IDENTIFICATION OF ATCPK11 SUBSTRATES BY YEAST TWO-HYBRID ANALYSIS

¹ Department of Biochemistry, University of Nevada, Reno, NV 89557-0014, USA

² Molecular Interaction Facility/Biotechnology Center, University of Wisconsin, Madison, WI 53706,USA

Complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by protein kinases, which change enzyme activities and protein conformations. Calcium-dependent protein kinases (CDPKs) are Ser/Thr protein kinases present in plants and some protozoans. The Arabidopsis genome encodes 34 CDPKs, but only a few substrates of these enzymes have been found. In this study, we have used a yeast two-hybrid approach (YTH) to identify substrates and/or interacting proteins of AtCPK11. This project is part of a systematic YTH screening initiated to identify kinase substrates. The initial YTH screening using AtCPK11 mutants revealed a total of 13 redundant clones that may encode interacting proteins. Interestingly, two of these proteins, Di19 (an ABA-independent drought inducible protein) and AtTOC33 (a GTP-binding protein involved in chloroplast protein import) were also detected in a YTH screening of AtCPK4 substrates, and in vitro kinase assays demonstrated that they were phosphorylated by AtCPK4. Other potential substrates include histone H2A (known to be phosphorylated in mammals), ADF3 (an actin depolymerizing factor also known to be phosphorylated in plants), PB1 domain-containing protein, myosin-related protein, geranylgeranyl reductase, ubiquitin c-terminal hydrolase, and acyl-carrier protein. Our results suggest that AtCPK4 and AtCPK11, two very closely related CDPKs, share common substrates and at the same time control the activity of different proteins. In vitro kinase assays using recombinant AtCPK11 and its putative substrates are in progress to validate the YTH data.