Project Number: 3611-21000-020-08
Project Type: Reimbursable

Start Date: Dec 01, 2006
End Date: Nov 30, 2009

Objective:
Characterize the phosphorylation of 14-3-3 proteins. Examine the effects of in vivo NR:14-3-3 interactions on enzyme activity and stability. Characterize in vivo BRI1/BAK1:14-3-3 interactions. Expand identification of in vivo 14-3-3 clients.

Approach:
Native 14-3-3 proteins, or recombinant 14-3-3s phosphorylated in vitro, will be digested with trypsin, phosphopeptides enriched by metal-affinity chromatography, and phosphopeptides identified by MALDI-ToF mass spectrometry. Potential sites will be confirmed by expression of directed 14-3-3 mutant proteins in Arabidopsis. An Arabidopsis mutant carrying T-DNA insertions within five different 14-3-3 isoforms will be used for this purpose, and also for characterizing the impact of depletion of 14-3-3s on nitrate reductase activity and stability. The association of 14-3-3s with plasma membrane-bound receptor-like kinases will be examined using the brassinosteroid receptor (BRI1) as a model system. Transgenic plants expressing an epitope-tagged BRI1 will be used to immunoprecipitate the signaling complex that forms in the presence of brassinosteroid and proteins will be identified by immunoblotting. Transgenic plants expressing an array of directed mutants of BRI1 will be evaluated for ability to bind 14-3-3 proteins and to respond to brassinosteroids. Finally, transgenic Arabidopsis expressing a mutant 14-3-3 that has increased affinity for client proteins will be used to develop a novel in vivo approach to identify binding partners.