HORMONAL REGULATION OF CELLULAR METABOLISM

通过磷酸化和氧化对蛋白质进行共价修饰， 调节许多细胞功能的重要机制。 蛋白激酶C（PKC）引起的蛋白磷酸化与细胞凋亡有关。 调节过多的细胞活动。 的生理靶标 PKC，神经颗粒蛋白（Ng），也被一氧化氮和其他氧化剂修饰。 Ng在低水平存在下以高亲和力结合钙调蛋白（CaM） 钙。 大鼠、牛、鸡和人Ng具有相同的PKC 磷酸化位点/CaM结合结构域。 PKC磷酸化降低 它们对CaM的结合亲和力，因此有利于激活CaM依赖性 内切酶 大鼠脑Mg含有4个半胱氨酸，分别位于3，4，9位， 和51. 定点突变显示Cys 9和Cys 51是必需的 对于氧化剂介导的分子内二硫键桥接， Ng的CaM结合亲和力。 牛Ng和人类变体（Ng-2）缺乏 Cy 351和鸡Ng和另一种人变体（Ng-1），缺乏Cys 9和Cys 10。 Cys51 基于这些结构变化，我们预测氧化剂- 与PKC不同，Ng功能的介导调节在 物种 一种新的28 kDa PKC/CK 2底物，称为HASPP 28，已被发现。 鉴定并克隆了其cDNA。 这种蛋白质是CK 2的底物， NIE 115神经母细胞瘤细胞，其磷酸化和蛋白水平， 细胞周期调节。 基因组克隆含有超过90%的 编码区和转录起始位点上游1.2kb， 没有内含子序列，已经被表征。 启动子活性 用与荧光素酶报告基因融合的巢式缺失构建体定义 基因; RGF，dBu-cAMP和PMA引起2-3倍的刺激，而PDGF， bFGF和胰岛素没有影响。 免疫沉淀的HASPP 28含有 磷酸化内源性HASPP 28或外源性HASPP 28的CK 2样激酶 重组蛋白 HASPP 28相关蛋白正在研究中 酵母双杂交系统。 各种PKC的功能作用 在NIE 115中分析了控制细胞分化的同工酶 神经母细胞瘤细胞 PKC激活佛波酯抵消cAMP- 通过选择性易位诱导这些细胞的轴突生长（NOG 和PKC α或δ的下调， 相应的PKC对NOG的dBu-alphaAMP更敏感。 这两 反义PKC稳定转染子对PMA的反应也较低， 抵消cAMP诱导的NOG，这表明PKC α和δ是 参与监管。

Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of numerous cellular functions. Protein phosphorylation by protein kinase C (PKC) has been linked to the regulation of a plethora of cellular events. A physiological target of PKC, neurogranin (Ng), is also modified by nitric oxide and other oxidants. Ng binds calmodulin (CaM) with high affinity in the presence of low levels of calcium. Rat bovine, chicken, and human Ng have identical PKC phosphorylation site/CaM-binding domains. Phosphorylation by PKC reduces their binding affinity for CaM and thus favors activation of CaM-dependent enzymes. Rat brain Mg contains four cysteines located at positions 3,4,9, and 51. Site-directed mutagenesis showed Cys9 and Cys51 to be essential for the oxidant-mediated intramolecular disulfide bridging that attenuates the CaM-binding affinity of Ng. Bovine Ng and a human variant (Ng-2) lack Cy351, and chicken Ng and another human variant (Ng-1), lack both Cys9 and Cys51. Based on these structural variations we predict that the oxidant- mediated regulation of Ng function, unlike that by PKC, is variable among species. A novel 28 kDa PKC/CK2 substrate, termed HASPP28, has been identified and its cDNA cloned. This protein is a substrate of CK2 in NIE115 neuroblastoma cells, and its phosphorylation and protein level are cell cycle-regulated. Genomic clones containing more than 90 percent of the coding region and 1.2 kb upstream of the transcription start site, without intron sequences, have been characterized. Promoter activity was defined with nested deletion constructs fused to a luciferase reporter gene; RGF, dBu-cAMP, and PMA caused 2-3 fold stimulation whereas PDGF, bFGF, and insulin had no effect. Immunoprecipitated HASPP28 contains a CK2-like kinase that phosphorylates endogenous HASPP28 or exogenous recombinant protein. The HASPP28-associated protein is being investigated by yeast two-hybrid system. The functional roles of the various PKC isozymes in the control of cellular differentiation were analyzed in NIE115 neuroblastoma cells. PKC-activating phorbol ester counteracts the cAMP- induced neurite outgrowth (NOG) of these cells by selective translocation and downregulation of PKC alpha or delta, with reduced levels of the respective PKCs, were more sensitive to dBu-alphaAMP for NOG. These two antisense PKC stable transfectants were also less responsive to PMA in counteracting cAMP-induced NOG, suggesting that PKC alpha and delta are involved in regulating NOG.