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1 Importance of tyrosine phosphorylation in receptor kinase complexes 2 Alberto P. Macho1, Rosa Lozano-Durán1 and Cyril Zipfel 3 4 The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, 5 United Kingdom. 6 1 7 Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 8 201602, China. 9 Corresponding authors: Macho, A.P. ([email protected]); Zipfel, C. Present Address: Shanghai Center for Plant Stress Biology, Shanghai 10 ([email protected]). 11 Keywords: 12 brassinosteroids, PAMPs. tyrosine phosphorylation, 13 1 receptor kinases, immunity, 1 2 Tyrosine 3 modification, known to regulate receptor kinase-mediated signaling in 4 animals. Plant receptor kinases are annotated as serine/threonine 5 kinases, but recent work revealed that tyrosine phosphorylation is also 6 critical for the activation of receptor kinase-mediated signaling in plants. 7 These initial observations paved the way for subsequent detailed 8 studies on the mechanism of activation of plant receptor kinases and 9 the biological relevance of tyrosine phosphorylation for plant growth 10 and immunity. In this opinion article, we review recent reports on the 11 contribution of receptor kinase tyrosine phosphorylation in plant growth 12 and immunity, and propose a major regulatory role of tyrosine 13 phosphorylation for the initiation and transduction of receptor kinase- 14 mediated signaling in plants. phosphorylation is an 15 16 2 important post-translational 1 Receptor kinases 2 Receptor kinases (RKs) are plasma membrane-localized proteins that initiate 3 signaling in a multitude of plant processes. RKs are composed of an 4 ectodomain potentially involved in ligand binding, a single-pass trans- 5 membrane domain, and an intracellular kinase domain. Well-studied 6 examples of RK-mediated signaling pathways are those triggered by plant 7 perception of the steroid hormones brassinosteroids (BR), which promote 8 plant growth [1], or by perception of pathogen-associated molecular patterns 9 (PAMPs), leading to immune responses that constitute the first active layer of 10 plant immunity [2]. 11 Plant RKs have been traditionally classified as serine/threonine kinases [3]. 12 This is in contrast with most animal receptor kinases (RKs), where tyrosine 13 phosphorylation is important for their activation and subsequent signaling, 14 leading to the onset of a multitude of cellular processes [4,5]. Phosphorylation 15 on tyrosine residues was originally observed in plant RKs after auto- 16 phosphorylation in vitro [6-8], although until recently it was not clear whether 17 this also occurred in vivo. Reports over the past five years have shown that, in 18 addition to serine/threonine phosphorylation, several RKs in Arabidopsis 19 thaliana (hereafter referred to as Arabidopsis) can undergo tyrosine 20 phosphorylation, and that this modification plays an important role for their 21 activation and the initiation of subsequent signaling [8-10] (Figure 1, Table 1). 22 Additionally, 23 phosphorylation, uncovering an additional role of this post-translational 24 modification in downstream signal transduction [11-13] (Figure 1; Table 1). In 25 this opinion article, we propose that tyrosine phosphorylation plays a major RK-associated proteins 3 are also regulated by tyrosine 1 role in the activation, transduction and, potentially, specificity of receptor 2 kinase-mediated signaling in plants. 3 4 Tyrosine phosphorylation in the brassinosteroids receptor complex 5 The BR signaling pathway is one of the best studied in plants [1]. In a seminal 6 article published by Oh and co-workers [8], the authors observed that the BR 7 receptor, 8 INSENSITIVE1 (BRI1), is able to auto-phosphorylate on tyrosine residues, in 9 addition to serine and threonine residues [8]. This was unexpected, because 10 BRI1 was classified as a serine/threonine kinase, alongside other plant RKs 11 [14,15]. Strikingly, the authors also reported an unanticipated difficulty on the 12 identification of specific BRI1 phosphorylated tyrosines in vivo when using 13 liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, 14 through a thorough analysis using site-directed mutagenesis and modification- 15 and sequence-specific antibodies, the authors identified a single tyrosine 16 residue in the juxta-membrane domain of BRI1 (Y831; Figure 1; Table 1), 17 which plays an important role in BR signaling in vivo, without affecting the 18 overall BRI1 kinase activity [8]. Additional tyrosine residues, Y956 and Y1072, 19 were found to be phosphorylated in vivo and in vitro, respectively, although 20 mutations on those residues abolished BRI1 kinase activity [8] (Figure 1; 21 Table 1). 22 Moreover, it was observed that the LRR-RK BRI1-ASSOCIATED KINASE1 23 (BAK1), which acts as a BR co-receptor with BRI1 [16], was able to auto- 24 phosphorylate on tyrosine residues, which prompted a follow-up study on the 25 relevance of different tyrosine residues in BAK1 [10]. Using site-directed the leucine-rich repeat 4 (LRR)-RK BRASSINOSTEROID- 1 mutagenesis and modification- and sequence-specific antibodies, a single 2 tyrosine residue in the C-terminal domain of BAK1 (Y610; Figure 1; Table 1) 3 was identified as a major auto-phosphorylation site upon BR perception, being 4 required for BR-triggered signaling [10]. 5 Altogether, these two reports unequivocally demonstrated that both BRI1 and 6 BAK1 are dual-specificity kinases, and that tyrosine phosphorylation plays a 7 prominent role in the activation of the BR receptor complex and subsequent 8 signal transduction. 9 Later work added more complexity to the role of tyrosine 10 phosphorylation in the regulation of BR receptor activation. One of the 11 mechanisms that keep BRI1 in an inactive state in the absence of the 12 hormone is mediated by the interaction with the plasma membrane-localized 13 protein BRI1-KINASE INHIBITOR1 (BKI1) [17]. BR perception leads to 14 phosphorylation of a specific tyrosine residue in BKI1 (Y211), followed by 15 release of the protein into the cytosol [11] (Figure 1; Table 1). BKI1 16 dissociation relieves the inhibition of BRI1 kinase activity and increases the 17 affinity between BRI1 and BAK1 kinase domains, therefore allowing for the 18 formation of a fully active BR receptor complex [11,18] (Figure 1). Thus, 19 tyrosine phosphorylation appears as an essential regulatory mechanism for 20 the robustness of the multi-layered regulation of BR signaling initiation in 21 plants. 22 23 Tyrosine phosphorylation in plasma membrane immune receptor 24 complexes 5 1 In 2 understanding of the initiation of immune signaling triggered by plant pattern 3 recognition receptors (PRRs), which mediate the perception of PAMPs 4 (reviewed in [19]). Several PRRs are RKs, such as the LRR-RKs FLAGELLIN- 5 SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which perceive bacterial 6 flagellin (or the elicitor peptide flg22) and elongation factor Tu (EF-Tu; or the 7 elicitor peptide elf18), respectively [20,21]. Although protein phosphorylation 8 within PRR complexes was known to be critical for the initiation of immune 9 signaling [22-24], the exact phosphorylation events occurring within these 10 complexes and their exact biological roles were unknown. Recently, we 11 reported that EFR is activated upon ligand binding by phosphorylation on 12 tyrosine residues [9]. A combination of site-directed mutagenesis and targeted 13 mass spectrometry analysis identified a single tyrosine residue on EFR 14 (Y836) that is phosphorylated in vivo after elf18 perception and is required for 15 the activation of EFR and downstream immune responses [9] (Fig. 1; Table 1). 16 Interestingly, this residue is not required for the overall kinase activity of EFR, 17 but only for its ligand-induced activation [9]. An additional demonstration of 18 the biological relevance of tyrosine phosphorylation for PRR-triggered 19 immunity (PTI) comes from the finding that the phytopathogenic bacterium 20 Pseudomonas syringae pv. tomato DC3000 injects a tyrosine phosphatase 21 inside the plant cell to target EFR and FLS2. The interaction of this bacterial 22 protein with these PRRs reduces their level of tyrosine phosphorylation, 23 subsequently inhibiting immune signaling and thereby increasing susceptibility 24 to P. syringae [9]. recent years, several reports 6 have significantly increased our 1 Similar to BRI1, PRR-associated kinases also undergo tyrosine 2 phosphorylation. It is important to note that the co-receptor BAK1 also plays 3 an essential role in PTI, by acting as co-receptor in the perception of PAMPs 4 and a positive regulator of subsequent signaling [23-28]. Importantly, 5 phosphorylation of Y610 on BAK1 seems to play a role in the basal 6 expression of immune-related genes, beyond its contribution to the initiation of 7 BR-signaling [10,29] (Fig. 1; Table 1). The involvement of this and additional 8 BAK1 tyrosine residues on PTI signaling will require further investigation. 9 The cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) interacts with 10 FLS2, EFR and BAK1, and plays an important role in the activation of specific 11 downstream responses after PAMP perception [30-33]. It was recently shown 12 that BIK1 is phosphorylated on several tyrosine residues in vitro, including 13 Y23, Y150, Y168, Y214, Y234, Y243 and Y250 [12,13,34] (Figure 1; Table 1). 14 Notably, several BIK1 tyrosine residues are required for the BIK1-mediated 15 phosphorylation of substrates in vitro [13] (Table 1) and for BIK1-dependent 16 PTI responses in planta [12]. 17 Altogether, these reports indicate that tyrosine phosphorylation is a key 18 regulatory mechanism of the activation of innate immune complexes and the 19 subsequent signal transduction. 20 21 Role of tyrosine phosphorylation in early RK-mediated signaling: a 22 multi-functional mechanism for signaling initiation? 23 In animal RKs, auto-phosphorylation of specific tyrosine residues is required 24 to increase the catalytic efficiency of the RK itself, whereas phosphorylation of 25 additional tyrosine residues creates docking sites for downstream signaling 7 1 molecules [4,5]. Similarly, a BRI1 tyrosine residue phosphorylated in vivo, 2 Y956, is required for BRI1 kinase activity [8], although this is most likely due 3 to the role of BRI1 Y956 as gatekeeper residue at the back of the ATP binding 4 site, allowing the active conformation of the kinase domain [35]. Consequently, 5 the interpretation of results obtained by aminoacid substitutions should be 6 done carefully, considering that they may have direct or indirect structural 7 consequences. Another BRI1 tyrosine residue phosphorylated in vivo, Y831, 8 is not essential for BRI1 kinase activity and plays an inhibitory role in BR- 9 triggered signaling [8]. Also, two phosphorylated tyrosine residues in BAK1 10 (Y610) and EFR (Y836) were found to be essential for BR- and elf18-triggered 11 response, respectively, while being non-essential for their respecive kinase 12 activities [9,10]. It is therefore possible that, similarly to what is observed in 13 animals, phosphorylation on specific tyrosine residues is required for kinase 14 activation (and subsequent phosphorylation of relevant serine, threonine or 15 tyrosine residues), while other phosphorylated tyrosine residues are involved 16 in dynamic interactions with downstream kinase substrates or other signaling 17 components. A similar situation was found for phosphorylated tyrosine 18 residues in the cytoplasmic kinase BIK1 in vitro [12,13]. 19 Additionally, as seen for BKI1 [11], tyrosine phosphorylation of negative 20 regulators, such as kinase inhibitors or protein phosphatases, could disrupt 21 their inhibition of RK activation. This would provide a prompt and transient 22 switch, contributing to the formation of active receptor complexes in a robust 23 ligand-dependent manner. 24 It is important to note that several central signaling components are 25 present in both BR and PAMP receptor complexes. The most prominent 8 1 example is BAK1, which acts as co-receptor for BRI1 and several PRRs, and 2 is a positive regulator for both BR- and PAMP-triggered responses. A different 3 case is that of BIK1, which acts as a positive regulator of PTI, but is a 4 negative regulator of BR signaling. How these proteins mediate specific 5 signaling outputs upon perception of different ligands is still unknown [36]. It is 6 tempting to speculate that phosphorylation of particular, distinct tyrosine (as 7 well as serine or threonine) residues could contribute to ligand-dependent 8 outputs mediated by these shared components, therefore providing signaling 9 specificity. 10 11 Concluding remarks 12 Recent work has uncovered tyrosine phosphorylation as an important 13 mechanism for the initiation of signaling mediated by plasma membrane- 14 localized RK complexes in plants. Remarkably, the relevance of tyrosine 15 phosphorylation for BR- and PAMP-triggered signaling goes beyond receptor 16 complexes and extends to downstream signaling cascades, since several 17 downstream regulatory components, including glycogen synthase kinase 3 18 (GSK3)-like kinases, mitogen-activated protein kinases (MAPKs) or calcium- 19 dependent protein kinases (CDPKs), have been shown to phosphorylate on 20 tyrosine residues [37-39]. 21 Further work will be required to gain insight into the specific hierarchy 22 of phosphorylation of tyrosine, serine and threonine residues in plant RK 23 complexes and assess their relative contribution to the recruitment and 24 release of additional regulators and downstream interactors, and hence to the 25 onset of signaling. 9 1 2 Acknowledgements 3 Research in the Zipfel laboratory on this topic is funded by the Gatsby 4 Charitable Foundation and the European Research Council. APM was funded 5 by a long-term post-doctoral fellowship from the Federation of European 6 Biochemical Societies. RL-D was funded by a long-term post-doctoral 7 fellowship from the Fundación Ramón Areces. We thank all members of the 8 Zipfel laboratory, as well as Steven Huber, Vardis Ntoukakis and Benjamin 9 Schwessinger for helpful discussions. We apologize to colleagues whose 10 work could not be cited due to space limitations. 11 12 10 1 References 2 1. Zhu JY, Sae-Seaw J, Wang ZY: Brassinosteroid signalling. Development 3 2013, 140:1615-1620. 4 2. Boller T, Felix G: A renaissance of elicitors: perception of microbe- 5 associated molecular patterns and danger signals by pattern-recognition 6 receptors. Annu Rev Plant Biol 2009, 60:379-406. 7 3. Shiu SH, Bleecker AB: Receptor-like kinases from Arabidopsis form a 8 monophyletic gene family related to animal receptor kinases. Proc Natl Acad 9 Sci U S A 2001, 98:10763-10768. 10 4. Lemmon MA, Schlessinger J: Cell signaling by receptor tyrosine kinases. 11 Cell 2010, 141:1117-1134. 12 5. Lim WA, Pawson T: Phosphotyrosine signaling: evolving a new cellular 13 communication system. Cell 2010, 142:661-667. 14 6. Mu JH, Lee HS, Kao TH: Characterization of a pollen-expressed receptor- 15 like kinase gene of Petunia inflata and the activity of its encoded kinase. Plant 16 cell 1994, 6:709-721. 17 7. Shah K, Vervoort J, de Vries SC: Role of threonines in the Arabidopsis 18 thaliana somatic embryogenesis receptor kinase 1 activation loop in 19 phosphorylation. J Biol Chem 2001, 276:41263- 41269. 20 8. Oh MH, Wang X, Kota U, Goshe MB, Clouse SD, Huber SC: Tyrosine 21 phosphorylation of the BRI1 receptor kinase emerges as a component of 22 brassinosteroid signaling in Arabidopsis. Proc Natl Acad Sci U S A 2009, 23 106:658-663. 24 9. Macho AP, Schwessinger B, Ntoukakis V, Brutus A, Segonzac C, Roy S, 25 Kadota Y, Oh MH, Sklenar J, Derbyshire P, et al.: A bacterial tyrosine 11 1 phosphatase inhibits plant pattern recognition receptor activation. Science 2 2014, 343:1509-1512. 3 10. Oh MH, Wang X, Wu X, Zhao Y, Clouse SD, Huber SC: 4 Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in 5 brassinosteroid signaling and basal defense gene expression. Proc Natl Acad 6 Sci U S A 2010, 107:17827-17832. 7 11. Jaillais Y, Hothorn M, Belkhadir Y, Dabi T, Nimchuk ZL, Meyerowitz EM, 8 Chory J: Tyrosine phosphorylation controls brassinosteroid receptor activation 9 by triggering membrane release of its kinase inhibitor. Genes Dev 2011, 10 25:232-237. 11 12. Lin W, Li B, Lu D, Chen S, Zhu N, He P, Shan L: Tyrosine phosphorylation 12 of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity. 13 Proc Natl Acad Sci U S A 2014, 111:3632-3637. 14 13. Xu J, Wei X, Yan L, Liu D, Ma Y, Guo Y, Peng C, Zhou H, Yang C, Lou Z, 15 et al.: Identification and functional analysis of phosphorylation residues of the 16 Arabidopsis BOTRYTIS-INDUCED KINASE1. Prot cell 2013, 4:771-781. 17 14. Dievart A, Clark SE: LRR-containing receptors regulating plant 18 development and defense. Development 2004, 131:251-261. 19 15. Shiu SH, Bleecker AB: Expansion of the receptor-like kinase/Pelle gene 20 family and receptor-like proteins in Arabidopsis. Plant physiol 2003, 132:530- 21 543. 22 16. Sun Y, Han Z, Tang J, Hu Z, Chai C, Zhou B, Chai J: Structure reveals 23 that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell res 24 2013, 23:1326-1329. 12 1 17. Wang X, Chory J: Brassinosteroids regulate dissociation of BKI1, a 2 negative regulator of BRI1 signaling, from the plasma membrane. Science 3 2006, 313:1118-1122. 4 18. Wang J, Jiang J, Chen L, Fan SL, Wu JW, Wang X, Wang ZX: Structural 5 insights into the negative regulation of BRI1 signaling by BRI1-interacting 6 protein BKI1. Cell res 2014, 24:1328-1341. 7 19. Macho AP, Zipfel C: Plant PRRs and the Activation of Innate Immune 8 Signaling. Mol cell 2014, Mol Cell 24; 263-72 9 20. Gómez-Gómez L, Boller T: FLS2: an LRR receptor-like kinase involved in 10 the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 2000, 11 5:1003-1011. 12 21. Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G: 13 Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts 14 Agrobacterium-Mediated Transformation. Cell 2006, 125:749-760. 15 22. Chen X, Chern M, Canlas PE, Jiang C, Ruan D, Cao P, Ronald PC: A 16 conserved threonine residue in the juxtamembrane domain of the XA21 17 pattern recognition receptor is critical for kinase autophosphorylation and 18 XA21-mediated immunity. J Biol Chem 2010, 285:10454-10463. 19 23. Schulze B, Mentzel T, Jehle AK, Mueller K, Beeler S, Boller T, Felix G, 20 Chinchilla D: Rapid heteromerization and phosphorylation of ligand-activated 21 plant transmembrane receptors and their associated kinase BAK1. J Biol 22 Chem 2010, 285:9444-9451. 23 24. Schwessinger B, Roux M, Kadota Y, Ntoukakis V, Sklenar J, Jones A, 24 Zipfel C: Phosphorylation-dependent differential regulation of plant growth, 13 1 cell death, and innate immunity by the regulatory receptor-like kinase BAK1. 2 PLoS Genet 2011, 7:e1002046. 3 25. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger T, Jones 4 JD, Felix G, Boller T: A flagellin-induced complex of the receptor FLS2 and 5 BAK1 initiates plant defence. Nature 2007, 448:497-500. 6 26. Heese A, Hann DR, Gimenez-Ibanez S, Jones AM, He K, Li J, Schroeder 7 JI, Peck SC, Rathjen JP: The receptor-like kinase SERK3/BAK1 is a central 8 regulator of innate immunity in plants. Proc Natl Acad Sci U S A 2007, 9 104:12217-12222. 10 27. Roux M, Schwessinger B, Albrecht C, Chinchilla D, Jones A, Holton N, 11 Malinovsky FG, Tor M, de Vries S, Zipfel C: The Arabidopsis leucine-rich 12 repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for 13 innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 2011, 14 23:2440-2455. 15 28. Sun Y, Li L, Macho AP, Han Z, Hu Z, Zipfel C, Zhou JM, Chai J: Structural 16 basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune 17 complex. Science 2013, 342:624-628. 18 29. Oh MH, Wu X, Clouse SD, Huber SC: Functional importance of BAK1 19 tyrosine phosphorylation in vivo. Plant Signal Behav 2011, 6:400-405. 20 30. Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, 21 Jones JD, Shirasu K, Menke F, Jones A, et al.: Direct regulation of the 22 NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant 23 immunity. Mol cell 2014, 54:43-55. 14 1 31. Li L, Li M, Yu L, Zhou Z, Liang X, Liu Z, Cai G, Gao L, Zhang X, Wang Y, 2 et al.: The FLS2-associated kinase BIK1 directly phosphorylates the NADPH 3 oxidase RbohD to control plant immunity. Cell host microbe 2014, 15:329-338. 4 32. Lu D, Wu S, Gao X, Zhang Y, Shan L, He P: A receptor-like cytoplasmic 5 kinase, BIK1, associates with a flagellin receptor complex to initiate plant 6 innate immunity. Proc Natl Acad Sci U S A 2010, 107:496-501. 7 33. Zhang J, Li W, Xiang T, Liu Z, Laluk K, Ding X, Zou Y, Gao M, Zhang X, 8 Chen S, et al.: Receptor-like cytoplasmic kinases integrate signaling from 9 multiple plant immune receptors and are targeted by a Pseudomonas 10 syringae effector. Cell Host Microbe 2010, 7:290-301. 11 34. Wang Y, Li Z, Liu D, Xu J, Wei X, Yan L, Yang C, Lou Z, Shui W: 12 Assessment of BAK1 activity in different plant receptor-like kinase complexes 13 by quantitative profiling of phosphorylation patterns. J Proteom 2014, 14 108:484-493. 15 35. Bojar D, Martinez J, Santiago J, Rybin V, Bayliss R and Hothorn M: 16 Crystal structures of the phosphorylated BRI1 kinase domain and implications 17 for brassinosteroid signal initiation. Plant J 2014, 78: 31–43 18 36. Lozano-Duran R, Zipfel C: Trade-off between growth and immunity: role of 19 brassinosteroids. Trends in plant science 2014, 20,12-19. 20 37. Kim TW, Guan S, Sun Y, Deng Z, Tang W, Shang JX, Burlingame AL, 21 Wang ZY: Brassinosteroid signal transduction from cell-surface receptor 22 kinases to nuclear transcription factors. Nature cell biol 2009, 11:1254-1260. 23 38. Mithoe SC, Boersema PJ, Berke L, Snel B, Heck AJ, Menke FL: Targeted 24 quantitative phosphoproteomics approach for the detection of phospho- 25 tyrosine signaling in plants. J proteome res 2012, 11:438-448. 15 1 39. Oh MH, Wu X, Kim HS, Harper JF, Zielinski RE, Clouse SD, Huber SC: 2 CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation 3 attenuates kinase activity. FEBS letters 2012, 586:4070-4075. 4 5 6 16 1 2 3 Figure legends 4 involved in early brassinosteroids (BR)- (A) and PAMP (here, elf18)- (B) 5 triggered signaling. (A) In the absence of BR, BKI1 and BIK1 negatively 6 regulate the activation of BRI1. BR perception leads to phosphorylation of 7 BKI1 Y211, followed by the release of the protein into the cytosol, which 8 relieves the inhibition of BRI1 kinase activity and increases the affinity 9 between BRI1 and BAK1 kinase domains. BR perception also leads to the 10 release of BIK1. The dissociation of both negative regulators allows for the 11 formation of a fully active BR receptor complex. (B) BIK1 associates with EFR 12 and BAK1 in unelicited conditions. Upon elf18 perception, EFR Y836 is 13 phosphorylated and contributes to the ligand-induced activation of the EFR- 14 BAK1 complex. PAMP perception also leads to the phosphorylation of BIK1, 15 which is released from the receptor complex and activate downstream 16 immune responses. Abbreviations: BAK1, BRI1-ASSOCIATED KINASE1; 17 BIK1, BOTRYTIS-INDUCED KINASE1; BKI1, BRI1-KINASE INHIBITOR1; 18 BRI1, 19 PAMP, pathogen-associated molecular pattern. Figure 1. Simplified diagram of tyrosine-phosphorylated proteins BRASSINOSTEROID-INSENSITIVE1; 20 21 17 EFR, EF-TU RECEPTOR; Table 1. Tyrosine-phosphorylated residues in components of RK complexes Evidence of phosphorylation Protein Residue Y831 BRI1 Y956 Identif ication in vivo in vitro in vivo in vitro Detection by LC/MS-MS Sequenceand phosphory lationspecific antibodies Sitedirected mutagenes is Auto Yes Yes Nd Auto No Liganddependency Auto-/transphosphorylation Nd Mutation to Phe abolishes kinase activity Refs Yes No [7] Yes Yes Yes [7] Y1072 in vitro Nd Auto No Yes Yes Yes [7] BAK1 Y610 in vivo in vitro BL-induced Auto No Yes Yes No [9] BKI1 Y211 in vivo BL-induced Trans No No Yes N/A [10] EFR Y836 in vivo elf18-induced Nd Yes No Yes No [8] Y23 in vitro Nd Auto Yes No No No [11] Y150 in vitro Nd Nd No No Yes Yes [11] Y168 in vitro Nd Auto/Trans Yes No No No [12,33] Y214 in vitro Nd Auto/Trans Yes No Yes No [12,33] Y234 in vitro Nd Auto Yes No Yes No [11] Y243 in vitro Nd Auto/Trans No No Yes No [11] Y250 in vitro Nd Auto/Trans Yes No Yes No [11,12,33] BIK1 Abbreviations: BAK1, BRI1-ASSOCIATED KINASE1; BIK1, BOTRYTIS-INDUCED KINASE1; BKI1, BRI1-KINASE INHIBITOR1; BRI1, BRASSINOSTEROID-INSENSITIVE1; EFR, EF-TU RECEPTOR. Nd, not determined. 18