Study of p38MAPK biological activity using flurorescence biosensor

[Objective]The process of p38MAPKs(p38 mitogen-activated protein kinases) phosphorylation was observed under the physiological state in the living MG-63 cells.[Methods]The ECFP-p38MAPK-Citrine(p38 MAPK biosensor) fusion protein expression vector was constructed and identified.Then the p38 MAPK biosensor was transfected into the MG-63 cells.The transfection efficiency and the expressions of fusion proteins were observed in 24 hours.Thirdly,FRET(flurorescence resonance energy transfer,FRET) of p38 MAPK biosensor was observed by the inversion fluorescence microscope and measured by the Meta Flour FRET 4.6 software.[Results]p38MAPK biosensor transfection efficiency was nearly 30%-40%.The p38 MAPK was presented in both cytoplasmic and nucleus compartments of MG-63 cells.And the FRET ratio of p38 MAPK biosensor was increased rapidly after stimulation with the ligand of TGFβ1 on MG-63 cells.The period of the FRET ratio reaching its zenith was about 30 minutes.The FRET ratio decreased gradually to its nadir in40 minutes after cells incubation with the p38 MAPK speecific inhibitor SB-203580.[Conclusion]With the technology of FRET,the p38 MAPK phosphorylation process could be realtime monitored dynamically under the physiological state in the living cells.