Visualizing dynamic interaction between calmodulin and calmodulin-related kinases via a monitoring method in live mammalian cells.
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. This method is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. We visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. We observed that CaMKK1 and CaMKIIalpha bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
Lee
Kyoung Hu
KH
Department of Biological Sciences, Graduate School of Nanoscience & Technology, Daejeon 305-701, Korea.
Lee
Sangkyu
S
Lee
Woo Yong
WY
Yang
Hee Won
HW
Heo
Won Do
WD
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal; D020798:Two-Hybrid System Techniques
D002147:Calmodulin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium