Dose-dependent proteome profiling of KRAS pathway inhibitors sheds new light on drug mechanism of action and KRAS signaling

Kras is a molecular switch controlling the activity of the MAPK pathway. As such, it determines ERK activation state. In this study we identified many novel p-sites that were regulated upon KRAS inhibition. Many of these regulated p-sites were located within a SP/TP motif, the recognition motif of proline directed Kinases such as ERK. In this study we used parallel reaction monitoring (PRM) to determine the ability of ERK to phosphorylate synthetic peptides carrying a subset of these p-sites in a time-resolved manner. We identified that ERK was able to phosphorylate 10 of the tested sequences of which only one was phosphorylated on a mutated control peptide.

KRAS是调控丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）通路活性的分子开关，可决定细胞外调节蛋白激酶（Extracellular Regulated Protein Kinase, ERK）的激活状态。本研究中，我们鉴定出诸多受KRAS抑制调控的新型磷酸化位点（phosphorylation sites，简称p-sites），其中大量调控位点位于SP/TP基序——这是ERK等脯氨酸定向激酶的识别基序。本研究采用平行反应监测（Parallel Reaction Monitoring, PRM）技术，以时间分辨的方式检测了ERK对携带部分上述p-sites的合成肽的磷酸化能力。我们发现，ERK可对10条受试序列进行磷酸化，而其中仅一条序列对应的突变型对照肽上的位点被磷酸化。