HORMONAL REGULATION OF CELLULAR METABOLISM

蛋白质通过磷酸化和氧化进行的共价修饰有 调节多种细胞功能的重要机制。 蛋白激酶C(PKC)的蛋白磷酸化与 对过多的细胞事件的调控。其中一个生理学的 PKC的靶标，神经颗粒素(Ng)，已经被证明也被 一氧化氮和其他氧化剂。NG是一种PKC选择性底物，它 在低水平的钙调素存在下与钙调素(CaM)高亲和力结合 钙。Ng的磷酸化和氧化都减弱了它的结合 对CaM的亲和力，从而使CaM对其他依赖CaM的酶产生游离性。 转基因Ng细胞对Ng氧化作用的研究 COS细胞和大鼠脑片。脑片中的NG含量更高 对NO供体和过氧化氢氧化的敏感性比 在COS细胞中的表达与来自两个来源的Ng相同 易被氯化汞氧化的。脑组织中Ng的氧化 切片产生的主要是分子内二硫化物形式的Ng和它 可被二硫苏糖醇、抗坏血酸和 还原型谷胱甘肽。在体外，Ng也可以被 谷胱甘肽，高达4摩尔/摩尔的谷胱甘肽被引入到Ng中。 谷胱甘肽Ng以与还原形式类似的方式与CaM结合， 然而，与还原形式相比，它是较差的PKC底物。入内 另一种PKC底物的氧化和磷酸化的活体状态， 神经调制素/GAP-43(Nm)的电喷雾质谱研究 光谱分析。NM主要(80%)以磷酸化形式存在，向上 至4摩尔磷酸盐/摩尔Nm。缺氧或缺血可促进 在不改变Nm氧化状态的情况下使Nm去磷酸化 两个半胱氨酸残基。一种新型的28 kDa PKC/CK2底物 HASPP28基因已被鉴定，并克隆了其基因。这种蛋白质是一种 N1E115神经母细胞瘤细胞中CK2的底物。其影响范围 磷酸化和蛋白质水平受细胞周期调节。HeLa细胞 在细胞周期的有丝分裂阶段停滞显示一条额外的条带 通过免疫印迹检测；这种略高的分子的出现 重量蛋白条带暗示额外的磷酸化状态 HASPP28。包含90%以上编码区的基因组克隆和1.2 Kb位于转录起始点的上游，不受 内含子序列，对其进行了表征。启动子活性为 用融合到荧光素酶报告基因的嵌套删除结构定义 将DBU-cAMP和PMA分别导入293细胞，可使细胞增殖倍数增加2-4倍 而EGF则不起作用。相比之下，当这些记者 将构建的基因导入COS细胞，所有这些效应物 引起相同程度的刺激，表明特定类型的细胞 HASPP28基因表达对表皮生长因子的响应近端启动子，最高可达 -251，包含p50 NFkappaB和p37 TATA盒结合的结合位点 蛋白。HASPP28相关蛋白正在由 酵母双杂交系统。

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 plethora of cellular events. One of the physiological targets of PKC, neurogranin (Ng), has been shown to be modified also by nitric oxide and other oxidants. Ng is a PKC-selective substrate which binds calmodulin (CaM) with high affinity in the presence of low level of calcium. Both phosphorylation and oxidation of Ng attenuate its binding affinity for CaM and thus free CaM for other CaM-dependent enzymes. Oxidation of Ng in intact cells was investigated with Ng cDNA-transfected COS cells and with rat brain slices. Ng in the brain slices was more sensitive to oxidation by NO donors and hydrogen peroxide than that expressed in the COS cells and Ng from both sources were equally susceptible to oxidation by mercuric chloride. Oxidation of Ng in brain slices generates predominantly intramolecular disulfide form of Ng and it can be reduced by reductants such as dithiothreitol, ascorbic acid, and reduced glutathione. In vitro, Ng can also be modified by glutathionation, up to 4 mol/mol of glutathione are incorporated into Ng. The glutathionated Ng binds CaM in a similar manner as the reduced form, however, it is a poorer substrate for PKC than the reduced form. The in vivo states of oxidation and phosphorylation of another PKC substrate, neuromodulin/GAP-43 (Nm), was investigated by electrospray mass spectrometry. Nm was found largely (>80%) in the phosphorylated form, up to 4 mol phosphate/mol of Nm. Hypoxia or ischemia promotes the dephosphorylation of Nm without changing the state of its oxidation at the two cysteine residues. A novel 28 kDa PKC/CK2 substrate, named HASPP28, has been identified and its cDNA cloned. This protein is a substrate of CK2 in N1E115 neuroblastoma cells. The extent of phosphorylation and protein level are cell cycle-regulated. HeLa cells arrested at the mitotic stage of cell cycle displayed an additional band detected by immunoblot; the appearance of this slightly higher molecular weight protein band implying an additional phosphorylation state of HASPP28. Genomic clones containing over 90% of the coding region and 1.2 kb upstream of the transcription start site, without interruption by intron sequence, has been characterized. The promoter activity was defined with nested deletion constructs fused to luciferase reporter gene; when transfected into 293 cells, dBu-cAMP and PMA caused 2-4-fold stimulation whereas EGF had no effect. In contrast, when these reporter gene constructs were transfected into COS cells, all these effectors caused the same degree of stimulation, suggesting a cell type-specific response of HASPP28 gene expression to EGF. The proximal promoter, up to -251, contains binding sites for p50 NFkappaB and p37 TATA box-binding protein. The HASPP28-associated protein is being investigated by the yeast two-hybrid system.