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。 调节生长、分化、分泌、细胞代谢， 记忆和感觉传导。多个PKC亚型已被 通过分子克隆鉴定， 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。的职能作用 这些激酶在控制细胞分化中的作用用 N1 E115神经母细胞瘤细胞。PKC激活佛波醇酯抵消了 cAMP通过选择性移位诱导这些细胞的轴突生长 和PKC α和δ的下调，而PKC β和zeta是 没有受到太大影响。PMA刺激几种蛋白质的磷酸化 包括95 kDa延伸因子2，其磷酸化减弱 的cAMP。阐明PKC调节的事件也强调 基板的特性。一种新的28 kDa蛋白激酶C/酪蛋白激酶 II底物已被鉴定并克隆了其cDNA。这种蛋白质是 N1 E115神经母细胞瘤细胞中酪蛋白激酶II的底物。程度 磷酸化和蛋白质水平是细胞周期调节的。另一 p14.4是一种潜在的PKC底物，已被鉴定并克隆了其cDNA。 该蛋白也可以被p34 cdc 2激酶磷酸化。神经颗粒 (Ng)是钙调蛋白（CaM）结合PKC底物。 其磷酸化 通过PKC降低了对CaM的亲和力，从而使CaM释放为CaM。 依赖酶此外，Ng在空气中或通过过氧化物或 一氧化氮导致分子内二硫键的形成， 降低了这种蛋白对钙调素的亲和力。定点诱变 Ng中的四个Cys残基导致鉴定了一对 参与二硫键形成的半胱氨酸残基。Ng是一个优秀的 一氧化氮受体; Ng巯基的亚硝化速率 基团和随后的二硫键形成甚至比一个更快， 最著名的还原剂DTT。通过氧化对Ng进行改性， 是一氧化氮在中枢神经系统中的重要信号机制。的 分析CNS特异性PKC γ和Ng的基因组结构， 定义组织特异性和发育调控的目的 这两个基因的表达。Ng基因的启动子活性可以 通过佛波酯或通过与PKC cDNA共转染来刺激。一 与PKC γ和Ng的AT富集区结合的20 kDa蛋白质 基因启动子已被鉴定;这种蛋白质的磷酸化， PKC减弱其与含有ATTA、ATAA和AATA的DNA元件的结合 图案

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 growth, differentiation, secretion, cellular metabolism, memory, and sensory transduction. Multiple PKC subspecies have been identified by molecular cloning and the various isozymes have been shown to have distinct tissue, cellular, and subcellular distributions and are differentially expressed during development. The functional roles of these kinases in the control of cellular differentiation were tested with N1E115 neuroblastoma cells. PKC-activating phorbol ester counteracts the cAMP-induced neurite outgrowth of these cells by selective translocation and downregulation of PKC alpha and delta, whereas PKC beta and zeta are not greatly affected. PMA stimulates phosphorylation of several proteins including a 95 kDa elongation factor 2, its phosphorylation is attenuated by cAMP. Elucidation of the PKC-regulated events also places emphasis in the characterization of the substrate. A novel 28 kDa PKC/casein kinase II substrate has been identified and its cDNA cloned. This protein is a substrate of casein kinase II in N1E115 neuroblastoma cells. The extent of phosphorylation and protein level are cell cycle-regulated. Another potential PKC substrate, p14.4, has been identified and its cDNA cloned. This protein can also be phosphorylated by p34cdc2 kinase. Neurogranin (Ng) is a calmodulin (CaM)-binding PKC substrate. Its phosphorylation by PKC reduces the affinity for CaM and thus frees CaM for the CaM- dependent enzymes. In addition, oxidation of Ng in air or by peroxide or nitric oxide results in intramolecular disulfide bond formation and reduces the affinity of this protein for CaM. Site-directed mutagenesis of the four Cys residues in Ng has lead to identification of a pair of Cys residues involved in disulfide bond formation. Ng is an excellent acceptor for nitric oxide; the rate of nitrosation of Ng sulfhydryl groups and subsequent disulfide bond formation is even faster than one of the best known reductants, DTT. Modification of Ng by oxidation could be an important signaling mechanism of nitric oxide in the CNS. The genomic structures of the CNS-specific PKC gama and Ng were analyzed for the purpose of defining tissue-specific and development-regulated expression of these two genes. The promoter activity of the Ng gene can be stimulated by phorbol ester or by co-transfection with PKC cDNAs. A 20 kDa protein that binds to the AT-rich regions of the PKC gamma and Ng gene promoters has been identified; phosphorylation of this protein by PKC attenuates its binding to DNA elements containing ATTA, ATAA and AATA motifs.