Wend Sabrina, Wagner Hanna J, Müller Konrad, Zurbriggen Matias D, Weber Wilfried, Radziwill Gerald
Faculty of Biology, University of Freiburg , 79104 Freiburg, Germany.
ACS Synth Biol. 2014 May 16;3(5):280-5. doi: 10.1021/sb400090s. Epub 2013 Oct 4.
Light-dependent dimerization is the basis for recently developed noninvasive optogenetic tools. Here we present a novel tool combining optogenetics with the control of protein kinase activity to investigate signal transduction pathways. Mediated by Arabidopsis thaliana photoreceptor cryptochrome 2, we activated the protein kinase C-RAF by blue light-dependent dimerization, allowing for decoupling from upstream signaling events induced by surface receptors. The activation by light is fast, reversible, and not only time but also dose dependent as monitored by phosphorylation of ERK1/2. Additionally, light-activated C-RAF controls serum response factor-mediated gene expression. Light-induced heterodimerization of C-RAF with a kinase-dead mutant of B-RAF demonstrates the enhancing role of B-RAF as a scaffold for C-RAF activity, which leads to the paradoxical activation of C-RAF found in human cancers. This optogenetic tool enables reversible control of protein kinase activity in signal duration and strength. These properties can help to shed light onto downstream signaling processes of protein kinases in living cells.
光依赖二聚化是最近开发的非侵入性光遗传学工具的基础。在此，我们展示了一种将光遗传学与蛋白激酶活性控制相结合的新型工具，用于研究信号转导通路。由拟南芥光受体隐花色素2介导，我们通过蓝光依赖二聚化激活蛋白激酶C-RAF，从而使其与表面受体诱导的上游信号事件解偶联。如通过ERK1/2磷酸化监测的那样，光激活快速、可逆，不仅具有时间依赖性，还具有剂量依赖性。此外，光激活的C-RAF控制血清反应因子介导的基因表达。光诱导C-RAF与B-RAF激酶失活突变体的异源二聚化证明了B-RAF作为C-RAF活性支架的增强作用，这导致了在人类癌症中发现的C-RAF的反常激活。这种光遗传学工具能够在信号持续时间和强度方面对蛋白激酶活性进行可逆控制。这些特性有助于阐明活细胞中蛋白激酶的下游信号转导过程。
