Poster: Protein modification

Abs # 811: Identification of potential substrates of AtCPK11: a calcium-dependent protein kinase induced by drought and salt stress

Presenter:	Rodriguez, Miguel A, rodrigm@unr.edu
Authors	Rodriguez, Miguel A (A)   Uno, Yuichi  (A)   Townsend, Jared  (A)   Maher, Eileen  (B)   Cushman, John C (A)
Affiliations:	(A): Department of Biochemistry, University of Nevada, Reno, NV 89557-0014 (B): Molecular Interaction Facility, Biotechnology Center, University of Wisconsin, Madison, WI 53706

Calcium is a ubiquitous and pivotal second messenger in the signal transduction networks that plants use to respond to a wide variety of physiological stimuli. Ca2+-dependent or calmodulin-like domain protein kinases (CDPKs) are an important group of Ser/Thr protein kinases present in plants and some protozoans that decode Ca2+ signals. The Arabidopsis thaliana genome encodes 34 CDPKs, but only a few substrates of these enzymes have been identified. As part of a large, systematic yeast two-hybrid (YTH) screening program to identify CDPK substrates, we used a YTH approach to identify substrates and/or interacting proteins of AtCPK11, a CDPK induced by drought and salt stress. Six independent YTH screens using three catalytically impaired AtCPK11 mutants as baits and two different prey libraries were performed using a high throughout procedure. In vitro kinase assays using a recombinant His-AtCPK11 and several GST-fusions showed that the top three most redundant prey proteins, H2A.Z (a histone variant involved in transcription regulation in yeast), Di19 (an ABA-independent, drought-inducible protein), and a PB1 domain-containing protein (probably involved in protein-protein interactions in the cytoplasm) were phosphorylated in vitro by recombinant 6x-His tagged AtCPK11 in a calcium-dependent manner. In addition, AtTOC33 (a GTP-binding protein involved in chloroplast protein import) was also phosphorylated by AtCPK11. Currently, we are studying the subcellular co-localization of AtCPK11 with its in vitro substrates as well as phosphorylation specificity observed among other members of the CDPK superfamily