Regulation of Sequential Protein Kinase Pathways

顺序蛋白激酶途径的调节

• 批准号: 8038148
• 负责人: GARY L. JOHNSON
• 金额: $ 10.14万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038148
• 关键词: Ablation; Animals; Antibodies; Back; Binding; Biological Assay; Biological Factors; Biological Models; Blood Vessels; Breast Cancer Cell; Breast Fat Pad; Cancer cell line; Cell Communication; Cell Line; Cell Nucleus; Cell membrane; Cell physiology; Cells; Chromatin; Chronic; Complex; Cultured Cells; Data; Data Set; Databases; Development; Disease; EGF gene; Endosomes; Enhancers; Extracellular Matrix; FOS gene; Family; Fatty acid glycerol esters; Fibroblasts; Focal Adhesions; Funding; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Grant; Granulocyte-Macrophage Colony-Stimulating Factor; Homeostasis; Human; Hypoxia; IL8 gene; Image Analysis; Immigration; Immunoblotting; In Vitro; Inflammation; Injection of therapeutic agent; Ischemia; JUN gene; Laboratories; Least-Squares Analysis; Left ventricular structure; Libraries; Location; Lung; MAP3K1 gene; MAP3K7 gene; MAPK11 gene; MAPK14 gene; MAPK7 gene; MAPK8 gene; MCF7 cell; MEKKs; Mammalian Cell; Mammary gland; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Measurement; Measures; Metalloproteases; Metric; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Modeling; Mus; Neoplasm Metastasis; Output; Pathway Analysis; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphotransferases; Physiological; Physiology; Placenta; Play; Primary Neoplasm; Promoter Regions; Property; Protease Gene; Protein Family; Protein Kinase; Proteins; RNA Interference; Receptor Gene; Regulation; Research Personnel; Role; Serine; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Stimulus; Stromal Cell-Derived Factor 1; System; Tail; Testing; Threonine; Time; Tissues; Transcription Factor AP-1; Tumor Cell Line; Ubiquitination; United States National Institutes of Health; Urokinase; Urokinase Plasminogen Activator Receptor; Vascular Endothelial Growth Factors; Veins; Work; base; bone; cell growth; cytokine; drug discovery; fos-related antigen-2; human disease; in vivo; knockout gene; malignant breast neoplasm; matrigel; member; migration; model development; neoplastic cell; promoter; protein expression; receptor; receptor expression; research study; response; small hairpin RNA; soft tissue; spatiotemporal; tool; transcription factor; tumor; tumor progression

Mitogen activated protein kinases (MAPKs) are serine-threonine protein kinases that play critical roles in regulating cellular homeostasis. MAPKs are members of a three kinase relay system composed of the MARK, MARK kinase (MKK) and MARK kinase kinase (MKKK). Four MARK families, ERK1/2, p38, JNK and ERK5 are members of an integrated MARK signaling network. There are 7 MKKs but at least 20 MKKKs in the MARK signaling network. We have made gene knockouts to ablate expression or knockins to inactivate the kinase activity of four MKKKs (MEKK1, 2, 3, 4). Ablation or kinase inhibition of each MEKK results in a unique physiological phenotype in the mouse. Different MEKKs were found to regulate neurulation, vascular and placenta! development, and dissemination of tumor cells during metastasis, tissue remodelingfollowing ischemia or hypoxia, and specific aspects of chronic inflammation. The hypothesis of the current proposal is that MKKKs differentially integrate the activation of specific MAPKs in cellular responses to diverse stimuli. Despite the importance of MARK signaling networks in cell and animal physiology there is no comprehensive understanding of how different MKKKs coordinately regulate the MARK network for cellular homeostasis. We have shown that RNAi faithfully recapitulates targeted gene knockout of MKKKs in cells. Therefore,to define the function of MKKKs, an RNAi screen to ablate expression of a test set of 8 MKKKs that have particular relevance in control of homeostasis, will be performed in four human breast cancer cell lines. The aims of the proposal will define: 1. The functional consequence of MKKK knockdown on the signaling dynamics of the MARK network. 2. Role of MKKKs in the spatiotemporal activation of ERK1/2, JNK, p38 and ERK5. 3. To understand the transcriptional mechanism for changes in cytokine and protease expression resulting from specific MKKK knockdown. 4. To analyze the consequence of specific MKKK knockdown on orthotopic tumor cell growth and metastasis. 5. From these data a multivariate model based on Partial Least Squares will be developed to define the function of specific MKKKs in controlling signaling networks and physiological outputs. The creation of this systems-level model, along with iterative cycling between model and experiment, will allow us to define the function of specific MKKKs in controlling signaling networks related to tumor cell phenotype including metastasis-related physiological outputs.

丝裂原活化蛋白激酶(MAPKs)是丝氨酸-苏氨酸蛋白激酶，在 调节细胞动态平衡。MAPK是一个由三个激酶组成的传递系统的成员 MARK、MKK和MKKK。四个MARK家族，ERK1/2，p38，JNK和 ERK5是一个集成的MARK信令网络的成员。有7个MKK，但至少有20个MKK Mark信令网络。我们已经进行了基因敲除来削弱表达，或者敲打来灭活 四种MKKK(MEKK1、2、3、4)的激酶活性。消融或抑制每个MEKK会导致 小鼠独特的生理表型。不同的MEKK被发现调节神经形成、血管 还有胎盘！转移过程中肿瘤细胞的发展和扩散，随后的组织重塑 缺血或缺氧，以及慢性炎症的特定方面。目前提案的假设是 MKKKs在细胞对不同刺激的反应中不同地整合了特定MAPKs的激活。 尽管MARK信号网络在细胞和动物生理学中很重要，但目前还没有全面的 了解不同的MKKK如何协调调节MARK网络的细胞内稳态。 我们已经证明，RNAi忠实地概括了细胞中MKKK的靶向基因敲除。因此，要 定义MKKK的功能，这是一个RNAi屏幕，用于去除8个MKKK的测试集的表达 在动态平衡的控制方面，将在四个人类乳腺癌细胞系中进行特别相关的研究。这个 该提案的目的将明确：1.MKKK基因敲除对信号的功能后果 Mark网络的动态。2.MKKK在ERK1/2、JNK、p38和ERK1/2的时空激活中的作用 ERK5。3.了解细胞因子和蛋白酶表达变化的转录机制 由特定的MKKK基因敲除所致。4.分析特定MKKK基因敲除的后果 原位肿瘤细胞生长和转移。5.利用这些数据建立了基于偏最小二乘法的多元模型 将开发方块来定义特定MKKK在控制信令网络和 生理输出。这个系统级模型的创建，以及模型之间的迭代循环 和实验，将使我们能够定义特定的MKKK在控制信令网络中的功能 与肿瘤细胞表型相关，包括与转移相关的生理输出。