Expression, Structure/function And Regulation Of Phospho

Phospho的表达、结构/功能和调控

• 批准号: 6541694
• 负责人: VINCENT MANGANIELLO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541694
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; 3T3 cells; B lymphocyte; T lymphocyte; adipocytes; cAMP response element binding protein; cell differentiation; cell line; enzyme activity; esterase inhibitor; gene targeting; human tissue; insulin; insulinlike growth factor; isozymes; laboratory mouse; leukocyte activation /transformation; macrophage; nucleotide metabolism; protein kinase; protein structure function

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cyclic nucleotides. Understanding cellular expression and regulation of PDE isoforms [which belong to eleven gene families (PDEs 1-11)] will be of increasing importance for targeting specific PDEs in treating various diseases, including pulmonary disorders. PDE3B appears (or greatly increases in activity) during differentiation of cultured 3T3-L1 adipocytes or cultured human monocyte-derived macrophages. To study regulation of PDE3B expression, a genomic fragment of the 5'-flanking region of the mouse(M) PDE3B gene was isolated; two promoter regions were identified, a distal promoter located ~4 kb upstream, and a proximal region, ~500 bp upstream of the translation initiation ATG codon. Transfection of 3T3-L1 fibroblasts and differentiating 3T3-L1 adipocytes with a series of reporter plasmids containing the luciferase gene coupled to different fragments of the 5'-UTR of the MPDE3B gene indicated that the distal region induced about 50-fold higher promoter activity than did the proximal region. Transfection with distal and proximal promoters indicated that (1) a strong negative regulatory region was present between the distal and proximal promoter regions; and (2) transfection with constructs containing CRE elements of the distal promoter markedly increased promoter activity in differentiating adipocytes. Differentiation of 3T3-L1 adipocytes is induced by medium containing DMI (dexamethasone, isobutylmethylxanthine and insulin). Induction of PDE3B was markedly enhanced by M (which presumably increased cAMP, leading to phosphorylation/activation of CREB proteins)and was dissociated from lipid accumulation, which was more pronounced in the presence of DI. Taken together, these results suggest that activation of CREB proteins might play a crucial role in the expression of MPDE3B in 3T3-L1 differentiating adipocytes. Studies with 3T3-L1 adipocytes and FDCP2 cells indicated that insulin- and IGF-1-induced phosphorylation/activation of PDE3B was mediated via PI3-K- and PKB-dependent signals, and that PDE3B is most likely a direct substrate of PKB. MPDE3B contains a consensus phosphorylation site (Ser 273) for PKB; after mutation of Ser 273 Ala, PKB does not activate MPDE3B in vitro. To study activation of microsomal MPDE3B by insulin in 3T3-L1 cells, we examined the subcellular distribution of components of the insulin-signaling pathway involved in activation of PDE3B. In 3T3-L1 adipocytes, PDE3B is primarily, but not exclusively, associated with intracellular membranes (LDM and HDM fractions). Incubation with insulin results in tyrosine phosphorylation of IRS-1 and activation of PI3-K associated with intracellular membranes. In addition, a portion of the intracellular pool of PKB is also found in association with intracellular membranes, co-elutes with PDE3B during size exclusion chromatography on AcA34, and co-immunoprecipitates with MPDE3B. The structural determinants for this later interaction reside in the N-terminal portion of MPDE3. These results suggest that signals to activate membrane-associated PDE3B are directed via IRS/PI3-K to intracellular membranes where activation of PI3-K and production of phosphoinositides results in activation of PDK, PKB and, consequently, PDE3B. To further examine the functional role of PDE3B isoforms, null PDE3A and PDE3B mice were generated. As PDE3B null mice age, the males demonstrate abnormal glucose and insulin tolerance tests. Adipocytes from PDE3B null mice are smaller than those from WT animals, and they are less responsive to lipolytic stimuli, including isoproterenol and adenosine deaminase. Studies are in progress to understand the role of PDE3B in these and other phenotypic changes.

通过催化 cAMP 和 cGMP 的水解，环核苷酸磷酸二酯酶 (PDE) 是环核苷酸的细胞内浓度和生物效应的关键调节剂。了解 PDE 亚型 [属于 11 个基因家族 (PDE 1-11)] 的细胞表达和调节对于靶向特定 PDE 治疗各种疾病（包括肺部疾病）将变得越来越重要。 PDE3B 在培养的 3T3-L1 脂肪细胞或培养的人单核细胞来源的巨噬细胞的分化过程中出现（或活性大大增加）。为了研究 PDE3B 表达的调控，分离了小鼠 (M) PDE3B 基因 5'-侧翼区域的基因组片段；鉴定出两个启动子区域，远端启动子位于翻译起始 ATG 密码子上游约 4 kb 的上游，近端区域位于翻译起始 ATG 密码子上游约 500 bp。用一系列含有与 MPDE3B 基因 5'-UTR 不同片段偶联的荧光素酶基因的报告质粒转染 3T3-L1 成纤维细胞并分化 3T3-L1 脂肪细胞，表明远端区域诱导的启动子活性比做了近端区域。远端和近端启动子转染表明：（1）远端和近端启动子区之间存在强负调控区； (2)用含有远端启动子CRE元件的构建体转染显着增加分化脂肪细胞中的启动子活性。 3T3-L1 脂肪细胞的分化由含有 DMI（地塞米松、异丁基甲基黄嘌呤和胰岛素）的培养基诱导。 M 显着增强了 PDE3B 的诱导（可能增加了 cAMP，导致 CREB ​​蛋白的磷酸化/激活），并且与脂质积累分离，这在存在 DI 的情况下更为明显。综上所述，这些结果表明 CREB ​​蛋白的激活可能在 3T3-L1 分化脂肪细胞中 MPDE3B 的表达中发挥至关重要的作用。对 3T3-L1 脂肪细胞和 FDCP2 细胞的研究表明，胰岛素和 IGF-1 诱导的 PDE3B 磷酸化/激活是通过 PI3-K 和 PKB 依赖性信号介导的，并且 PDE3B 最有可能是 PKB 的直接底物。 MPDE3B 包含 PKB 的共有磷酸化位点 (Ser 273)； Ser 273 Ala 突变后，PKB 在体外不会激活 MPDE3B。为了研究 3T3-L1 细胞中胰岛素对微粒体 MPDE3B 的激活，我们检查了参与 PDE3B 激活的胰岛素信号通路成分的亚细胞分布。在 3T3-L1 脂肪细胞中，PDE3B 主要（但不完全）与细胞内膜（LDM 和 HDM 部分）相关。与胰岛素一起孵育会导致 IRS-1 的酪氨酸磷酸化以及与细胞内膜相关的 PI3-K 的激活。此外，还发现细胞内 PKB 库的一部分与细胞内膜相关，在 AcA34 上的尺寸排阻色谱过程中与 PDE3B 共洗脱，并与 MPDE3B 共免疫沉淀。此后相互作用的结构决定因素位于 MPDE3 的 N 端部分。这些结果表明，激活膜相关 PDE3B 的信号通过 IRS/PI3-K 引导至细胞内膜，其中 PI3-K 的激活和磷酸肌醇的产生导致 PDK、PKB 以及 PDE3B 的激活。为了进一步检查 PDE3B 亚型的功能作用，生成了无效 PDE3A 和 PDE3B 小鼠。随着 PDE3B 缺失小鼠年龄的增长，雄性小鼠表现出异常的葡萄糖和胰岛素耐量测试。 PDE3B 缺失小鼠的脂肪细胞比 WT 动物的脂肪细胞小，并且它们对脂肪分解刺激（包括异丙肾上腺素和腺苷脱氨酶）的反应较差。正在进行研究以了解 PDE3B 在这些和其他表型变化中的作用。