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     

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.     

Receptor-like protein kinases,BAK1 and BKK1, regulate a light-dependent cell-death control pathway

BAK1 and BKK1 are two functionally redundant leucine-rich repeat receptor-like protein kinases (LRR-RLKs) involved in brassinosteroid signal transduction by their direct interactions with the BR receptor, BRI1. Recent studies from our group and others indicated that the two RLKs also play critical roles in regulating pathogen-related and pathogen-unrelated cell-death controls. Genetic data suggest that the two kinases are essential for plant survival because the double mutants show spontaneous cell-death and seedling lethality phenotypes. Physiological analyses further suggest that the cell-death of the double mutant is triggered by the light, as dark-grown seedlings do not show any cell-death symptoms. These observations indicate that BAK1 and BKK1 regulate a novel signaling pathway to detoxify or to limit the production of a yet unknown toxin/toxins produced by plants under light conditions.Key words: receptor-like kinases, cell-death, light, reactive oxygen speciesPlant receptor-like protein kinases (RLKs) are transmembrane proteins essential for cell-to-cell communications. A typical RLK is composed of a cell-surface receptor domain which can sense and perceive diversified signaling molecules within the extracellular space, a transmembrane domain anchoring the protein to the plasma membrane, and a cytoplasmic kinase domain whose activity can often be regulated by the conformation change in the receptor domain upon the binding of the signaling molecules to the receptor. The unique structure of RLKs suggests that these kinases may act as key switches in triggering many signal transduction cascades which greatly influence plant growth and development. Recent studies support this notion, as the functions of more and more RLKs have been revealed, and these RLKs indeed play critical roles in mediating many physiological processes such as steroidal hormone action, pathogenesis responses, and recognition of various peptide signals.^1–3 There are more than 600 RLKs in the Arabidopsis genome.^4,5 Based on the structure of the receptor domains, RLKs can be divided into more than 10 subfamilies. Among them, LRR-RLKs belong to the largest group consisting of at least 220 members. The functions of only a small fraction of RLKs have been revealed.BAK1 is a typical LRR-RLK, identified via an activation tagging genetic screen for suppressors of a weak BR receptor (BRI1) mutant called bri1–5, and via a yeast two-hybrid screen for BRI1 kinase domain physical interactors.^6,7 Although the detailed molecular mechanisms of BAK1 in activating the BR signaling pathway is still mysterious, the in vivo interaction between BAK1 and BRI1 is clearly ligand (BR)-dependent.⁸ The mutual phosphorylation of the two kinases is also BR-dependent.⁸ BKK1, the closest homolog of BAK1, was identified as a functionally redundant protein of BAK1.⁹ Interestingly, the double null mutant of BAK1 and BKK1, bak1–4 bkk1-1, did not show a typical bri1 phenotype but showed a spontaneous cell-death phenotype under a normal growth condition. This unexpected result suggests that BAK1 and BKK1 may have more roles than their functions in BR signal transduction. This hypothesis is supported by the recent discovery of BAK1 in mediating pathogen-related signaling pathways in order to regulate innate immunity and cell-death control.^10–12 The spontaneous cell-death seen in the bak1–4 bkk1-1 double mutant, however, is not caused by the challenges from pathogens;¹⁰ it is also unlikely to be the result from the disruption of the FLS2-dependent innate immunity pathway,^11,12 as overexpression or T-DNA disruption of the RLK gene, FLS2, does not show a phenotype similar to that of the bak1–4 bkk1-1 double mutant. In addition, the cell-death phenotype of the double mutant occurs even in a sterile growth condition, suggesting that the pathogens are not the key triggers of cell-death in the bak1–4 bkk1-1 double mutant. Early results indicated that the double mutant seedlings are indistinguishable from the wild-type seedlings during the first 4–5 days after germination but quickly show terminating growth and cotyledon necrosis phenotypes a week after germination.⁹ This observation prompted a test of whether light is a true trigger for cell-death seen in the double mutant. Both wild-type and the double mutant were planted in the dark and long-day lighting conditions. Cotyledons from eight-day-old seedlings were stained with Trypan blue to examine cell-death symptoms of the seedlings grown under different illumination conditions.¹³ Both the dark-grown wild-type and the double mutant seedlings showed no cell-death symptoms on their cotyledons at any time during a three-week experimental period (Fig. 1A and B). Under a long-day lighting condition, on the other hand, cotyledons from the double mutant, but not from the wild-type, exhibited severe cell-death symptoms (Fig. 1C and D). Three weeks after germination, the double mutant seedlings growing under a long-day lighting condition was completely dead, the ones under the dark condition were still healthy and showed no cell-death symptoms (data not shown).Open in a separate windowFigure 1BAK1 and BKK1 regulate a light-dependent cell-death control pathway. (A) A representative Trypan blue stained wild-type cotyledon from a dark grown seedling on a ½ MS plate; (B) A representative Trypan blue stained bak1–4 bkk1-1 cotyledon from a dark grown seedling on a ½ MS plate; (C) A representative Trypan blue stained wild-type cotyledon from a long-day light-grown seedling on a ½ MS plate; (D) A representative Trypan blue stained bak1–4 bkk1-1 cotyledon from a long-day light-grown seedling on a ½ MS plate; (E) A hypothetical model of BAK1 and BKK1 in regulating both the BR signaling pathway to promote cell growth, and a novel light-dependent cell-death control pathway to prevent plants from unnecessary cell-death. Under a light condition, plants naturally produce unknown toxins (phototoxins), whose accumulation can lead to the cell-death. BAK1 and BKK1 likely mediate a signaling pathway to constantly check and limit the levels of these toxins.Based on our current results, it is apparent that the double mutant is more vulnerable to light. It is probable that the mutant lost its capability to detoxify or to restrict the production of an unknown toxin/toxins naturally generated by plants under a light condition. The wild-type plants may also produce the toxin/toxins, but BAK1 and BKK1 can direct a signal transduction pathway to constantly check and eliminate extra amount of the toxin/toxins (Fig. 1E). Under a sterile growth condition, BAK1 and BKK1 are likely involved in both the BR signaling pathway to positively regulate cell growth and in a novel pathway to negatively control cell-death. Under normal environmental conditions (not sterile condition), BAK1 might also be recruited to participate in the innate immunity pathway via its interaction with FLS2 and other RLKs. Based on the model from the BRI1/BAK1 signal transduction, there might be another RLK which can pair with BAK1 or BKK1 in controlling the light-dependent cell-death process. An unknown “survival signal” could be an unknown metabolite or the toxin/toxins causing the cell-death. Under the current model, the “survival signal” may activate the BAK1- and BKK1-associated stress defense pathway and constantly check the levels of the light-dependent toxin/toxins in the plants. The homeostasis of the toxin/toxins is therefore strictly under control. If both BAK1 and BKK1 are removed, as in the case of the double mutant, the plant loses its capability to check the levels of the toxin/toxins. The uncontrolled accumulation of the toxin/toxins is likely the ultimate cause of the spontaneous cell-death observed in the double mutant.     

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.     

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.     

Negative control of BAK1 by protein phosphatase 2A during plant innate immunity

Recognition of pathogen‐associated molecular patterns (PAMPs) by surface‐localized pattern‐recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP‐triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co‐receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B’η/ζ inhibits immune responses triggered by several PAMPs and anti‐bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A‐based regulation leads to increased steady‐state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface‐localized immune receptor complexes.     

Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1

Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.     

SLK1, a yeast homolog of MAP kinase activators, has a RAS/cAMP-independent role in nutrient sensing

The Saccharomyces cerevisiae SLK1 protein is implicated in nutrient sensing and growth control. Under nutrient-limiting conditions, slk1 mutants fail to undergo cell cycle arrest. The role of the SLK1 protein in nutrient sensing was examined with respect to the cAMP-dependent protein kinase (PKA) pathway, which has a well characterized role in growth control in yeast, and by the analysis of dominant SLK1 alleles that affect the nutrient response of wild-type cells. Interactions with the PKA pathway were examined by phenotypic analysis of double mutants of slk1 and various PKA pathway mutants. Combining the slk1- mutation with a mutation that is thought constitutively activate the PKA pathway, pde2, resulted in enhanced growth control defects. The combination of slk1- with mutations that inhibit the PKA pathway, cdc25 and ras1 ras2, failed to alleviate the slk1 cell cycle arrest defect and lowered the permissive temperature for growth. Furthermore bcy1 tpk1 tpk2 tpk3 ^w (bcyl tpk ^w) mutants, which have constitutive, low-level, cAMP-independent kinase activity, exhibit nutrient sensing, which is eliminated in the slk1 bcy1 tpk ^w mutants. These results implicated SLK1 in PKA-independent growth control in yeast. The amino-terminal, noncatalytic region of the SLK1 protein may be important in the regulation of SLK1 function in growth control. Overexpression of this region caused starvation sensitivity in wild-type cells by interfering with SLK1 protein function.     

The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization

Interactor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the first plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green fluorescent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1DeltaN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1DeltaN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1DeltaN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.     

CHRK1, a chitinase-related receptor-like kinase,plays a role in plant development and cytokinin homeostasis in tobacco

CHRK1 encodes a receptor-like kinase that contains a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. In this study, we showed that CHRK1 is mainly expressed in the shoot apex region including leaf primordia and young leaves, and germinating seedlings and vascular tissues, based on GUS activity of transgenic tobacco plants carrying the CHRK1 promoter-GUS fusion gene. Transgenic tobacco plants in which CHRK1 expression was suppressed exhibited pleiotrophic developmental abnormality, including formation of proliferating shooty calli from emerging seedlings and severely altered seedling development. At the cellular level, ectopic cell proliferation, reduced cell specificity, and aberrant chloroplast development were observed. The transgenic lines contained 3-fold higher level of cytokinin than the wild-type plants. Consistently, the transgenic seedlings exhibited a typical cytokinin response in the absence of hormone, such as deetiolation under the dark. Based on these results, we propose that CHRK1 is involved in a developmental signaling pathway regulating cell proliferation/differentiation and the endogenous cytokinin levels in tobacco.     

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.     

Death-associated protein kinase 1 has a critical role in aberrant tau protein regulation and function

The presence of tangles composed of phosphorylated tau is one of the neuropathological hallmarks of Alzheimer''s disease (AD). Tau, a microtubule (MT)-associated protein, accumulates in AD potentially as a result of posttranslational modifications, such as hyperphosphorylation and conformational changes. However, it has not been fully understood how tau accumulation and phosphorylation are deregulated. In the present study, we identified a novel role of death-associated protein kinase 1 (DAPK1) in the regulation of the tau protein. We found that hippocampal DAPK1 expression is markedly increased in the brains of AD patients compared with age-matched normal subjects. DAPK1 overexpression increased tau protein stability and phosphorylation at multiple AD-related sites. In contrast, inhibition of DAPK1 by overexpression of a DAPK1 kinase-deficient mutant or by genetic knockout significantly decreased tau protein stability and abolished its phosphorylation in cell cultures and in mice. Mechanistically, DAPK1-enhanced tau protein stability was mediated by Ser71 phosphorylation of Pin1, a prolyl isomerase known to regulate tau protein stability, phosphorylation, and tau-related pathologies. In addition, inhibition of DAPK1 kinase activity significantly increased the assembly of MTs and accelerated nerve growth factor-mediated neurite outgrowth. Given that DAPK1 has been genetically linked to late onset AD, these results suggest that DAPK1 is a novel regulator of tau protein abundance, and that DAPK1 upregulation might contribute to tau-related pathologies in AD. Therefore, we offer that DAPK1 might be a novel therapeutic target for treating human AD and other tau-related pathologies.     

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.     

The effect of ICK1, a plant cyclin-dependent kinase inhibitor,on mitosis in living plant cells

The inhibitory activity of Arabidopsis thaliana ICK1, a plant cyclin-dependent kinase inhibitor, has previously been characterised by its effect on plant cyclin-dependent kinase activity in vitro and its effect on growth in transgenic plants. Herein, we examine cyclin-dependent kinase-driven cell-cycle events, probed by testing the sensitivity of living cells to introduced ICK1 protein. The microinjection of ICK1 into individual Tradescantia virginiana stamen hair cells during late prophase and prometaphase resulted in a clear protein-specific increase in the metaphase transit time (time from nuclear envelope breakdown to the onset of anaphase) in a manner dependent on load and injection time. The results indicate a continuing role for cyclin-dependent kinases in mitotic progression and provide in vivo evidence at the cellular level that ICK1 can restrict growth in the plant by inhibiting cell division.