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In situ studying RSK2 kinase activity in single living cell by combining single molecule spectroscopy with activity-based probes

Abstract

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and determination of protein kinase activity plays a key role in pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in single living cell due to shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label to active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitor and ultraviolet irradiation, we observed that endogenous RSK2 showed different behaviors in the cytoplasm and nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.

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Publication details

The article was received on 25 Jan 2019, accepted on 12 Apr 2019 and first published on 15 Apr 2019


Article type: Paper
DOI: 10.1039/C9AN00178F
Citation: Analyst, 2019, Accepted Manuscript

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    In situ studying RSK2 kinase activity in single living cell by combining single molecule spectroscopy with activity-based probes

    S. Yu, Z. Du, C. Dong and J. Ren, Analyst, 2019, Accepted Manuscript , DOI: 10.1039/C9AN00178F

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