Systems Approach to Dynamic Interplay between cAMP/PKA and ERK Signaling

cAMP/PKA 和 ERK 信号传导之间动态相互作用的系统方法

• 批准号: 10389956
• 负责人: Clara Anne Posner
• 金额: $ 3.97万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389956
• 关键词: Affect; Biosensor; Cancer Etiology; Cell Line; Cell membrane; Cell model; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Colorectal; Colorectal Cancer; Complex; Computer Models; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Differential Equation; Epidermal Growth Factor; Extracellular Signal Regulated Kinases; Fluorescence Resonance Energy Transfer; GNAS gene; Genes; Guanosine Triphosphate Phosphohydrolases; Human; Image; In Vitro; KRAS2 gene; Kinetics; Knock-in; Liquid substance; Malignant Neoplasms; Malignant neoplasm of appendix; Malignant neoplasm of pancreas; Measures; Mediating; Microscope; Mitogen-Activated Protein Kinases; Modeling; Monitor; Mutate; Mutation; Names; PC12 Cells; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Rattus; Reaction; Regulation; Resolution; Signal Pathway; Signal Transduction; System; Testing; Variant; Work; base; cancer therapy; colon cancer cell line; experimental study; flexibility; imaging platform; improved; in silico; melanoma; migration; multiplexed imaging; mutant; new therapeutic target; predictive modeling; response; sensor; spatiotemporal; targeted cancer therapy; therapy development

Project Summary Extracellular-Signal Regulated Kinase (ERK), the terminal master kinase in the mitogen-activated protein kinase (MAPK) signaling pathway, regulates a variety of critical cell processes, and its aberrant activation contributes to the development of various cancers. In appendiceal, pancreatic, and colorectal cancers, there is significant co-occurrence of activating mutations in the ERK pathway with activating mutations in the cAMP/cAMP-dependent kinase (PKA) pathway, indicating cooperation between these two pathways in cancer development. The exact mechanism of crosstalk between the two canonical signaling pathways has been elusive. This proposal focuses on investigating the mechanisms by which PKA regulates ERK activity in a context dependent manner. Preliminary results in PC12 cells, a model cell line used for spatiotemporal ERK activity studies, indicate both inhibitory and stimulatory regulation by PKA on plasma membrane localized ERK (pmERK) activity depending on the initial activation of the two pathways. When PKA is activated before EGF activation of the ERK pathway, the inhibitory effects of PKA on pmERK seem to dominate. On the other hand, when ERK signaling is already active, PKA activation seems to sustain EGF stimulated pmERK activity. Previous studies have shown PKA phosphorylation of the GTPase Rap1 causes two effects. One effect is that Rap1 leaves the plasma membrane, which decreases pmERK activity. The other effect is that phosphorylated Rap1 interacts with B-Raf, which sustains pmERK activity. Our preliminary results support the hypothesis that the GTPase Rap1 mediates crosstalk between cAMP/PKA and pmERK activity. To test this hypothesis, PKA and Rap1 biosensors will be co-imaged in single cells using an in-house high-throughput, automated liquid handling microscope system. Various perturbations of the cAMP/PKA pathway before and after EGF stimulation will be used to determine the temporal relationship between PKA and Rap1. These experiments will then be repeated with Rap1 biosensors containing a mutated PKA phosphorylation site to test whether removing PKA phosphorylation of Rap1 alone impedes the crosstalk. We will develop a computational model to investigate whether the proposed mechanism is sufficient to replicate the ERK activity observed in preliminary results and to predict how cancer-specific activating mutations in upstream components of both pathways affect ERK activity. These model predictions will then be tested in human colorectal cancer cell lines. The results of this work will uncover a previously unresolved mechanism of PKA regulation of ERK activity, providing more information for the development of therapies for cancers driven by aberrant ERK signaling.

项目摘要 细胞外信号调节激酶（ERK），丝裂原激活中的末端主激酶 蛋白激酶（MAPK）信号通路，调节多种关键细胞过程，其异常 激活有助于各种癌症的发展。在阑尾，胰腺和结直肠 癌症，ERK途径中激活突变与激活突变存在显着的共发生 在营地/营地依赖性激酶（PKA）途径中，表明这两个途径之间的合作 癌症发展。两个规范信号通路之间的串扰的确切机制具有 难以捉摸。该提案重点是研究PKA调节ERK活性的机制 上下文依赖方式。 PC12细胞（用于时空ERK活性研究的模型细胞系）的初步结果表明 PKA对质膜局部ERK（PMERK）活性的抑制和刺激调节 取决于两种途径的初始激活。当EGF激活之前激活PKA时 ERK途径，PKA对PMERK的抑制作用似乎占主导地位。另一方面，当erk 信号传导已经有效，PKA激活似乎维持EGF刺激的PMERK活性。先前的研究 已显示GTPase RAP1的PKA磷酸化会引起两种影响。一个效果是Rap1离开 质膜，可降低PMERK活性。另一个效果是磷酸化的RAP1相互作用 与B-RAF一起维持PMERK活动。我们的初步结果支持GTPase的假设 RAP1介导营地/PKA和PMERK活动之间的串扰。 为了检验该假设，PKA和RAP1生物传感器将在单个细胞中使用内部共同成像 高通量，自动液体处理显微镜系统。营地/PKA的各种扰动 EGF刺激前后的途径将用于确定PKA之间的时间关系 和Rap1。然后将使用包含突变PKA的RAP1生物传感器重复这些实验 磷酸化位点以测试单独去除RAP1的PKA磷酸化是否会阻碍串扰。我们 将开发一个计算模型来研究提出的机制是否足以复制 在初步结果中观察到的ERK活性，并预测癌症特异性激活突变如何 两种途径的上游成分都会影响ERK活性。这些模型预测将在 人结直肠癌细胞系。这项工作的结果将发现以前未解决的机制 PKA对ERK活动的调节，为开发癌症驱动的疗法提供更多信息 通过异常的ERK信号传导。