Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions
Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions
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DOI：
10.1038/srep38155
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发表时间：
2016-11-30
影响因子：
4.6
通讯作者：
Radziwill, Gerald
中科院分区：
文献类型：
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作者：
Fischer, Adrian;Warscheid, Bettina;Radziwill, Gerald
多结构域蛋白 CNK1 等支架蛋白通过整合和控制底层通路来协调信号网络。使用光遗传学方法刺激与上游效应器解偶联的 CNK1，我们确定了选择性的 CNK1 簇，它们根据所施加的光强度刺激 RAF-MEK-ERK 或 AKT 信号传导。在 MCF7 细胞中实施的 OptoCNK1 在低光强度下诱导分化，刺激 ERK 活性，而通过较高光强度刺激 AKT 信号传导则促进细胞增殖。 CNK1 响应 EGF 浓度增加而聚集，表明 CNK1 与 RAF 结合，并与低 EGF 剂量下的 ERK 激活相关。在较高 EGF 剂量下，活性 AKT 与 CNK1 结合并磷酸化并抑制 RAF。 CNK1 的敲低可保护 CNK1 免受 AKT/RAF 串扰的影响。在 C2 骨骼肌细胞中，CNK1 表达随着分化的开始而被诱导。因此，AKT 结合的 CNK1 会抵消分化细胞中的 ERK 刺激，但不会抵消增殖细胞中的 ERK 刺激。异位表达的 CNK1 促进 C2 细胞分化，敲低 CNK1 会损害 C2 细胞分化的转录网络。因此，CNK1表达、CNK1聚类和与其相关的差异信号传导过程通过协调AKT和RAF信号传导以细胞类型和细胞阶段依赖的方式决定增殖和分化。
Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type-and cell stage-dependent manner by orchestrating AKT and RAF signalling.