PROTEIN KINASE II AND PROTEIN PHOSPHATASE IN CALCIUM STIMULATED GENE EXPRESSION

该项目采用多方面的方法评估以下方面的作用： 钙/钙调蛋白依赖性蛋白激酶II（CaM-激酶II）和蛋白质 磷酸酶在调节Ca ~（2+）刺激的基因转录中的作用 PC 12细胞。 我们将集中在几个基因，利用增强子 蛋白质CREB和/或CREB相关元件，如在5 ′-侧翼 c-fos、努尔/77（NGF 1-B）和酪氨酸羟化酶基因的序列。 这三个内源基因的Ca 2+依赖性转录将被抑制。 由三种“Ca 2+激动剂”引发：去极化（KCl加BAY K8644）， 离子霉素或毒胡萝卜素。 最初的实验将决定 对CaM-激酶II（KN-62）的细胞渗透性抑制剂的mRNA水平的影响，或 蛋白磷酸酶（冈田酸）或转染与显性 CREB阴性突变体。 PC 12细胞也将用CRE-或 CaRE-CAT构建体，以及用于评估CAT表达的相同范例。 细胞也将用CaM的组成型活性突变体脂质转染。 激酶II，其应提供Ca 2+非依赖性基因表达。 合成 将使用几种蛋白激酶和磷酸酶的肽抑制剂 作为探针，通过显微注射到用CRE-1稳定转染的PC 12细胞中， 或CaRE-lacZ报告基因，和β-半乳糖苷酶表达， 细胞对Ca 2 +-激动剂的反应。" 该项目的第二个主要重点是研究CREB的磷酸化 和缺乏88-102位残基的delta-CREB。 特别 关注Ser（133）和Ser（96）的磷酸化（缺乏 在delta-CREB中），CaM-激酶II的共有位点。 除了动能 这些底物对CaM-激酶II的特性（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 CREB和delta-CREB，在Ser（133）上硫代磷酸化，和/或 将Ser（98）显微注射到CaRE-lacZ的细胞质或细胞核中 转染PC 12细胞，并测定β-半乳糖苷酶表达。 最后，将畸胎瘤F9细胞用CREB或δ- CREB和组成型活性CaM激酶II突变体测定CAT 表情 这些互补的方法，被认为是必要的，因为“串扰” 多功能蛋白激酶与细胞内的简并性之间的关系 转录调控基因的增强子结合元件，应 使我们能够剖析钙调蛋白激酶II，蛋白磷酸酶， 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.