Regulation of Sequential Protein Kinase Pathways

丝裂原活化蛋白激酶（MAPK）是丝氨酸-苏氨酸蛋白激酶，其在细胞分裂中起关键作用。 调节细胞内稳态MAPKs是三个激酶中继系统的成员， MARK、MARK激酶（MKK）和MARK激酶激酶（MKKK）。四个MARK家族，ERK 1/2，p38，JNK和 ERK5是整合的MARK信号网络的成员。有7个MKK，但至少有20个MKKK在 MARK信号网络我们已经通过基因敲除来消除表达，或者通过基因敲入来消除表达。 四种MKKK（MEKK 1、2、3、4）的激酶活性。每种MEKK的消融或激酶抑制导致 小鼠中独特的生理表型。发现不同的MEKKs调节神经形成、血管生成和血管生成。 胎盘!发展和转移过程中的肿瘤细胞的传播， 缺血或缺氧，以及慢性炎症的特定方面。目前提案的假设是 MKKK在对不同刺激的细胞反应中差异地整合了特异性MAPK的激活。 尽管MARK信号网络在细胞和动物生理学中的重要性， 了解不同的MKKK如何协调调节MARK网络以实现细胞内稳态。 我们已经证明，RNAi忠实地再现了细胞中MKKK的靶向基因敲除。因此 定义MKKK的功能，RNAi筛选以消除具有以下特征的8个MKKK的测试集的表达： 特别是在体内平衡控制方面的相关性，将在四种人乳腺癌细胞系中进行。的 该提案的目标将确定：1。MKKK敲低对信号传导的功能性后果 MARK网络的动态。2. MKKKs在ERK 1/2、JNK、p38和p53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 由特异性MKKK敲除引起。4.为了分析特异性MKKK敲低对 原位肿瘤细胞生长和转移。5.从这些数据中，基于偏最小二乘的多变量模型 将开发方块来定义特定MKKK在控制信令网络中的功能， 生理输出。这种系统级模型的创建，沿着着模型之间的迭代循环 和实验，将允许我们定义特定MKKK在控制信令网络中的功能 与肿瘤细胞表型相关，包括转移相关的生理输出。

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.