Rapid detection of calmodulin/target interaction via the proximity biotinylation method

• Published in: Biochemical and biophysical research communications 2023-06, Vol.659, p.29-33
• Main Authors: Nakamura, Kento Nlandu, Yamauchi, Haruki, Mima, Hiroyuki, Chen, Yerun, Ohtsuka, Satomi, Magari, Masaki, Morishita, Ryo, Tokumitsu, Hiroshi
• Format: Article
• Language: English
• Subjects: AirID; AirID-CaM; Animals; Biotinylation; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Kinase - metabolism; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Calmodulin; Calmodulin - metabolism; CaM targets; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Phosphorylation; protein kinases; Protein-protein interaction; Proximity biotinylation; rapid methods; Rats; signal transduction
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2023.03.072

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity. •Expression and purification of biotinylation enzyme fused with calmodulin.•AirID-CaM is capable of biotinylating multiple CaM kinases in a Ca2+-dependent manner.•Biotinylation of CaMKKα/1 by AirID-CaM likely occurs in interaction-dependent manner.•AirID-CaM can be useful for the rapid detection of CaM/target interaction.