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

项目摘要 细胞外信号调节激酶(ERK)--丝裂原激活的终末主激酶 蛋白激酶(MAPK)信号通路，调节多种关键的细胞过程，及其异常 激活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存 在cAMP/cAMP依赖的激酶(PKA)通路中，表明这两个通路在 癌症的发展。两条典型的信号通路之间的串扰的确切机制是 一直难以捉摸。这项建议侧重于研究PKA调节ERK活性的机制。 依赖于上下文的方式。 在用于时空ERK活性研究的模型细胞系PC12细胞中的初步结果表明 PKA对质膜定位ERK(PmERK)活性的抑制和刺激调节 这取决于两条通路的初始激活情况。当在EGF激活之前激活PKA时 ERK途径中，PKA对pmERK的抑制作用似乎占主导地位。另一方面，当ERK 信号已经激活，PKA的激活似乎维持了EGF刺激的pmERK活性。以前的研究 已经证明PKA对GTPase Rap1的磷酸化有两个原因。一种效果是Rap1离开了 质膜，降低pmERK活性。另一种作用是磷酸化的RAP1相互作用 与维持pmERK活性的B-Raf结合。我们的初步结果支持这样的假设，即GTP酶 RAP1介导cAMP/PKA和pmERK活性之间的串扰。 为了验证这一假设，PKA和Rap1生物传感器将在单个细胞中使用内部成像 高通量、自动化液体处理显微镜系统。营地/库尔德工人党的各种扰动 EGF刺激前后的通路将被用来确定PKA之间的时间关系 和Rap1.然后用含有突变的PKA的Rap1生物传感器重复这些实验 磷酸化位点来测试单独去除Rap1的PKA磷酸化是否阻碍串扰。我们 将开发一个计算模型，以调查拟议的机制是否足以复制 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 两条通路的上游成分都会影响ERK的活性。然后，这些模型预测将在 人结直肠癌细胞株。这项工作的结果将揭示一个以前未解决的机制 PKA对ERK活性的调节，为癌症治疗的发展提供更多信息 通过异常的ERK信号。

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.