Visualizing dynamic interaction between calmodulin and calmodulin-related kinases via a monitoring method in live mammalian cells

Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3412-7. doi: 10.1073/pnas.0911262107. Epub 2010 Feb 3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism
  • Calcium Signaling*
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / chemistry
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
  • Calmodulin / chemistry
  • Calmodulin / metabolism*
  • HeLa Cells
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Microscopy, Confocal
  • Two-Hybrid System Techniques*

Substances

  • Calmodulin
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium