REGULATION OF THE CAMP SPECIFIC PHOSPHODIESTERASES

营地特定磷酸二酯酶的调节

基本信息

批准号：
2729097
负责人：
GRAEME B BOLGER
金额：
$ 22.56万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2003-12-31
项目状态：
已结题

项目摘要

The cAMP-specific cyclic nucleotide phosphodiesterases (PDE4 enzymes) are a diverse family of enzymes that control degradation of the second messenger cAMP and thus are important regulators of intracellular signaling. They are also the specific targets for an important class of drugs with therapeutic potential in humans, especially in disorders of the central nervous system and of the immune/inflammatory systems, such as asthma. Long-Term Goals: To determine which cellular signaling pathways regulate the PDE4 enzymes, as follows: 1. To characterize the regulation of the PDE4 enzymes by novel protein-protein interactions. 2. To characterize the regulation of the PDE4 enzymes by novel phosphorylation events. Specific Aims: 1. To characterize the interactions between the human cAMP-specific PDE4D5 isoform and the RACK1 anchoring protein. 2. To characterize the regulation of the cAMP-specific PDE4A5 isoform by the AIP/ARA9 protein, a newly identified immunophilin. 3. To characterize the phosphorylation of the cAMP-specific PDE4A5 isoform by three different kinases: cAMP- dependent protein kinase (PKA), MAPK and p70rsk. 4. To isolate novel cDNAs that encode cAMP-specific PDEs, derived from transcripts in the mammalian brain and olfactory system. 5. To isolate cDNAs enoding novel proteins that interact with additional human cAMP-specific PDE isoforms. Research Plan and Methods: 1. The ability of RACK1 and PDE4D5 to interact in mammalian cells will be tested by co- immunoprecipitation. To determine the site(s) on each protein responsible for their interaction, mutations in RACK1 or PDE4D5 will be tested for interaction using the two-hybrid system and by expression in mammalian cells. The effect of these mutations on the targeting of PDE4D5 to membranes will also be tested. 2. To test for an interaction between PDE4A5 and ARA9/AIP, they will be co- immunoprecipitated from cells. Mutant forms of the PDE4A5 isoform and the ARA9/AIP protein will also be tested for their interaction in cells. The interaction between bacterially-expressed proteins will also be studied. Interactions between ARA9/AIP and p70rsk and other kinases will be tested by co-immunoprecipitation. 3. Site-directed mutagenesis will be used to create mutations at putative MAPK, p70rsk and PKA sites in the PDE4A5 protein. These will be expressed in mammalian cells and assayed for changes in enzymatic activity. The phosphorylation status of PDE4A5 isolated from cells will also be studied. 4. cDNA libraries from brain and olfactory tissue will be screened for new PDE4 isoforms, and their tissue distribution and enzymatic properties will be determined. 5. The two-hybrid system will be used to clone cDNAs encoding proteins that interact with 4 novel PDE4 isoforms.

cAMP特异性环状核苷酸磷酸二酯酶（PDE4酶）是一个多样的酶家族，控制第二质阵营的降解，因此是细胞内信号传导的重要调节剂。它们也是人类具有治疗潜力的重要药物的特定靶标，尤其是中枢神经系统和免疫/炎症系统（例如哮喘）的疾病。长期目标：确定哪种细胞信号通路调节PDE4酶，如下所示：1。通过新型蛋白质 - 蛋白质相互作用来表征PDE4酶的调节。 2。通过新型的磷酸化事件来表征PDE4酶的调节。具体目的：1。表征人类营地特异性PDE4D5同工型与RACK1锚定蛋白之间的相互作用。 2。表征通过AIP/ARA9蛋白（一种新鉴定出的免疫蛋白）对CAMP特异性PDE4A5同工型的调节。 3。表征三种不同激酶的cAMP特异性PDE4A5同工型的磷酸化：cAMP依赖性蛋白激酶（PKA），MAPK和P70RSK。 4。隔离编码cAMP特异性PDE的新型cDNA，这些ccam是从哺乳动物大脑和嗅觉系统中的转录本衍生而来的。 5。分离cDNA构造新型蛋白质，这些蛋白质与其他人类营地特异性PDE同工型相互作用。研究计划和方法：1。RACK1和PDE4D5在哺乳动物细胞中相互作用的能力将通过共沉淀测试。为了确定每个负责其相互作用的蛋白质的位点，将使用两个杂交系统和在哺乳动物细胞中的表达来测试RACK1或PDE4D5中的突变。这些突变对PDE4D5靶向膜的影响也将进行测试。 2。为了测试PDE4A5和ARA9/AIP之间的相互作用，它们将从细胞中进行免疫沉淀。 PDE4A5同工型的突变形式和ARA9/AIP蛋白在细胞中的相互作用也将测试。还将研究细菌表达的蛋白质之间的相互作用。 ARA9/AIP和P70RSK与其他激酶之间的相互作用将通过共免疫沉淀进行测试。 3。定点诱变将用于在PDE4A5蛋白中的推定MAPK，P70RSK和PKA位点创建突变。这些将在哺乳动物细胞中表达，并测定酶活性的变化。还将研究从细胞分离的PDE4A5的磷酸化状态。 4。将筛选来自大脑和嗅觉组织的cDNA文库以进行新的PDE4同工型，并确定其组织分布和酶特性。 5。两个杂交系统将用于克隆cDNA，编码与4种新型PDE4同工型相互作用的蛋白质。