Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1-5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.
BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P Ϫ 3, P Ϫ 4, and P ϩ 5 (relative to the phosphorylated Ser at P ϭ 0).
Lysine acetylation (LysAc), a form of reversible protein posttranslational modification previously known only for histone regulation in plants, is shown to be widespread in Arabidopsis (Arabidopsis thaliana). Sixty-four Lys modification sites were identified on 57 proteins, which operate in a wide variety of pathways/processes and are located in various cellular compartments. A number of photosynthesis-related proteins are among this group of LysAc proteins, including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins (LHCb), Rubisco large and small subunits, and chloroplastic ATP synthase (b-subunit). Using two-dimensional native green/sodium dodecyl sulfate gels, the loosely PSII-bound LHCb was separated from the LHCb that is tightly bound to PSII and shown to have substantially higher level of LysAc, implying that LysAc may play a role in distributing the LHCb complexes. Several potential LysAc sites were identified on eukaryotic elongation factor-1A (eEF-1A) by liquid chromatography/mass spectrometry and using sequence-and modification-specific antibodies the acetylation of Lys-227 and Lys-306 was established. Lys-306 is contained within a predicted calmodulin-binding sequence and acetylation of Lys-306 strongly inhibited the interactions of eEF-1A synthetic peptides with calmodulin recombinant proteins in vitro. These results suggest that LysAc of eEF-1A may directly affect regulatory properties and localization of the protein within the cell. Overall, these findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism of a large number of nonhistone proteins affecting a wide range of pathways and processes in Arabidopsis and likely in all plants.
Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.
BAK1 is a leucine-rich repeat receptor-like kinase that functions as a coreceptor with the brassinosteroid (BR) receptor BRI1 and the flagellin receptor FLS2, and as a negative regulator of programmed cell death. BAK1 has been shown to autophosphorylate on numerous serine/threonine sites in vitro as well as to transphosphorylate associated receptor kinases both in vitro and in planta. In the present study we identify Tyr-610 in the carboxyl-terminal domain of BAK1 as a major site of autophosphorylation that is brassinolide-induced in vivo and requires a kinase-active BAK1. Expression of BAK1(Y610F)-Flag in transgenic plants lacking the endogenous bak1 and its functional paralogue, bkk1 , produced plants that were viable but extremely small and generally resembled BR signaling mutants, whereas an acidic substitution for Tyr-610 to mimic phosphorylation restored normal growth. Several lines of evidence support the notion that BR signaling is impaired in the BAK1(Y610F)-Flag plants, and are consistent with the recently proposed sequential transphosphorylation model for BRI1/BAK1 interaction and activation. In contrast, the FLS2-mediated inhibition of seedling growth by the flg22 elicitor occurred normally in the Y610F-directed mutant. However, expression of many defense genes was dramatically reduced in BAK1(Y610F) plants and the nonpathogenic hrpA mutant of Pseudomonas syringae was able to grow rapidly in the mutant. These results indicate that phosphorylation of Tyr-610 is required for some but not all functions of BAK1, and adds significantly to the emerging notion that tyrosine phosphorylation could play an important role in plant receptor kinase signaling.
The type-A ARRs of cytokinin two-component signaling system act as negative regulators for cytokinin signaling except for ARR4, but the molecular mechanism by which the A-type ARRs regulate cytokinin signaling remain elusive. To get insights into the molecular function of A-type ARR in cytokinin response, we sought to find the components that function downstream of A-type ARR protein by investigating the effects of ARR7 overexpression on cytokinin-regulated gene expression with the Affymetrix full genome array. To examine early cytokinin response, plants were treated with cytokinin for 30 min or 2 h, followed by GeneChip analysis. The hierarchical clustering analysis of our GeneChip data showed that ARR7 overexpression had distinctively repressive impacts on various groups of the cytokinin-regulated genes. In particular, the induction of all A-type ARRs except for ARR22, and AHK(ARABIDOPSIS HISTIDINE KINASE)1 and AHK4 was suppressed by ARR7. Cytokinin-induced expression of most of 12 expansin genes were repressed by ARR7, indicating potential involvement of ARR7 in cell expansion and plant development. Up-regulation of five cytokinin oxidase genes by cytokinins was negatively affected by ARR7. Our GeneChip analysis suggest that ARR7 mainly acts as a transcriptional repressor for a variety of early cytokinin-regulated genes encoding transcription factors, signal transmitters, plant development, and cellular metabolism, which may be responsible for reduced sensitivity of Arabidopsis transgenic plants overexpressing ARR7 to exogenous cytokinins.
The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P+4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534 in vivo. First, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). These results are consistent with the notion that oxidation of surface-exposed Met residues in kinase substrate proteins, such as NR, can inhibit the phosphorylation of nearby sites and thereby couple oxidative signals to changes in protein phosphorylation.
Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis.
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