Note: This project was renewed by NSF on July 1, 2010. To see the abstract of the renewal project, "Arabidopsis 2010: Protein Interacting Networks and Site-Specific Phosphorylation in Leucine-Rich Repeat Receptor-Like Kinase Function", please go to the NSF website and view award abstract 1021363.

Arabidopsis 2010: Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases in Arabidopsis

National Science Foundation (MCB)0419819

PI:                      Steve Clouse, North Carolina State University

Co-PI:                 Michael Goshe, North Carolina State University

Co-PI:                 Jia Li, University of Oklahoma

Co-PI:                 Steve Huber, University of Illinois

Project Abstract

The Arabidopsis genome encodes more than 200 Leucine-Rich Repeat Receptor-like kinases (LRR RLKs) with an organization of functional domains similar to that of animal receptor kinases. Several LRR RLKs are known to be critical elements in signaling pathways regulating plant development and response to the environment, but the biological functions of most members of this large family of putative receptors remain unknown. This project will acquire fundamental biochemical knowledge of the kinase domains of all Arabidopsis LRR RLKs by using Gateway cloning and high-throughput technology for in vitro analysis of autophosphorylation activity, substrate preference and pair-wise interactions. A proteomic analysis of membrane proteins isolated from Arabidopsis plants grown under a variety of physiological conditions will be used to identify a subset of 30 LRR RLKs for detailed analysis of in vivo autophosphorylation sites by mass spectrometry. The functional significance of selected phosphorylation sites will be examined using genetic and biochemical approaches, and interactions between selected LRR RLKs in planta will also be examined. Significance to 2010 Project Objectives-- Identification of specific phosphorylation sites and their functional significance will advance our understanding of RLK signaling mechanisms and provide a comparative database of phosphorylation sites for membrane localized kinases. The family-wide analysis of LRR RLK kinase domains may reveal new aspects of the evolution of function in multigene families and studies of in vivo interactions may uncover novel heterodimers, suggesting possible cross-talk between signaling pathways.  Broader Impact of the Proposed Activity--Phosphorylation sites determined in this study will be posted regularly on this site, creating a unique community resource for comparative study. A variety of tagged constructs in Gateway vectors for in vitro and in vivo studies of Arabidopsis LRR RLKs will be deposited in the Arabidopsis Biological Resource Center, along with numerous lines of transgenic plants. Given the known importance of several LRR RLKs to plant development, it is likely that increased knowledge of this gene family could have practical agricultural impact. The project will provide excellent training in protein biochemistry, mass spectrometry, and Arabidopsis molecular genetics at all levels from high school student interns through postdoctoral scientists.