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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

基本信息

批准号：
10570165
负责人：
Clara Anne Posner
金额：
$ 2.29万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10570165
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 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 Proliferating Rattus Reaction Regulation Resolution Signal Pathway Signal Transduction System Testing Variant Work 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

项目摘要

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.

项目总结