POWRE: A Bacterial Two-Hybrid System for Studying CDPK-Substrate Interaction.

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This is a POWRE award. The scientific focus of this project will be the establishment of a bacterial two-hybrid system for analysis of the interaction between plant calcium-dependent protein kinases (CDPKs) and their substrates. The PI will spend the first six months of the project in Dr. Jeffery Harper's lab where the basic system will be set up. This work will require extensive use of recombinant DNA techniques and will provide the opportunity to improve cloning skills and streamline laboratory procedures. The bacterial two-hybrid system will be useful for identifying which members of the large Arabidopsis CDPK family interact with and phosphorylate a given protein. Materials generated in this project may be developed in the future into a system for screening cDNA libraries for substrates of CDPKs. This work will contribute basic knowledge that will help reveal details of this large family of multifunctional protein kinases whose physiological roles are not well defined. In the second six months of the project, the PI will prepare samples of phosphorylated CDPKs and substrates and analyze these for the sites of phosphorylation in the laboratory of Dr. Michael Sussman using electrospray mass spectrometry.