Poster: Intracellular Signaling

Abs # 844: Calcium-dependent protein kinase (CDPK) substrates and interacting proteins in Arabidopsis.

Presenter:	Uno, Yuichi , yuno@unr.edu
Authors	Uno, Yuichi  (A) (B)  Etheridge, Naomi  (C)   Chan, Catherine  (D)   Maher, Eileen  (D)   Hrabak, Estelle M (C)   Cushman, John C (A)
Affiliations:	(A): Department of Biochemistry, University of Nevada, Reno, NV 89557-0014, USA (B): Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan (C): Department of Plant Biology, University of New Hampshire, Durham, NH 03824, USA (D): Molecular Interaction Facility/Biotechnology Center, University of Wisconsin, Madison, WI 53706, USA

CDPKs (calcium-dependent protein kinase or calmodulin-like domain protein kinases) are essential sensor-transducers of calcium signaling pathways in plants. CDPKs and four closely related families, the CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PPCK-related kinases (PPCRKs), and SNF-1 related kinases (SnRKs) include 84 kinases and represent about 9% of all the protein kinases in the Arabidopsis genome. Of these, 59 CDPKs and SnRK3s (subgroup 3 of the SNF-1 related kinase) kinases are predicted to be regulated by calcium. Functional characterization of CDPKs is of great interest because there are no animal or fungal homologs for the majority of these kinases. To understand the specific functional roles of each protein kinase of this super family, a systematic yeast two-hybrid screening project has been used to identify CDPK substrates and other interacting protein. Wildtype, catalytically impaired, and constitutively active versions of bait constructs were used for a high-throughput, robotic-based interaction-screening program in which more than 18 million prey clones from two different cDNA libraries are tested for each bait. Results from selected CDPK and PPCK screens show that catalytically impaired and constitutively active CPK baits behave more efficiently in yeast two-hybrid screens for the isolation of putative CPK substrates and interacting proteins. Results from the interaction-screening program are then confirmed by in vitro interaction and kinase assays. Identification of CDPK substrates and interacting proteins that direct their subcellular locations should provide novel insights into the complex calcium signaling processes of plants. This research is funded by the NSF 2010 program (MCB- 0114769).