3P-28 PDF
細胞膜透過性キナーゼ基質ペプチドを用いた細胞内キノーム活性計測
Protein phosphorylation catalyzed by kinases plays significant roles in cellular signal transduction. Monitoring cellular kinome activities is important to detect the dysregulation of protein phosphorylation associated with various diseases. Therefore, profiling kinome activities is useful for diagnostic and therapeutic purposes. Recent advances in phosphoproteomics allow identifying thousands of phosphosites. However, it is still difficult to obtain the kinome profiles because the information of kinase-substrate relationship is mostly missing. Alternatively, it has been reported that in vitro kinase activities can be measured by spiking kinase-selective substrate peptides into the lysed cells, although kinase activities within intact cells cannot be monitored. In this study, we aimed to develop a method to monitor in vivo kinome activities. Firstly, we experimentally collected the data of in vitro substrates for each kinase using approximately 400 kinases and HeLa proteins dephosphorylated prior to in vitro kinase reaction. Using this dataset, we selected peptides with unique sequences phosphorylated by each kinase exclusively. Then, the kinase-specific substrate peptides were concatenated with octaarginine sequence. We confirmed that these peptides penetrated the cell membrane with high efficiency.