Minisymposium 4: Signaling

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Abs # M0404: Correlation between cytoplasmic domain sequence and autophosphorylation among Arabidopsis leucine-rich repeat receptor-like kinases

Presenter:	Hardin, Shane C.       Contact Presenter
Authors	Hardin, Shane C. (A)   Dhundaydham, Murali  (B)   Clouse, Steven D (B)   Huber, Steven C (C) (A)
Affiliations:	(A): University Of Illinois, Dept. of Plant Biology (B): North Carolina State University, Dept. of Horticultural Science (C): USDA-ARS Photosynthesis Research Unit
Web Site:	http://www.life.uiuc.edu/plantbio/PlantBioWeb/FacultyPages/Huber.html

The leucine-rich repeat receptor-like kinases (LRR-RLKs) are implicated in signaling roles during plant growth, development and defense; conditions which include changes in cellular redox. A paradigm for receptor kinase activation involves dimerization and auto- or trans-phosphorylation within the cytoplasmic domain. Our goals are to identify intracellular domain phosphorylation sites and their effects on the activity and protein-protein interactions of Arabidopsis LRR-RLKs. The cytoplasmic domains of many of these kinases have been expressed in E. coli. The LRR-RLKs can be sub-divided into "RD" and "non-RD" types based on a signature sequence motif amino-terminal to the kinase domain activation loop that is postulated to correlate with the requirement for activation loop phosphorylation. All of the RD-type kinases tested to date displayed strong autophosphorylation activity, while non-RD kinases that had CD- and LD-sequences displayed weak but measurable autophosphorylation, using ProQ-Diamond stain. In marked contrast, the non-RD type kinases did not autophosphorylate anywhere within the cytoplasmic domain suggesting the necessity for transphosphorylation during activation. Analysis of peptide kinase activities demonstrated that distinct substrate preferences existed even within the autophosphorylated RD-type receptor-like kinases, suggesting that different downstream proteins may be uniquely targeted by different members of the family. Numerous novel autophosphorylation sites have been identified in LRR-RLKs through a combination of immobilized metal chelate resin phosphopeptide purification, phosphatase treatments and MALDI-ToF mass and post-source decay spectrometry. Signaling and aerobic metabolism also involve reactive oxygen species that may alter proteins. Methionine residues in proteins are particularly susceptible to oxidation to methionine sulfoxide (MetSO) converting its side chain from hydrophobic to hydrophilic. We postulated that this could have a significant impact on target protein phosphorylation. Using synthetic peptides, we found that MetSO at critical positions in phosphorylation motifs could inhibit phosphorylation by CDPKs. Conversely, protein phosphatase activity is seemingly unaffected by neighboring MetSO. In silico analysis predicts numerous proteins may be dually regulated by both reversible phosphorylation and methionine oxidation, potentially influencing LRR-RLK signaling, autophosphorylation and substrate phosphorylation.