BAK1, an Arabidopsis LRR receptor-like protein kinase,interacts with BRI1 and modulates brassinosteroid signaling

Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling.     

BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling

The Arabidopsis BAK1 (BRI1 Associated receptor Kinase 1) was identified by a yeast two-hybrid screen as a specific interactor for BRI1, a critical component of a membrane brassinosteroid (BR) receptor. In yeast, BAK1/BRI1 interaction activates their kinase activities through transphosphorylation. BAK1 and BRI1 share similar gene expression and subcellular localization patterns and physically associate with each other in plants. Overexpression of the BAK1 gene leads to a phenotype reminiscent of BRI1-overexpression transgenic plants and rescues a weak bri1 mutant. In contrast, a bak1 knockout mutation gives rise to a weak bri1-like phenotype and enhances a weak bri1 mutation. We propose that BAK1 and BRI1 function together to mediate plant steroid signaling.     

Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.     

Brassinosteroid-independent functions of the BRI1-associated kinase BAK1/SERK3

Eukaryotes have evolved programmed cell death (PCD) mechanisms that play important roles in both, development and immunity.^1–3 We demonstrated a requirement for the Arabidopsis thaliana leucine-rich repeat receptor-like kinase (LRR-RLK), BAK1/SERK3 (BRI1-Associated receptor Kinase 1/Somatic Embryogenesis Receptor Kinase 3) in regulating the containment of microbial infection-induced necrosis. BAK1-deficient plants showed constitutive expression of defense-related genes and developed spreading cell death upon infection by necrotizing pathogens that result in enhanced susceptibility to necrotrophic pathogens. This reaction was not inducible by exposition of bak1 mutants to general stresses but appeared to be solely inducible by necrotizing pathogen infection. BAK1 is known to interact with the brassinosteroid receptor, BRI1, and thereby facilitates plant growth and development in a brassinolide (BL)-dependent manner.^4,5 Surprisingly, the cell death-related phenotype in bak1 mutants is brassinolide-independent. In this addendum we want to present recent new data on BAK1 and discuss its role as a general regulator in plant processes being as diverse as brassinosteroid signaling in development, perception of pathogen associated molecular patterns (PAMPs), and cell-death control in innate immunity.Key words: LRR-RLK, cell-death control, immunity, brassinosteroids, BAK1, SERK3, BRI1, FLS2     

BAK1 Directly Regulates Brassinosteroid Perception and BRI1 Activation

Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid （BR） signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRII. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.     

Heterodimerization of BAK and MCL-1 activated by detergent micelles

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.     

The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarization of the plasma membrane, which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarization and wall expansion, the activation of the plasma membrane P-type H⁺-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyze long-distance BL signaling in Arabidopsis seedlings.Key words: BRI1, fluorescence lifetime, membrane potential, P-ATPase, cell wall expansionUsing spectro-microscopic technologies, we recently started the quantitative analysis of the properties and subcellular function of GFP fusion of the plasma membrane-localized brassinosteroid (BR) receptor, BRI1, in living plant cells of Arabidopsis thaliana and tobacco (Nicotiana benthamiana) leaf cells.^1,2 Brassinosteroids, such as brassinolide (BL), are involved in responses to biotic and abiotic stresses and developmental processes, including cell elongation.³ The present model of the BR response pathway includes the binding of BRs to BRI1, resulting in the autophosphorylation of the receptor and the subsequent recruitment of the co-receptor BRI1-associated receptor kinase 1 (BAK1). This association is followed by trans-phosphorylation between BRI1 and BAK1 and results in the activation of downstream BR signaling processes leading to differential gene expression and, finally, to the execution of the specific responses.⁴ However, the molecular events that take place in the plasma membrane immediately after the perception of BL and initiate cell elongation still have to be included in this model.⁵ We recently reported a rapid BRI1-GFP-dependent cell wall expansion in Arabidopsis seedlings, which is attributed to wall loosening and water incorporation into the wall, and precedes cell elongation.^1,2 This expansion response was accompanied by a change in the FLT of BRI1-GFP, which reflects an alteration in the plasma membrane potential (E_m).^2,6 For both the FLT change in BRI1-GFP and the wall expansion, the activity of the plasma membrane P-ATPase is crucial. Notably, H⁺-pump activation was shown to depend on the kinase activity of BRI1.² This suggests a fast BRI1-dependent response pathway in the plasma membrane which links BL perception via P-ATPase activation and E_m hyperpolarization to wall expansion. In this report, we demonstrate that the phosphorylation of a conserved threonine in the auto-inhibitory domain of AHA1 is not required for the enzymatic activation by BRI1 suggesting a novel mechanism by which BRI1 may initiate the activation of the P-ATPase. Furthermore, we show that the FLT of BRI1-GFP is a useful and senstitive probe for the non-invasive analysis of systemic signaling processes in living plants.     

BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are perceived by the plasma membrane-localized leucine-rich-repeat-receptor kinase BRI1. Based on sequence similarity, we have identified three members of the BRI1 family, named BRL1, BRL2 and BRL3. BRL1 and BRL3, but not BRL2, encode functional BR receptors that bind brassinolide, the most active BR, with high affinity. In agreement, only BRL1 and BRL3 can rescue bri1 mutants when expressed under the control of the BRI1 promoter. While BRI1 is ubiquitously expressed in growing cells, the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Loss-of-function of brl1 causes abnormal phloem:xylem differentiation ratios and enhances the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and also exhibit abnormal vascular differentiation. Thus, Arabidopsis contains a small number of BR receptors that have specific functions in cell growth and vascular differentiation.     

BAK7 displays unequal genetic redundancy with BAK1 in brassinosteroid signaling and early senescence in arabidopsis

BRI1-Associated kinase1 (BAK1), a five leucine-rich-repeat containing receptor-like serine/threonine kinase, has been shown to have dual functions: mediating brassinosteroid (BR) signaling and acting in the BR-independent plant defense response. Sequence analysis has revealed that BAK1 has two homologs, BAK7 and BAK8. Because BAK8 deviates from the canonical RD kinase motif, we focused on the functional analysis of BAK7. The expression pattern and tissues in which BAK7 appeared partially overlapped with those observed for BAK1. Expression levels of BAK7 increased in the bak1 mutant. Overexpression of BAK7 rescued the bri1 mutant phenotype, indicating that BAK7 can compensate for BAK1 in BR-mediated processes, especially in the absence of BAK1. However, root and hypocotyl elongation patterns of transgenic plants overexpressing BAK1 or BAK7 appeared to be different from the patterns observed in a BRI1 overexpressor. Furthermore, the sensitivity of transgenic plants overexpressing BAK7 to brassinazole, a biosynthetic inhibitor of brassinolide (BL), did not change compared to that of wild-type plants. In addition, we generated transgenic plants expressing BAK7 RNA interference constructs and found severe growth retardation and early senescence in these lines. Taken together, these results suggest that BAK7 is a component of the BR signaling pathway, with varying degrees of genetic redundancy with BAK1, and that it affects plant growth via BL-independent pathways in vivo.     

BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.     

AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana.

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.     

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1‐associated kinase 1 (BAK1)‐interacting receptor‐like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.     

拟南芥BAK1基因转化及防御作用

将拟南芥BAK1基因采用Gateway方法连接到植物表达载体,通过侵花粉管进行转化,从基因和蛋白表达水平检测转化是否成功。以不同BAK1表达水平植株作为试验材料,分析BAK1在芜菁缩叶病毒(Turnip crinkle virus,TCV)-拟南芥(Col-0)亲和互作系统中对植株防御的影响。结果显示,在接种TCV后,BAK1缺陷型植株对TCV较为感病,衰老相关基因表达水平增加,表明BAK1能够增强宿主对病毒的防御作用。     

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP‐dependent downstream signaling

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine‐rich repeat receptor‐like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co‐factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.