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），在促分裂原活化的细胞内的终末主激酶。 蛋白激酶（MAPK）信号通路，调节多种关键细胞过程，其异常 活化有助于各种癌症的发展。在阑尾、胰腺和结直肠 在癌症中，ERK通路中的激活突变与激活突变显著共现。 在cAMP/cAMP依赖性激酶（PKA）途径中，表明这两种途径在 癌症发展两个经典信号通路之间串扰的确切机制已经被证实。 一直难以捉摸。该建议的重点是研究PKA调节ERK活性的机制， 上下文相关的方式。 在用于时空ERK活性研究的模型细胞系PC 12细胞中的初步结果表明， PKA对质膜定位的ERK（pmERK）活性的抑制性和刺激性调节 这取决于两种途径的初始激活。当PKA在EGF激活之前被激活时， PKA对pmERK的抑制作用似乎占主导地位。另一方面，当ERK 信号已经活跃，PKA活化似乎维持EGF刺激的pmERK活性。以前的研究 已经显示GTdR Rap 1的PKA磷酸化导致两种效应。一个影响是Rap 1离开了 质膜，其降低pmERK活性。另一个作用是磷酸化Rap 1与 与B-Raf，维持pmERK活性。我们的初步研究结果支持了这一假设，即GTdR Rap 1介导cAMP/PKA和pmERK活性之间的串扰。 为了检验这一假设，PKA和Rap 1生物传感器将使用内部的 高通量自动液体处理显微镜系统。cAMP/PKA的各种扰动 EGF刺激前后的PKA通路将用于确定PKA与EGF刺激之间的时间关系。 Rap 1这些实验将用含有突变PKA的Rap 1生物传感器重复进行 磷酸化位点，以测试单独去除Rap 1的PKA磷酸化是否阻碍串扰。我们 将开发一个计算模型，以调查所提出的机制是否足以复制 在初步结果中观察到ERK活性，并预测癌症特异性激活突变如何在 这两种途径的上游组分影响ERK活性。这些模型预测将在 人结肠直肠癌细胞系。这项工作的结果将揭示一个以前未解决的机制， PKA对ERK活性的调控，为开发癌症驱动的治疗方法提供更多信息 通过异常的ERK信号