Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3)

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3(NPH3), with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.     

Mapping of the phosphorylation sites on the phototropic signal transducer, NPH3

The phototropic response in Arabidopsis thaliana is initiated by the blue-light photoreceptors, phototropin (phot)1 and phot2. A recent study has shown that one of the phototropic signal transducers, NPH3, is phosphorylated under dark conditions and dephosphorylated by blue-light irradiation. To further understand the function of phosphorylation and dephosphorylation of NPH3 during this phototropic response, we have mapped the phosphorylation sites of NPH3 in our current study. The NPH3 protein has at least three phosphorylation sites at serine residues, Ser212, Ser222, and Ser236, and these sites are dephosphorylated by blue-light irradiation. By immunoblotting analysis, these phosphorylation sites in phot1 mutants are not dephosphorylated following blue-light irradiation at both low and high fluence rates, even though such irradiations induce the phot2-dependent phototropic response in phot1. These results suggest that the dephosphorylated NPH3 is involved in the phot1-dependent phototropic response and is not essential for the phot2-dependent phototropic response. We generated two types of transgenic nph3 plants expressing a NPH3^{S212A/S222A/S232A/S236A} protein and a NPH3^Δ212–238 protein in which the phosphorylation region is deleted, and assessed the phototropic phenotype of these. Based upon our present findings, we discuss the role of dephosphorylated and phosphorylated NPH3 in mediating the phototropic response.     

FERONIA is involved in phototropin 1-mediated blue light phototropic growth in Arabidopsis

Plant shoot phototropism is triggered by the formation of a light-driven auxin gradient leading to bending growth. The blue light receptor phototropin 1(phot1) senses light direction, but how this leads to auxin gradient formation and growth regulation remains poorly understood. Previous studies have suggested phot1’s role for regulated apoplastic acidification, but its relation to phototropin and hypocotyl phototropism is unclear. Herein, we show that blue light can cause phot1 to interact with...     

Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli.

The phototropic response is an important component of seedling establishment in higher plants because it orients the young seedlings for maximal photosynthetic light capture. Despite their obvious importance, little is known about the mechanisms underlying the perception and transduction of the light signals that induce phototropic curvatures. Here, we report the isolation of eight mutants of Arabidopsis that lack or have severely impaired phototropic responses. These nph (for nonphototropic hypocotyl) mutants comprise four genetic loci: nph1, nph2, nph3, and nph4. Physiological and biochemical characterization of the nph1 allele series indicated that the NPH1 locus may encode the apoprotein for a dual-chromophoric or multichromophoric holoprotein photoreceptor capable of absorbing UV-A, blue, and green light and that this photoreceptor regulates all the phototropic responses of Arabidopsis. It appears that the NPH1 protein is most likely a 120-kD plasma membrane-associated phosphoprotein because all of the nph1 mutations negatively affected the abundance of this protein. In addition, the putative NPH1 photoreceptor protein is genetically and biochemically distinct from the HY4 protein, which most likely acts as a photoreceptor for blue light-mediated hypocotyl growth inhibition. Furthermore, the NPH1 and HY4 proteins are not functionally redundant because mutations in either gene alone affect only one physiological response but not the other, thus providing strong support for the hypothesis that more than one blue light photoreceptor is required for the normal growth and development of a seedling.     

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

Dynamics of conformational changes of Arabidopsis phototropin 1 LOV2 with the linker domain

Conformational changes of Arabidopsis phot1-LOV2 with the linker (phot1-LOV2-linker) were investigated from the viewpoint of the changes in molecular volume and molecular diffusion coefficient (D) by time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectrum change completes within a few microseconds, the D-value detected by the TG method decreased drastically with a time constant of 1.0 ms from 9.2(+/-0.4)x10(-11) m(2)/s to 5.0(+/-0.3)x10(-11) m(2)/s. This time-dependent D was interpreted in terms of the unfolding of alpha-helices in the linker region. The change of the alpha-helices was confirmed by observing the recovery of the circular dichroism intensity. The TrL signal showed that the molecular volume decreases with two time constants; 300 micros and 1.0 ms. The former time constant is close to the previously observed photo-dissociation reaction rate of the phot1-LOV2 (without the linker) dimer, and the latter one agrees well with the rate of the D-change. Considering a similar time constant of the dissociation reaction of the LOV2 dimer, we interpreted these kinetics in terms of the dissociation step of the linker region from the LOV2 domain (T(390)(pre) state). After this step, the protein volume and D are decreased significantly with the lifetime of 1.0 ms. The D decrease indicates the increase of the intermolecular interaction between the protein and water molecules. On the basis of these observations, a two-step mechanism of the linker unfolding is proposed.     

Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.     

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.     

Regulation of fragile X mental retardation 1 protein by C-terminus of Hsc70-interacting protein depends on its phosphorylation status

The fragile X mental retardation 1 (FMR1) protein binds mRNA and acts as a negative regulator of translation. Lack of FMR1 causes the most common neurological disorder, fragile X syndrome, while its overexpression is associated with metastasis of breast cancer. Its activity has been well-studied in nervous tissue, but recent evidence as well as its role in cancer indicates that it also acts in other tissues. We have investigated the expression of FMR1 in brain and other tissues of mouse and examined its regulation. We detected expression of FMR1 in liver and heart tissues of mice as well as in brain tissue, supporting other contentions that it acts in non-nervous tissue. Expression of FMR1 inversely correlated with expression of the C-terminus of Hsc70-interacting protein (CHIP) and, based on the known activity of CHIP in protein homeostasis, we suggest that CHIP regulates expression of FMR1. CHIP ubiquitinated FMR1 for proteasomal degradation in a molecular chaperone-independent manner. FMR1 expression was reduced following treatment with okadaic acid, a phosphatase inhibitor, but not in CHIP-depleted cells. Also, a non-phospho FMR1 mutant was much less efficiently ubiquitinated by CHIP and had a longer half-life compared to either wild-type FMR or a phospho-mimic mutant. Taken together, our results demonstrate that CHIP regulates the levels of FMR1 as an E3 ubiquitin ligase in phosphorylation-dependent manner, suggesting that CHIP regulates FMR1-mediated translational repression by regulating the levels of FMR1.     

Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding

Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.     

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex

Phototropin (phot1) is a blue light‐activated plasma membrane‐associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non‐Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non‐invasive approach where PHOT1–GFP (P1–GFP) expression was targeted to the hypocotyl apex of the phot‐deficient mutant using the promoters of CUP‐SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1–GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1–GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m^?2 sec^?1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1–GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de‐phosphorylation showed that CUC3::P1–GFP and ANT::P1–GFP mis‐express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1‐mediated NPH3 de‐phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.     

Phot2‐regulated relocation of NPH3 mediates phototropic response to high‐intensity blue light in Arabidopsis thaliana

Two redundant blue‐light receptors, known as phototropins (phot1 and phot2), influence a variety of physiological responses, including phototropism, chloroplast positioning, and stomatal opening in Arabidopsis thaliana. Whereas phot1 functions in both low‐ and high‐intensity blue light (HBL), phot2 functions primarily in HBL. Here, we aimed to elucidate phot2‐specific functions by screening for HBL‐insensitive mutants among mutagenized Arabidopsis phot1 mutants. One of the resulting phot2 signaling associated (p2sa) double mutants, phot1 p2sa2, exhibited phototropic defects that could be restored by constitutively expressing NON‐PHOTOTROPIC HYPOCOTYL 3 (NPH3), indicating that P2SA2 was allelic to NPH3. It was observed that NPH3‐GFP signal mainly localized to and clustered on the plasma membrane in darkness. This NPH3 clustering on the plasma membrane was not affected by mutations in genes encoding proteins that interact with NPH3, including PHOT1, PHOT2 and ROOT PHOTOTROPISM 2 (RPT2). However, the HBL irradiation‐mediated release of NPH3 proteins into the cytoplasm was inhibited in phot1 mutants and enhanced in phot2 and rpt2‐2 mutants. Furthermore, HBL‐induced hypocotyl phototropism was enhanced in phot1 mutants and inhibited in the phot2 and rpt2‐2 mutants. Our findings indicate that phot1 regulates the dissociation of NPH3 from the plasma membrane, whereas phot2 mediates the stabilization and relocation of NPH3 to the plasma membrane to acclimate to HBL.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway.

Four genetic loci were recently identified by mutations that affect phototropism in Arabidopsis thaliana (L.) Heyhn. seedlings. It was hypothesized that one of these loci, NPH1, encodes the apoprotein for a phototropic photoreceptor. All of the alleles at the other three mutant loci (nph2, nph3, and nph4) contained wild-type levels of the putative NPH1 protein and exhibited normal blue-light-dependent phosphorylation of the NPH1 protein. This indicated that the NPH2, NPH3, and NPH4 proteins likely function downstream of NPH1 photoactivation. We show here that, although the nph2, nph3, and nph4 mutants are all altered with respect to their phototropic responses, only the nph4 mutants are also altered in their gravitropic responsiveness. Thus, NPH2 and NPH3 appear to act as signal carriers in a phototropism-specific pathway, whereas NPH4 is required for both phototropism and gravitropism and thus may function directly in the differential growth response. Despite their altered phototropic responses in blue and green light as etiolated seedlings, the nph2 and nph4 mutants exhibited less dramatic mutant phenotypes as de-etiolated seedlings and when etiolated seedlings were irradiated with unilateral ultraviolet-A (UV-A) light. Examination of the phototropic responses of a mutant deficient in biologically active phytochromes, hy1-100, indicated that phytochrome transformation by UV-A light mediates an increase in phototropic responsiveness, accounting for the greater phototropic curvature of the nph2 and nph4 mutants to UV-A light than to blue light.     

Regulation of the autophagy protein LC3 by phosphorylation

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP⁺) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.     

Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.     

BAK1‐mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis

Heterotrimeric G proteins consisting of Gα, Gβ and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein‐coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr¹⁹ upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1^T19A and GPA1^T19D, suggesting that the dynamic Thr¹⁹ phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22‐induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1^T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22‐induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.     

LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1

Phototropin is a blue light receptor in plants and is thought to be a light-regulated protein kinase. Previously, we defined the role of the photoreceptive domains, LOV1 and 2, in the light activation of the kinase in Arabidopsis phototropin2 (phot2). In this study, photoregulation of the kinase in phototropin1 (phot1) was studied using LOV2-linker-kinase polypeptide. We designed a new substrate consisting of the N-terminal part of the phot1 with autophosphorylation sites. The LOV2-linker-kinase had the same spectroscopic properties as those of the LOV2 core and phosphorylated the substrate in a light-dependent manner. Amino acid substitution experiments proved that the phosphorylation comes from the activation of the kinase via photoreaction of LOV2.     

Structural insights into the negative regulation of BRI1 signaling by BRI1-interacting protein BKI1

Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.