Abstract
In order to understand the specific functional roles of CDPK, we have initiated a high-throughput yeast two-hybrid screening program to identify CDPK substrates and interacting protein. Wildtype, catalytically impaired, and constitutively active kinase mutants were used as baits in a robotic-based, interaction-screening program in which 18 million prey clones from two different libraries are tested for each bait. Using six different versions of AtCPK4 we have identified a total of 370 interactions and 30 different in-frame prey fusion proteins. The use of constitutively active AtCPK4 baits was found to improve the efficiency of recovering positive interactors relative to the wildtype kinase. To date, several candidate AtCPK4 substrates have been confirmed in vitro including Di19, an abscisic acid-independent, drought-inducible protein (Gosti et al., 1995), a GTPase type chloroplast outer envelope receptor, AtToc33, which is known to be phosphorylated at Ser181 (Jelic et al., 2003), and a serine-rich protein of unknown function.
