Identification of interacting proteins for calcium-dependent protein kinase 8 by a novel screening system based on bimolecular fluorescence complementation

Abstract

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. We developed a novel assay system, based on the bimolecular fluorescence complementation (BiFC) technique in Escherichia coli, for detecting transient interactions such as those between kinases and their substrates. This system detected the interaction between OsMEK1 and its direct target OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of calcium-dependent protein kinase 8 (OsCPK8), a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins (ARF-GEF and peptidyl prolyl isomerase) was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.

Graphical Abstract

A novel assay system, for detecting specific interactions was developed based on the BiFC technique in Escherichia coli.

Key words:

• protein kinase
• substrate
• protein–protein interaction
• BiFC
• high-throughput screening

Acknowledgments

We are grateful to Ms. Hiromi Morii for excellent technical support. This work was supported in part by a Grant-in-Aid for Scientific Research (B) (25292067) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the Program for the Promotion of Basic and Applied Research for Innovation in Bio-Oriented Industry.