Progress 09/01/04 to 08/31/09
Outputs
OUTPUTS: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development through a signal transduction pathway that is initiated by BR binding to the transmembrane receptor kinase BRI1. Activated BRI1 associates with a second receptor kinase, BAK1, leading to enhanced signaling output. The detailed molecular mechanisms of phosphorylation, kinase activation and oligomerization of the BRI1/BAK1 pair in response to BR were unknown at the start of this project. Using immunoprecipitation of transgenic BRI1-Flag epitope-tagged Arabidopsis plants followed by LC/MS/MS analysis, we identified at least 11 sites of in vivo phosphorylation of BRI1 and the results have been published. Using a similar approach combining immunoprecipitation from BR-treated Arabidopsis plants followed by LC/MS/MS analysis, we confirmed that BAK1 is phosphorylated on at least three residues in the activation loop in vivo, T-446, T-449 and T-455 (equivalent to BRI1 T-1049). A fourth activation loop site, T-450 is also consistent with our in vivo data, and two other in vivo sites, S-290 and T-312 were also confirmed. These results have also been published. With respect to tomato BRI1 phosphorylation sites, we identified 9 in vitro phosphorylation sites in tomato BRI1 compared to 11 we previously identified in Arabidopsis. Interestingly, seven of the tomato sites were conserved in Arabidopsis, but two were not suggesting significant conservation, but also possible differences in BRI1 downstream signaling between the two species. The functional significance of each of the identified and predicted phosphorylation sites in BRI1 was assessed by site-directed mutagenesis of each specific Ser or Thr to Ala followed by biochemical analysis in vitro and testing for the ability of the altered construct to rescue the weak bri1-5 BR-insensitive mutant in planta. For biochemical function we assessed the effect of mutagenesis on autophosphorylation of the BRI1 cytoplasmic domain in vitro, and on phosphorylation of a synthetic peptide (BR13) containing the previously determined consensus sequence for optimum BRI1 substrate phosphorylation. A similar analysis with mutagenized lines of BAK1 in which phosphorylated Thr or Ser residues were substituted with Ala, Glu, or Asp was also completed. In vitro kinase assays showed that S286D and T455A mutations were completely inactive, similar to the K317E mutant, while T446, T449 and T450 had somewhat reduced kinase function. PARTICIPANTS: Dr. Steven Clouse, PI Dr. Michael Goshe, Co-PI, Dr. Xiaofeng Wang, Postdoctoral Scientist, and Mr. Vikramjit Singh Bajwa, Graduate Student, worked on this project. Training in plant biochemistry, molecular genetics and protein analysis by mass spectrometry was provided to one postdoctoral scientist and one Ph.D. student. Furthermore, two high school summer interns worked on this project under the supervision of Dr. Wang. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Transgenic plants expressing epitope tagged BRI1 and BAK1 (either native or kinase-inactive mutants) in different genetic backgrounds were used to study BR-dependence of the physical association of the pair and the ability to auto- and trans-phosphorylate in the heterooligomer. We showed the association between BRI1 and BAK1 and their respective phosphorylation was BR dependent and the results have been published. We also expressed different combinations of kinase- inactive and wild-type tagged versions of BRI1 and BAK1 in the same transgenic plant and found that an active BRI1 kinase, but not BAK1 kinase, was required for BR-dependent association of the pair. Moreover, when BAK1-GFP was expressed in the bri1-1 null mutant background, phosphorylation levels were dramatically reduced in BAK1-GFP. A range of in vitro kinase assays also showed that BAK1 stimulates BRI1 activity and that both BRI1 and BAK1 can transphosphorylate each other on specific residues. Based on these findings we proposed a novel sequential transphosphorylation mechanism of BR signaling in which BRI1 can act independently from BAK1 (and its homolog BKK1) with respect to ligand binding, initial kinase activation, and participation in a basal level of BR signal transduction. However, the full potential of BR signaling is realized when ligand-activated BRI1 heterodimerizes with BAK1, leading to BAK1 kinase activation by BRI1-mediated transphosphorylation of BAK1 activation loop residues. The activated BAK1 in turn transphosphorylates BRI1 on juxtamembrane and carboxy terminal residues, increasing BR signaling by enhancing the phosphorylation of specific BRI1 substrates. This model has several similarities with, but also distinct differences from, mammalian receptor kinase function.
Publications
- Oh, M.-H., Wang, X., Kota, U., Goshe, M.B., Clouse, S.D., and Huber, S.C. 2009. Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proceedings of the National Academy of Sciences 106, 658-663.
- Malinowski, R., Higgins, R., Luo, Y., Piper, L., Nazir, A., Bajwa, V.S., Clouse, S.D., Thompson, P.R., and Stratmann, J.W. 2009. The tomato brassinosteroid receptor BRI1 increases binding of systemin to tobacco plasma membranes, but is not involved in systemin signaling. Plant Mol Biol.70:603-16.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development through a signal transduction pathway that is initiated by BR binding to the transmembrane receptor kinase BRI1. Activated BRI1 associates with a second receptor kinase, BAK1, leading to enhanced signaling output. The detailed molecular mechanisms of phosphorylation, kinase activation and oligomerization of the BRI1/BAK1 pair in response to BR are unknown. During the past year we examined in detail the interaction between BRI1 and BAK1 in Arabidopsis. We also expressed the cytoplasmic kinase domain of tomato BRI1 in E. coli, autophosphorylated the recombinant protein and determined the phosphorylation sites in tryptic peptides using LC/MS/MS analysis. PARTICIPANTS: Dr. Steven Clouse, PI Dr. Michael Goshe, Co-PI Dr. Xiaofeng Wang, Postdoctoral Scientist Mr. Vikramjit Singh Bajwa, Graduate Student. Training in plant biochemistry, molecular genetics and protein analysis by mass spectrometry was provided to one postdoctoral scientist and one Ph.D. student. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We demonstrated that BR activation of BRI1 precedes that of BAK1 in planta and that BRI1 transphopshorylates BAK1 on specific kinase domain residues, some of which are critical for BAK1 kinase function and optimal BR signaling. BAK1 also transphosphorylates BRI1 on juxtamembrane and carboxy terminal domain residues, increasing BRI1 kinase activity towards a specific substrate. Our results are consistent with a novel sequential transphosphorylation model that clarifies early events of BR signaling and allows a direct comparison of the molecular mechanisms of plant and animal receptor kinases. We also identified 8 in vitro phosphorylation sites in tomato BRI1 (verified by manual inspection of each MS/MS spectra) compared to 11 we previously identified in Arabidopsis. Interestingly, five of the tomato sites were conserved in Arabidopsis, but three were not suggesting significant conservation, but also possible differences in BRI1 downstream signaling between the two species. We also have bulked seeds for an advanced transgenic tomato line expressing full-length tomato BRI1-Flag and are beginning in vivo phosphorylation site analysis by immunoprecipitation of BR-treated tissue followed by LC/MS/MS analysis. The successful identification of tomato BRI1 phosphorylation sites will allow us to perform a comparative functional analysis of BRI1 and BAK1 phosphorylation sites in tomato and Arabidopsis.
Publications
- Wang, X., Kota, U., He, K., Blacburn, K., Li, J., Goshe, M.B., Huber, S.C., and Clouse, S.D. 2008. Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase Complex Impacts Early Events in Brassinosteroid Signaling. Developmental Cell 15:220-235.
- Clouse, S.D. 2008. The Molecular Intersection of brassinosteroid-regulated growth and flowering in Arabidopsis. Proceedings of the National Academy of Sciences USA 105:7345-7346.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) for hormone perception and signal transduction. In previous years of the project we examined early events in BR signaling by using co-immunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR-dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis plants followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS), identified multiple BRI1 in vivo phosphorylation sites and site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that two highly conserved activation loop residues were essential for kinase function in vitro and normal BRI1 signaling in planta. During the current
reporting period we have used a similar approach to study the in vivo sites of phosphorylation for BAK1 and have further examined the in planta function of these sites by mutational analysis. We also examined the association and phosphorylation of BRI1 and BAK1 in various genetic backgrounds, including bri1 and bak1 null mutants, to elucidate the detailed mechanisms of phosphorylation and dimerization in response to BR. These studies are designed to enhance our understanding of early events in BR signal transduction as well as general modes of action for plant receptor kinases.
PARTICIPANTS: Dr. Steven Clouse, PI Dr. Michael Goshe, Co-PI Dr. Xiaofeng Wang, Postdoctoral Scientist Mr. Vikramjit Singh Bajwa, Graduate Student Training in plant biochemistry, molecular genetics and protein analysis by mass spectrometry was provided to one postdoctoral scientist and on Ph.D. student.
Impacts
We identified at least 5 sites of in vivo phosphorylation of BAK1 by LC/MS/MS analysis of samples from immunoprecipitated BR-treated plants. The functional significance of each of the identified and predicted phosphorylation sites in BAK1 was assessed by site-directed mutagenesis of each specific phosphorylation site followed by biochemical analysis in vitro and testing for the ability of the altered construct to suppress the weak bri1-5 BR-insensitive mutant in planta. A wild-type BAK1 transgene suppresses the bri1-5 mutant phenotype but a specific activation loop substitution mutant fails to do so, and causes a dominant negative effective similar to the K317E kinase inactive mutant. A Ser or Thr at this position is found in over 100 Arabidopsis receptor kinases, suggesting this is a critical residue for kinase domain function and signal transduction pathways controlling plant growth and development. Transgenic plants expressing epitope tagged BRI1 and BAK1 (either
native or kinase-minus mutants) in different genetic backgrounds were used to study BR-dependence of the physical association of the pair and the ability to auto- and trans-phosphorylate in the heterodimer. We showed the association between BRI1 and BAK1 and their respective phosphorylation was BR dependent. We also expressed different combinations of kinase-inactive and wild-type tagged versions of BRI1 and BAK1 in the same transgenic plant and found that an active BRI1 kinase but not BAK1 kinase was required for BR-dependent association of the pair. Moreover, when BAK1-GFP was expressed in the bri1-1 null mutant background, phosphorylation levels were dramatically reduced in BAK1-GFP. A range of in vitro kinase assays showed that BAK1 stimulates BRI1 activity and that both BRI1 and BAK1 can transphosphorylate each other on specific residues. This work has advanced our understanding of the molecular mechanisms of these important receptor kinases and their role in regulating plant
growth and development.
Publications
- Clouse, S.D., Goshe, M.B., Huber, S.C., Li, J. 2007. Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases. In, GK Agrawal and R Rakwal (eds) Plant Proteomics: Technologies, Strategies and Applications. John Wiley & Sons (In press).
- Clouse, S.D. 2007. Brassinosteroid Signalling. In, L. Bogre, G. Beemster eds., Plant Growth Signalling. Springer (In Press).
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Progress 10/01/05 to 09/30/06
Outputs
Progress on Specific Objectives: (1) At least eight sites of in vitro or in vivo phosphorylation in BAK1 have been determined in the recombinant BAK1 kinase domain followed by MALDI-TOF MS or in Arabidopsis plants by immunoprecipitation of epitope-tagged BAK1 followed by LC/MS/MS analysis; (2) Thirty-five individual T3 transgenic lines of BRI1-FLAG were generated in which Ser or Thr residues were mutated to Ala, Asp or Glu, and the effect on in planta function is being examined by morphometric analysis. (3) Transgenic plants expressing epitope-tagged BRI1 and BAK1 (either native or kinase-minus mutants) have been generated and are being used to study BR-dependence of the physical association of the pair and the ability to auto- and trans-phosphorylate in the heterodimer. These lines have also been transferred to bri1 and bak1 null mutants to determine the effect of genetic background on in vivo phosphorylation of BRI1-FLAG and BAK1-GFP. Specific sites of
transphosphorylation between the BRI1/BAK1 pair have been determined by LC/MS/MS and MALDI-TOF MS analyses. (4) Transgenic tomato lines expressing tBRI1-FLAG have been generated and identification of in vivo phosphorylation sites is underway using immunoprecipitation and LC/MS/MS analysis.
Impacts
We have shown peviously that the interaction of BRI1 and BAK1 as well as phosphorylation of each receptor kinase is ligand dependent. During the last year we have clarified the mechanism of interaction of the two signaling proteins and have shown that transphosphorylation between the two receptor kinases occurs on specific Ser and Thr residues and is critical for normal BR signaling. This is advancing our understanding of the molecular mechanisms of these important receptor kinases and their role in regulating plant growth and development.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Objectives: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active Brassinosteroid Insensitive 1 (BRI1) receptor serine/threonine kinase for hormone perception and signal transduction. The recent discovery of BRI1-Associated Receptor Kinase 1 (BAK1), a second LRR-RLK that interacts with BRI1 in vitro and in vivo, suggests that receptor kinase heterodimerization may play an important role in BR signal transduction. Our objective is to examine early events in BR signal transduction by (1) Identifying the in vivo phosphorylation sites of BAK1; (2) Performing a functional analysis of BRI1 and BAK1 phosphorylation sites in vivo; (3) Studying factors that affect the BRI1/BAK1 in vivo interaction; (4) Undertaking a comparative analysis of early events in tomato and Arabidopsis BR signal transduction. Progress on Specific Objectives: (1) At least five sites of in vivo phosphorylation in BAK1 have been determined in Arabidopsis
by immunoprecipitation of epitope-tagged BAK1 followed by LC/MS/MS analysis; (2) Twenty-eight individual transgenic lines of BRI1- Flag were generated in which Ser or Thr residues were mutated to Ala, Asp or Glu, and the effect on kinase activity and in planta function were examined. Thr-1049 and Thr- 1044 in the activation loop of the kinase domain were shown to be essential for BRI1 receptor kinase function. Four such mutant lines have also been generated for BAK1 and are currently being evaluated. (3) Transgenic plants expressing epitope-tagged BRI1 and BAK1 (either native or kinase-minus mutants) have been generated and are being used to study BR-dependence of the physical association of the pair and the ability to auto- and trans-phosphorylate in the heterodimer. (4) Tomato BRI1 has been cloned into plant transformation vectors and the construct is being moved into tomato plants. The tomato BRI1 kinase domain has been cloned into a bacterial expression vector for biochemical
analysis of kinase function.
Impacts
Brassinosteroids (BRs) are now firmly established as essential regulators of plant growth and development affecting a broad spectrum of developmental processes. The identification of BR biosynthetic and insensitive mutants in tomato, rice, barley and pea, clearly extend the importance of these compounds from the experimental plant Arabidopsis thaliana to crop plants. Our experiments will test how conserved molecular mechanisms of BR signaling are between Arabidopsis and tomato, and should provide a model for comparison with other crops. It has been reported for decades that spraying many crops with BRs results in increased yields and tolerance to stress. Understanding molecular mechanisms of BR action could result in the same effects, without exogenous applications, by altering sensitivity to endogenous BR. This proposal addresses several issues important to USDA/NRI objectives including fundamental research relevant to plant growth and development, multidisciplinary
projects involving biology and chemistry, and applications of genomics and proteomics to future food and fiber production. With respect to the Developmental Mechansims of Crop Plants program area this proposal addresses two priorities, hormonal regulation of growth and development, and mechanisms of transducing internal and external signals required for normal growth and development.
Publications
- Wang, X., Goshe, M.B., Soderblom, E., Phinney, B., Kuchar, J.A., Li, J., Asami, T., Yoshida, S., Huber, S.C., Clouse, S.D. 2005. Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase. Plant Cell 17:1685-1703.
- Ehsan, H., Ray, W.K., Phinney, B., Wang, X., Huber, S.C., Clouse, S.D. 2005. Interaction of Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase with a homolog of mammalian TGF-? receptor interacting protein. Plant Journal 43:251-261.
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