Simultaneously monitoring endogenous MAPK members in single living cells by multi-channel fluorescence correlation spectroscopy

The mitogen-activated protein kinase (MAPK) pathway is a major module for cellular signal transduction. The dysregulation of the MAPK pathway has been involved in the pathogenesis of multiple diseases ranging from cancers to chronic inflammations. So far, we have not fully understood the influences of external factors and signaling networks on the MAPK pathway due to the lack of in situ methods for simultaneous detection of multiple kinases in the pathway in living cells. Herein, we present a new strategy for in situ and simultaneously monitoring MAPK pathway kinases in single living cells combining multi-channel fluorescence correlation spectroscopy (FCS) with affinity fluorescent probes. We chose rapidly growing fibrosarcoma kinase (RAF), mitogen-activated protein kinase (MEK), and extracellular signal-regulated kinase (ERK) as representative members in the MAPK pathway. We designed and synthesized three fluorescent affinity probes and experimental results demonstrated that the three probes specifically targeted endogenous BRAF, MEK1/2, and ERK1/2 in living cells. Based on the multi-channel FCS system, we studied the influences of biological substances, drugs and oxidative stress on the activities of endogenous MAPK kinases and the cross-talk between the MAPK and PI3K-mTOR pathways. We have found that serum, sorafenib, and hydrogen peroxide can regulate multiple MAPK kinases and the effects of external stimuli can transmit to the MAPK pathway; furthermore, we have observed that the MAPK pathway can be activated by modulating the PI3K-mTOR pathway. Our results illustrated the complexity of a cellular signal network and the necessity of in situ and simultaneous determination of biomolecules in living cells.