PROTEIN KINASE II AND PROTEIN PHOSPHATASE IN CALCIUM STIMULATED GENE EXPRESSION

该项目采用多方面的方法来评估 钙/钙调蛋白依赖性蛋白激酶II(CaM-Kinase II)与蛋白质 磷酸酶在钙离子刺激的基因转录调控中的作用 PC12细胞。我们将重点关注利用增强子的几个基因 在5‘-侧翼发现的蛋白CREB和/或Cre相关元件 C-fos、Nur/77(NGF1-B)和酪氨酸羟化酶基因序列。 依赖于钙离子的三个内源基因的转录将 由三种“钙激动剂”启动：去极化(Kc1+Bay K8644)， 离子霉素或他西格金。初步实验将确定其效果 细胞通透性CaM-Kinase II抑制物(KN-62)mRNA水平的研究 蛋白磷酸酶(冈田酸)或显性转染法 CREB阴性突变体。PC12细胞也将被Cre-or脂感染 CARE-CAT结构，以及用于评估CAT表达的相同范式。 细胞也将被具有构成活性的CaM-突变体脂染。 激酶II，它应该提供钙离子不依赖的基因表达。合成的 将使用几种蛋白激酶和磷酸酶的多肽抑制剂 作为探针通过显微注射到稳定转染Cre的PC12细胞中- 或CARE-LacZ报告基因，以及β-半乳糖苷酶在单个 确定细胞对“钙激动剂”的反应。 该项目的第二个主要重点将研究CREB的磷酸化。 和缺乏88-102残基的Delta-CREB，通过CaM-Kinase II。 注意Ser(133)和Ser(96)(缺少)的磷酸化 在Delta-CREB中)，这是CaM-Kinase II的一个共识位点。除了动力学 这些底物的表征(Km和Vmax)， (32)p-标记的CREB将作为几种蛋白质的底物进行测试 磷酸酶。硫代磷酸化的CREB和增量-CREB在Ser(133)和/或 Ser(98)，将被显微注射到CARE-LacZ的细胞质或核中 转染PC12细胞，并测定β-半乳糖苷酶的表达。 最后，将CREB或Delta-1基因导入畸胎癌F9细胞。 CREB和结构活性CaM-Kinase II突变体检测CAT 表情。 这些相辅相成的方法，被认为是必要的，因为“相声” 多功能蛋白激酶与细胞退行性变之间的关系 转录调控基因的增强子结合元件，应该 使我们能够剖析CaM-Kinase II、蛋白磷酸酶和 CREB在钙刺激基因转录中的作用。

This project employs a multifaceted approach to assess the roles of calcium/calmodulin-dependent protein kinase II (CaM-kinase II) and protein phosphatases in the regulation of Ca2+-stimulated gene transcription in PC12 cells. We will focus on several genes which utilize the enhancer protein CREB and/or CRE-related elements as found in the 5'-flanking sequences of c-fos, nur/77 (NGF1-B), and tyrosine hydroxylase genes. Ca2+-dependent transcription of the three endogenous genes will be initiated by three "Ca2+ agonists": depolarization (KC1 plus BAY K8644), ionomycin or thapsigargin. Initial experiments will determine the effect on MRNA levels of cell-permeable inhibitors of CaM-kinase II (KN-62) or protein phosphatases (okadaic acid) or transfection with a dominant negative mutant of CREB. PC12 cells will also be lipofected with CRE- or CaRE-CAT constructs, and the same paradigms used to assess CAT expression. Cells will also be lipofected with a constitutively-active mutant of CaM- kinase II which should give Ca2+-independent gene expression. Synthetic peptide inhibitors of several protein kinases and phosphatases will be used as probes by microinjection into PC12 cell stably-transfected with a CRE- or CaRE-lacZ reporter gene, and beta-galactosidase expression in single cells determined in response to the "Ca2+-agonists." The second major focus of the project will examine phosphorylation of CREB and delta-CREB, which lacks residues 88-102, by CaM-kinase II. Particular attention will be given to phosphorylation of Ser(133) and Ser(96) (lacking in delta-CREB), a consensus site for CaM-kinase II. In addition to kinetic characterization (Km and Vmax) of these substrates for CaM-kinase II, the (32)p-labeled CREB will be tested as substrate for several protein phosphatases. CREB and delta-CREB, thiophosphorylated on Ser (133) and/or Ser(98), will be microinjected into the cytoplasm or nucleus of CaRE-lacZ transfected PC12 cells, and beta-galactosidase expression determined. Lastly, teratocarcinoma F9 cells will be transfected with CREB or delta- CREB and the constitutively-active CaM-kinase II mutant to determine CAT expression. These complementary approaches, deemed necessary because of the "crosstalk" between the multifunctional protein kinases and the degeneracy in the enhancer binding elements of transcriptionally-regulated genes, should allow us to dissect the roles of CaM-kinase II, protein phosphatases, and CREB in calcium-stimulated gene transcription.