Identification of the phosphorylation targets of symbiotic receptor-like kinases using a high-throughput multiplexed assay for kinase specificity.
D. JAYARAMAN (1), A. Richards (1), M. Westphall (1), J. Coon (1), J. Ané (1) (1) University of Wisconsin-Madison, U.S.A.

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge and new techniques are constantly being developed to overcome this challenge. Here, we utilized a multiplexed assay for kinase specificity (MAKS) to directly identify the substrates and to map the phosphorylation site(s) of plant symbiotic receptor-like kinases. The symbiotic receptor-like kinases Does not Make Infections 2 (DMI2) and Lysin motif domain-containing receptor-like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor-like kinases, very little is known about their phosphorylation substrates. Using this high-throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor-like kinases. In particular, we also discovered the phosphorylation of LYK3 by DMI2 itself which was also confirmed by pair-wise kinase assays. Motif analysis of potential targets for these kinase revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high-throughput technique catalogues the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis. 

Abstract Number: P11-364
Session Type: Poster