Man Ho Oh

Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis

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.

Lysine Acetylation is a Widespread Protein Modification for Diverse Proteins in Arabidopsis

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 (β-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.

Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression

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.

Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis.

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.

A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

Move and Countermove Receptors on plant cell surfaces are tuned to recognize molecular patterns associated with pathogenic bacteria. Macho et al. (p. 1509; published online 13 March) found that activation of one of these receptors in Arabidopsis results in phosphorylation of a specific tyrosine residue, which in turn triggers the plants immune response to the phytopathogen Pseudomonas syringae. P. syringae counters by secreting a specifically targeted phosphatase, thus stalling the plants immune response. A plant pathogen and its host compete for control over a key phosphorylation site in an innate immune receptor. 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.

Deactivation of the Arabidopsis BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor kinase by autophosphorylation within the glycine-rich loop.

The activity of the dual-specificity receptor kinase, brassinosteroid insensitive 1 (BRI1), reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we elucidate a unique mechanism for deactivation that involves autophosphorylation of serine-891 in the ATP-binding domain. Serine-891 was identified previously as a potential site of autophosphorylation by mass spectrometry, and sequence-specific antibodies and mutagenesis studies now unambiguously establish phosphorylation of this residue. In vivo, phosphorylation of serine-891 increased slowly with time following application of brassinolide (BL) to Arabidopsis seedlings, whereas phosphorylation of threonine residues increased rapidly and then remained constant. Transgenic plants expressing the BRI1(S891A)–Flag-directed mutant have increased hypocotyl and petiole lengths, relative to wild-type BRI1–Flag (both in the bri1-5 background), and accumulate higher levels of the unphosphorylated form of the BES1 transcription factor in response to exogenous BL. In contrast, plants expressing the phosphomimetic S891D-directed mutant are severely dwarfed and do not accumulate unphosphorylated BES1 in response to BL. Collectively, these results suggest that autophosphorylation of serine-891 is one of the deactivation mechanisms that inhibit BRI1 activity and BR signaling in vivo. Many arginine-aspartate (RD)-type leucine-rich repeat receptor-like kinases have a phosphorylatable residue within the ATP-binding domain, suggesting that this mechanism may play a broad role in receptor kinase deactivation.

Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.

Enhancing Arabidopsis Leaf Growth by Engineering the BRASSINOSTEROID INSENSITIVE1 Receptor Kinase

The BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase has recently been shown to possess tyrosine kinase activity, and preventing autophosphorylation of the tyrosine-831 regulatory site by site-directed mutagenesis enhances shoot growth. In this study, we characterized the increased leaf growth of Arabidopsis (Arabidopsis thaliana) plants expressing BRI1(Y831F)-Flag compared with BRI1-Flag (both driven by the native promoter and expressed in the bri1-5 weak allele background) and provide insights into the possible mechanisms involved. On average, relative leaf growth rate was increased 16% in the Y831F plants (in the bri1-5 background), and the gain of function of the Y831F-directed mutant was dominant in the wild-type background. Leaves were larger as a result of increased cell numbers and had substantially increased vascularization. Transcriptome analysis indicated that genes associated with brassinolide biosynthesis, secondary cell wall biosynthesis and vascular development, and regulation of growth were altered in expression and may contribute to the observed changes in leaf architecture and whole plant growth. Analysis of gas exchange and chlorophyll fluorescence indicated that Y831F mutant plants had higher rates of photosynthesis, and metabolite analysis documented enhanced accumulation of starch, sucrose, and several amino acids, most prominently glycine and proline. These results demonstrate that mutation of BRI1 can enhance photosynthesis and leaf growth/vascularization and may suggest new approaches to increase whole plant carbon assimilation and growth.

Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a post-translational modification and is activated by the juxtamembrane domain

In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is Brassinosteroid Insensitive 1 (BRI1), which functions in brassinosteroid signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate in vitro exclusively on serine and threonine residues and subsequently numerous specific phosphoserine and phosphothreonine sites were identified. However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine) residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchical and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. This may explain why tyrosine autophosphorylation was not observed in some studies. Finally, we also show that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and that kinase specificity (serine/threonine versus tyrosine) can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.

Tyrosine phosphorylation in brassinosteroid signaling.

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases1. Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.