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Plant Signaling & Behavior 4:12, 1182-1185; December 2009; © 2009 Landes Bioscience
Tyrosine phosphorylation in brassinosteroid signaling Man-Ho Oh,1 Steven D. Clouse2 and Steven C. Huber1,* 1 US. Department of Agriculture; Agricultural Research Service; University of Illinois; Urbana, IL USA; 2Department of Horticultural Science; North Carolina State University; Raleigh, NC USA
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Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction Submitted: 09/10/09 Revised: 09/10/09 Accepted: 09/10/09 Previously published online: www.landesbioscience.com/journals/psb/ article/10046 *Correspondence to: Steven C. Huber; Email:
[email protected] Addendum to: Oh M-H, Wang X, Kota U, Goshe MB, Clouse SD, Huber SC. Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc Natl Acad Sci USA 2009; 106:658–63; PMID: 19124768; DOI: 10.1073/pnas.0810249106.
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rassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.1 Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants
Plant Signaling & Behavior
expressing BRI1(Y831F)-Flag may have applications for agriculture. Introduction Virtually every aspect of plant growth and development is regulated by hormones. Brassinosteroids (BRs) are essential for growth and brassinolide (BL) is the most biologically active member of the plant steroid hormone family. Brassinosteroid signal transduction is initiated when the hormone binds to the extracellular domain of the BRI1 receptor kinase,14 which leads to phosphorylation of the BRI1 cytoplasmic domain (CD) on Ser and Thr16,19 and also Tyr residues.1 The signaling complex that forms also includes the co-receptor, BAK1, that transduces the signal via intracellular protein kinases and phosphatases to regulate the phosphorylation status of down-stream transcription factors that alter the expression of many different genes promoting cell elongation, division, and differentiation.2-4 The Arabidopsis genome sequence has revealed that plants contain a much larger complement of receptor-like kinase (RLK) genes than other organisms.20 The RLKs are structurally similar to mammalian tyrosine kinases with an extracellular domain, a single-pass transmembrane domain, and an intracellular cytoplasmic domain consisting of a juxtamembrane region, Ser/Thr receptor kinase domain, and short carboxy-terminal polypeptide. Among receptor like-kinases (RLK) in Arabidopsis thaliana, the most common extracellular motif is the leucine-rich repeat (LRR), present in over half the Arabidopsis RLKs.20 LRRs are a common signal transduction motif thought
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contrast, substitution of Tyr at position 898, 945, 961, 1058 and 1070 with Phe enhanced autophosphorylation on Tyr residues as indicated by cross reactivity with anti-phosphotyrosine antibodies. While all of the site-directed mutants had active kinase domains, peptide kinase activity of the recombinant CD proteins was substantially reduced relative to the wild type BRI1-CD.1 Tyr-831 in the Juxtamembrane Domain is Important for Leaf Development and Flowering Time Figure 1. Effects of mutating Tyr residues in the BRI1 cytoplasmic domain on rescue of the weak bri1-5 mutant. (A) Ten different Tyr residues were individually substituted with Phe and representative plants (T2 generation) are shown after 40 days of growth under short days (8 h photoperiod). Similar growth phenotypes were observed with ten independent transgenic lines for each mutation. In the figure, ‘WT’ refers to the wild type BRI1-Flag expressed in the bri1-5 background.
to be involved in protein-protein interactions.21 They are not found on animal receptor kinases. Early analysis of the plant RLK genes revealed their involvement in a diverse array of developmental and defense functions that included gametophyte development,5 pollen-pistil interactions,6-9 shoot apical meristem equilibrium,10 hormone perception11 and cell morphogenesis.12,13 Autophosphorylation has been extensively studied in vitro using recombinant cytoplasmic domains of the plant RLKs. In early studies, phosphoamino acid analysis of autophosphorylated recombinant BRI1 kinase domain showed phosphorylation primarily on Ser residues, with a minor amount on Thr.16 Recently, we made the unexpected observation that recombinant BRI1-CD was recognized by anti-phosphotyrosine antibodies suggesting that these receptors may be dual-specificity kinases rather than Ser/ Thr kinases as generally classified.16-18 Tyr autophosphorylation was confirmed by several lines of evidence and further investigations used transgenic plants expressing site-directed mutants of BRI1Flag. In the present report, we describe the growth phenotypes associated with mutations (Phe substitutions) of individual Tyr residues in BRI1-CD and present a simplified model to describe different mechanisms that may underlie the role of Tyr-831.
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BRI1 is a Dual-Specificity Kinase As described in the previous report,1 a key observation was that BRI1 cross reacted strongly with anti-phosphotyrosine antibodies when immunoprecipitated from transgenic plants or expressed as a recombinant protein in E. coli. This was surprising because BRI1 is classified as a Ser/Thr protein kinase and had been shown previously to autophosphorylate in vitro only on Ser and Thr residues.16,19 However, in situ and in vivo the protein clearly contains phosphotyrosine. To understand the function of specific Tyr residues of BRI1 and as one approach to identify potential sites of Tyr autophosphorylation, we produced site-directed mutants of BRI1 by individually replacing each of the ten Tyr residues in the CD with Phe and expressing the soluble Flag-BRI1-CD proteins in E. coli or the full-length BRI1-Flag protein in transgenic plants. Of the ten Tyr residues in the BRI1-CD, substitution of Tyr at positions 956, 1052, 1057 and 1072 with Phe strongly inhibited autophosphorylation as indicated by almost complete loss of cross reactivity with anti-phosphothreonine and anti-phosphoTyr antibodies.1 These substitutions of BRI1-CD also strongly inhibited kinase activity on a peptide substrate (sequence: GRJRRIASVEJJK) indicating that these four Tyr residues are essential for BRI1 kinase activity. In
Plant Signaling & Behavior
To test the function of specific Tyr residues in BRI1 function in vivo, we transformed the weak bri1-5 mutant with wild type BRI1-Flag or site-directed mutants where ten different Tyr residues were individually substituted with Phe. The dwarf phenotype of the bri1-5 mutant was rescued by expression of wild-type BRI1Flag (Fig. 1), as expected.1 However, only slightly more growth of bri1-5 was observed in response to expression of the Y956F, Y1052F, Y1057F or Y1072F sitedirected mutants, which was expected based on the kinase-inactive phenotypes of the corresponding recombinant proteins.1 Expression of several BRI1-Flag site-directed mutants, in particular Y956F, Y961F, Y1058F or Y1070F in the bri1-5 mutant clearly rescued growth but many other characteristics of the bri1-5 mutant were not altered, including the round leaves, short petioles and short hypocotyl elongation. This is visually apparent in the plants grown for 40 days under short days (Fig. 1). Interestingly, expression of BRI1 with the Tyr at positions 831 or 898 substituted with Phe rescued growth but resulted in different phenotypes from the other mutants (Fig. 1). Based on individual Tyr to Phe site-directed mutants, we measured plant fresh weight, and the results show that BRI1(Y831)-Flag transgenic plants have the highest fresh weight and greatest plant growth among the ten different transgenic mutants (Fig. 2A and data not shown). Both clearly rescued growth of bri1-5, but the BRI1(Y898F)Flag transgenic plants had unusually elongated leaves and petioles (Fig. 1) similar to plants overexpressing a dominant negative mutant form of the transcription factor,
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BES1.23 The prominent feature of transgenic plants expressing BRI1(Y831F)Flag was a dramatically increased leaf size (Fig. 1) and shoot biomass (Fig. 2A). The ability of the various Tyr mutants to rescue growth of the bri1-5 mutant varied substantially and at least one of the contributing factors may be the capacity of the mutant BRI1 kinase domain to catalyze transphosphorylation, because as shown in Figure 2B there was an overall positive correlation between peptide kinase activity of the BRI1 site-directed mutants and shoot fresh weight for transgenic plants expressing the mutant BRI1. Interestingly, decreased kinase activity was generally associated with increased overall Tyr autophosphorylation and so we are speculating that increased autophosphorylation of one or more of the Tyr residues that are essential for kinase activity (i.e., Tyr-956, -1052, -1057 and -1072) may result in inhibition of kinase transphosphorylation activity. Current Working Model and Future Directions There are many important questions that remain to be answered. First, identifying the kinase domain motifs of the RLKs that control the ability to autophosphorylate on Tyr in addition to Ser/Thr (i.e., determine dual specificity) will be an exciting question to explore. The presently recognized dual specificity determinants1 do not predict BRI1 as a dual specificity kinase and hence additional determinants must exist. It is also clear that the ability of BRI1 to autophosphorylate on Tyr can be affected by minor changes in kinase domain primary structure as site-directed mutagenesis of several Tyr residues resulted in increased overall Tyr autophosphorylation1 and reduced ability to rescue growth of the bri1-5 mutant (Fig. 2B). It will be important to determine how the capacity for Tyr autophosphorylation is impacted by kinase domain structure and to determine which Tyr residues, when phosphorylated, result in inhibition of kinase transphosphorylation activity. Other remaining questions focus on the mechanisms responsible for increased growth and early flowering of plants expressing BRI1(Y831F)-Flag compared
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Figure 2. Rescue of the bri1-5 weak allele by BRI1-Flag site-directed mutants. (A) Shoot fresh weight of transgenic plants expressing BRI1-Flag with Phe substitutions for Tyr at positions indicated. In the figure, ‘WT’ refers to the wild type BRI1-Flag expressed in the bri1-5 background. (B) Overall positive correlation between shoot fresh weight for the ten different transgenics identified in Figure 1 and BRI1 peptide kinase activity of the cytoplasmic domains of the various mutants (kinase data taken from C of Oh et al. 2009). Shoot fresh weights in (A and B) were measured on plants grown for 25 d under long days (16 h photoperiod).
to wild type BRI1-Flag transgenic plants. BRI1 and BR signaling have been linked to growth24 and flowering,25 and it now appears that phosphorylation of Tyr-831 is a key determinant of these linkages. However, the molecular understanding of how Tyr 831 autophosphorylation affects flowering time, petiole elongation and leaf shape remain to be elucidated. To address these questions, we are currently conducting a genome-wide analysis of the BRI1(Y831)-responsive transcriptome using Affymetrix ATH1 gene arrays. As predicted from the divergent growth phenotypes of transgenic bri1-5 plants
Plant Signaling & Behavior
expressing BRI1-Flag versus BRI1(Y831F)Flag, the expression of many genes is altered suggesting that signal transduction mediated by BRI1 differs when Tyr-831 is in the phosphorylated versus unphosphorylated state. Phosphorylation could direct affect the conformation of the juxtamembrane domain (or its interaction with the kinase domain or other proteins in the signaling complex) or could mediate the interaction with one or more phosphotyrosine-binding proteins (see Fig. 3), which have not been explored in plants but are well known to play a critical role in signal transduction in animals.26
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Figure 3. Working model for the effect of Tyr-831 phosphorylation on BRI1-mediated signal transduction. Mutagenic analysis suggests that phosphorylation of Tyr-831 in the juxtamembrane domain affects BR signaling, with phosphorylation resulting in delayed flowering and inhibition of leaf growth. This alteration in signaling could be a direct result of phosphorylation, possibly including a conformational change in the juxtamembrane domain, or could involve presently unknown phosphotyrosine-binding proteins.
Acknowledgements
References
This work was supported in part by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant numbers 2004-35304-14930 and 2007-3531817801, National Science Foundation (MCB-0419819), and US Department of Agriculture (USDA)-Agricultural Research Service (ARS). Mention of a trademark of proprietary product does not constitute a guarantee or warranty by the USDA-ARS and does not imply its approval to the exclusion of other products that might also be suitable. Any opinions, findings and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Research Initiative, National Science Foundation or the USDA-ARS.
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