An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

Authors: MITRA, S - North Carolina State University; CHEN, R - North Carolina State University; DHUNDAYDHAM, M - North Carolina State University; WANG, X - North Carolina State University; BLACKBURN, R - North Carolina State University; KOTA, U - North Carolina State University; GOSHE, M - North Carolina State University; SCHWARTZ, D - University Of Connecticut; Huber, Steven; CLOUSE, S - North Carolina State University

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/2015
Publication Date: 6/1/2015
Citation: Mitra, S.K., Chen, R., Dhundaydham, M., Wang, X., Blackburn, R.K., Kota, U., Goshe, M.B., Schwartz, D., Huber, S.C., Clouse, S.D. 2015. An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Plant Journal. 82(6):1042-1060.

Interpretive Summary: Leucine-rich repeat receptor-like kinases (LRR RLKs) are anchored within plant membranes by a single-pass transmembrane sequence that connects an extracellular domain, often with specific ligand binding capabilities, to a cytoplasmic kinase domain that autophosphorylates specific Ser, Thr and in some cases Tyr residues. Although functions of the majority of LRR RLK family members remain unknown, several have proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress response. Because autophosphorylation of specific residues has been shown to be functionally critical for a number of LRR RLKs, we have employed a systems biology approach to identify and characterize sites of autophosphorylation in 73 representative LRR RLKs of the 223 member LRR RLK family in Arabidopsis thaliana. Analysis of the phosphosites identified general but discrete motifs associated with phosphoserine sites, including a strong discrimination against proline residues immediately adjacent to the phosphosite. The motifs have utility to predict phosphorylation sites in RLKs that have not yet been studied experimentally and may also have utility in modifying phosphosites by site-directed mutagenesis. Typically this is done by substituting a similar residue that lacks the –OH group necessary for phosphorylation; e.g., a Ser-to-Ala substitution but an alternative approach may be to modify positive recognition elements instead of the phosphorylated residue itself. The autophosphorylation database for the Arabidopsis LRR RLK family that will also serve as a useful entry point for family-wide analysis of receptor kinase phosphorylation. Many of the LRR RLKs in this database have known functions and are actively being investigated by numerous laboratories. The data generated here may guide site-directed mutagenesis of specific phosphorylation sites followed by expression in an LRR RLK mutant with a visible phenotype to determine the extent of rescue to wild type, which is a standard approach for LRR RLK functional phosphorylation site analyses.

Technical Abstract: We conducted a family-wide study to identify and characterize sites of autophosphorylation in 73 representative LRR RLKs of the 223 member LRR RLK family in Arabidopsis thaliana. His-tagged constructs of intact cytoplasmic domains (CDs) for 73 of 223 A. thaliana LRR RLKs were cloned into E. coli BL-21 cells. Following IPTG induction, His-LRR RLKs were purified using Ni-NTA beads, and then trypsinized following an optional in vitro kinase autophosphorylation reaction. Digests were then split, with a portion of each sample analyzed directly by LC/MS/MS and the remaining portion subjected to phosphopeptide enrichment using Fe-IMAC prior to LC/MS/MS analysis. Samples were analyzed using either a Q-Tof Premier and/or Orbitrap Elite LC/MS/MS system. Phosphorylation sites were identified by a combination of TAIR10 database searching with Mascot, automated phosphosite localization with Mascot or PhosphoRS, and manual curation. Motif analysis was conducted with the pLogo program. Although identification and functional characterization of in vivo phosphorylation sites is ultimately required for full understanding of LRR RLK biology and function, identification of in vitro sites provides a highly predictive, straightforward approach for the identification of putative sites of interest for further targeted in vivo or site-specific mutation studies. The present work describes identification of in vitro phosphorylation sites (pSer and pThr) in 73 A. thaliana LRR RLKs, development of an on-line database of these sites which includes supporting MS/MS spectral data, and motif analysis of the assigned sites. A total of 591 phosphorylation events were identified across the 73 LRR RLKs CDs studied, with 496 sites uniquely assigned to specific Ser (268 sites) or Thr (228 sites) residues in 68 LRR RLKs. For another 95 phosphorylation events, unambiguous site localization to a specific residue within the tryptic peptide was not possible. The average number of unique phosphorylation sites per LRR RLK was seven, with the highest number being 23 identified in At2g01820.1. A total of 22 LRR RLKs in this study had at least 10 or more unique autophosphorylation sites identified within their CDs. LRR RLK autophosphorylation sites identified using the in vitro approach were often found to identify not only sites previously reported from in vivo studies, but additional sites not yet reported from these in vivo analyses. For example, all five known in vivo autophosphorylation sites in BRASSINOSTEROID INSENSITIVE 1-associating receptor kinase 1 (BAK1) were detected using our in vitro approach, along with an additional 12 autophosphorylation sites not previously observed in vivo. Additional data will be presented with respect to trends in the localization of sites across various subdomains in the CDs from LRR RLKs utilized in this study, along with statistically significant sequence motifs observed for autophosphorylation sites in the in vitro datasets.