosphorylation of proteins is one of the most important mechanisms for the regulation of cellular functions. Protein kinase C (PKC) has emerged as a pivotal regulatory element for the regulation of many cellular functions. Multiple PKC subspecies have been identified by molecular cloning, however, the functional role of each of these enzymes is largely unknown. These enzymes were found to have distinct tissue, cellular, and subcellular distributions and were differentially expressed during development. Since the activation of PKC results in the phosphorylation of target proteins, we have isolated several PKC substrates from rat brain for analysis of their physiological functions. Among them, a 78-amino acid CNS-specific calmodulin (CaM)-binding protein, neurogranin, has been characterized as a specific PKC substrate. This proteins binds CaM in the absence of Ca2+ at a region adjacent to the phosphorylation site and thus becomes a poor substrate for PKC. Phosphorylation of neurogranin by Pkc promotes its dissociation from CaM. The formation of neurogranin/CaM complexes in the absence of Ca2+ was also observed in rat brain homogenate, suggesting that neurogranin is a specific Ca2+ -independent CaM-binding protein. A synthetic peptide corresponding to the site of phosphorylation and CaM- binding domain of neurogranin was found to be phosphorylated by PKC but not by other protein kinases. A substitution of tryptophan for phenylalanine enables this peptide to increase fluorescence upon phosphorylation, a characteristic useful for spectrofluorimetric assay of PKC. The CA2+-independent group B PKC delta and epsilon have been isolated from rat brain particulate and soluble fractions, respectively. These enzymes are free from contamination of the Ca2+-independent group A PKC alpha, beta, and gama. Two forms of PKC epsilon, a PS/DAG- dependent epsilon1 and independent epsilon2, have been isolated by chromatography on Mono Q column. Conversion of PKC epsilon1 and epsilon2 was achieved by incubation of the former with Ca2+ and PS/DAG, although the kinase activity inherent in PKC epsilon is independent of Ca2+. Thus, the receptor-mediated hydrolysis of phosphoinositides could trigger the activation of all PKCs and promote the conversion of PKC epsilon into the effector-independent enzyme for sustained activation when the stimulatory signal subsides. The genomic structures of CNS- specific PKC gama and neurogranin were analyzed for the purpose of defining tissue-specific and development-regulated expression of these two proteins. Multiple nuclear protein binding elements in PKC gamma gene have been identified by footprint and gel mobility shift assays. A potential negative regulatory element-binding protein, its expression in inversely related to the expression of PKC gamma, was identified in fetal rat brain nuclear extract. The structural feature of the neurogranin gene is currently under investigation.