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Opinion
Importance of tyrosine phosphorylation in receptor kinase complexes Alberto P. Macho*, Rosa Lozano-Dura´n*, and Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK
Tyrosine phosphorylation is an important posttranslational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Receptor kinases RKs are plasma membrane-localized proteins that initiate signaling in a multitude of plant processes. RKs are composed of an ectodomain potentially involved in ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain. Well-studied examples of RK-mediated signaling pathways are those triggered by plant perception of the steroid hormones brassinosteroids (BRs), which promote plant growth [1], or of pathogenassociated molecular patterns (PAMPs), leading to immune responses that constitute the first active layer of plant immunity [2]. Plant RKs have traditionally been classified as serine/ threonine kinases [3]. This is in contrast with most animal RKs where tyrosine phosphorylation is important for their activation and subsequent signaling, leading to the onset of a multitude of cellular processes [4,5]. Phosphorylation on tyrosine residues was originally observed in plant RKs after autophosphorylation in vitro [6–8], although until recently it was not clear whether this also occurred in vivo. Reports over the past 5 years have shown that, Corresponding authors: Macho, A.P. ([email protected]); Zipfel, C. ([email protected]). Keywords: tyrosine phosphorylation; receptor kinases; immunity; brassinosteroids; PAMPs. * Present addresses: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China. 1360-1385/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2015.02.005
in addition to serine/threonine phosphorylation, several RKs in Arabidopsis thaliana (hereafter referred to as Arabidopsis) can undergo tyrosine phosphorylation, and that this modification plays an important role in their activation and in the initiation of subsequent signaling [8–10] (Figure 1, Table 1). In addition, RK-associated proteins are also regulated by tyrosine phosphorylation, uncovering an additional role of this post-translational modification in downstream signal transduction [11–13] (Figure 1; Table 1). In this Opinion article we propose that tyrosine phosphorylation plays a major role in the activation, transduction, and, potentially, specificity of RKmediated signaling in plants. Tyrosine phosphorylation in the BR receptor complex The BR signaling pathway is one of the best studied in plants [1]. In a seminal article published by Oh and coworkers [8] it was observed that the BR receptor, the leucine-rich repeat (LRR)-RK BRASSINOSTEROID-INSENSITIVE 1 (BRI1), is able to autophosphorylate on tyrosine residues, in addition to serine and threonine residues [8]. This was unexpected because BRI1 was classified as a serine/threonine kinase, alongside other plant RKs [14,15]. Strikingly, the authors also reported an unanticipated difficulty in the identification of specific BRI1 phosphorylated tyrosines in vivo when using liquid chromatography–tandem mass spectrometry (LC–MS/ MS). However, through a thorough analysis using sitedirected mutagenesis and modification- and sequence-specific antibodies, the authors identified a single tyrosine residue in the juxtamembrane domain of BRI1 (Y831; Figure 1; Table 1) which plays an important role in BR signaling in vivo, without affecting overall BRI1 kinase activity [8]. Additional tyrosine residues, Y956 and Y1072, were found to be phosphorylated in vivo and in vitro, respectively, although mutations of these residues abolished BRI1 kinase activity [8] (Figure 1; Table 1). Moreover, it was observed that the LRR-RK BRI1ASSOCIATED KINASE 1 (BAK1), which acts as a BR coreceptor with BRI1 [16], was able to autophosphorylate on tyrosine residues, and this prompted a follow-up study on the relevance of different tyrosine residues in BAK1 [10]. Using site-directed mutagenesis and modificationand sequence-specific antibodies, a single tyrosine residue in the C-terminal domain of BAK1 (Y610; Figure 1; Table 1) was identified as a major autophosphorylation site upon Trends in Plant Science xx (2015) 1–4
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Opinion
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(A)
(B)
BRI1
BRI1
EFR
EFR elf18
BAK1
BAK1
BAK1
BR
PM
BAK1
PM P
BKI1 BIK1
P
BIK1
P
BKI1 BIK1
BIK1
P BIK1
BIK1 P
BIK1
BR signaling
PTI signaling Cytoplasm
Cytoplasm TRENDS in Plant Science
Figure 1. Simplified diagram of tyrosine-phosphorylated proteins involved in early brassinosteroid (BR)- and PAMP (here, elf18)-triggered signaling. (A) In the absence of BR, BKI1 and BIK1 negatively regulate the activation of BRI1. BR perception leads to phosphorylation (P) of BKI1 Y211, followed by the release of the protein into the cytosol, which relieves the inhibition of BRI1 kinase activity and increases the affinity between BRI1 and BAK1 kinase domains. BR perception also leads to the release of BIK1. The dissociation of both negative regulators allows the formation of a fully-active BR receptor complex. (B) BIK1 associates with EFR and BAK1 in unelicited conditions. Upon elf18 perception, EFR Y836 is phosphorylated and contributes to the ligand-induced activation of the EFR–BAK1 complex. PAMP perception also leads to the phosphorylation of BIK1, which is released from the receptor complex and activates downstream immune responses. Abbreviations: BAK1, BRI1-ASSOCIATED KINASE 1; BIK1, BOTRYTIS-INDUCED KINASE 1; BKI1, BRI1-KINASE INHIBITOR 1; BRI1, BRASSINOSTEROID-INSENSITIVE 1; EFR, EF-TU RECEPTOR; PAMP, pathogen-associated molecular pattern; PM, plasma membrane.
BR perception that is required for BR-triggered signaling [10]. Together, these two reports unequivocally demonstrated that both BRI1 and BAK1 are dual-specificity kinases, and that tyrosine phosphorylation plays a prominent role
in the activation of the BR receptor complex and subsequent signal transduction. Later work added more complexity to the role of tyrosine phosphorylation in the regulation of BR receptor activation. One of the mechanisms that keeps BRI1 in an inactive state
Table 1. Tyrosine-phosphorylated residues in components of RK complexesa Protein
BRI1
Residue
Y831 Y956
BAK1 BKI1 EFR BIK1
a
Y1072 Y610 Y211 Y836 Y23 Y150 Y168 Y214 Y234 Y243 Y250
Identification
In In In In In In In In In In In In In In In In
vivo vitro vivo vitro vitro vivo vitro vivo vivo vitro vitro vitro vitro vitro vitro vitro
Liganddependency
Auto-/transphosphorylation
Evidence of phosphorylation
Mutation to Phe abolishes kinase activity
Nd
Auto
Yes
Nd
Auto
Nd BL-induced BL-induced elf18-induced Nd Nd Nd Nd Nd Nd Nd
Detection by LC/ MS-MS
Refs
Sitedirected mutagenesis
Yes
Sequence- and phosphorylationspecific antibodies Yes
No
[7]
No
Yes
Yes
Yes
[7]
Auto Auto
No No
Yes Yes
Yes Yes
Yes No
[7] [9]
Trans Nd Auto Nd Auto/trans Auto/trans Auto Auto/trans Auto/trans
No Yes Yes No Yes Yes Yes No Yes
No No No No No No No No No
Yes Yes No Yes No Yes Yes Yes Yes
N/A No No Yes No No No No No
[10] [8] [11] [11] [12,33] [12,33] [11] [11] [11,12,33]
Abbreviations: BAK1, BRI1-ASSOCIATED KINASE 1; BIK1, BOTRYTIS-INDUCED KINASE 1; BKI1, BRI1-KINASE INHIBITOR 1; BRI1, BRASSINOSTEROID-INSENSITIVE 1; EFR, EF-TU RECEPTOR. Nd, not determined.
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Opinion in the absence of the hormone operates via an interaction with the plasma membrane-localized protein BRI1-KINASE INHIBITOR 1 (BKI1) [17]. BR perception leads to phosphorylation of a specific tyrosine residue in BKI1 (Y211), followed by release of the protein into the cytosol [11] (Figure 1; Table 1). BKI1 dissociation relieves the inhibition of BRI1 kinase activity and increases the affinity between BRI1 and BAK1 kinase domains, therefore allowing the formation of a fully-active BR receptor complex [11,18] (Figure 1). Thus, tyrosine phosphorylation appears to be an essential regulatory mechanism for the robustness of the multi-layered regulation of BR signaling initiation in plants. Tyrosine phosphorylation in plasma membrane immune receptor complexes In recent years, several reports have significantly increased our understanding of the initiation of immune signaling triggered by plant pattern-recognition receptors (PRRs), which mediate the perception of PAMPs (reviewed in [19]). Several PRRs are RKs, such as the LRR-RKs FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which perceive bacterial flagellin (or the elicitor peptide flg22) and elongation factor Tu (EF-Tu; or the elicitor peptide elf18), respectively [20,21]. Although protein phosphorylation within PRR complexes was known to be crucial for the initiation of immune signaling [22–24], the exact phosphorylation events occurring within these complexes and their exact biological roles were unknown. Recently, we reported that EFR is activated upon ligand binding by phosphorylation on tyrosine residues [9]. A combination of site-directed mutagenesis and targeted MS analysis identified a single tyrosine residue on EFR (Y836) that is phosphorylated in vivo after elf18 perception and which is required for the activation of EFR and downstream immune responses [9] (Figure 1; Table 1). Interestingly, this residue is not required for the overall kinase activity of EFR, but only for its ligand-induced activation [9]. An additional demonstration of the biological relevance of tyrosine phosphorylation for PRR-triggered immunity (PTI) comes from the finding that the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 injects a tyrosine phosphatase inside the plant cell to target EFR and FLS2. The interaction of this bacterial protein with these PRRs reduces their level of tyrosine phosphorylation, subsequently inhibiting immune signaling and thereby increasing susceptibility to P. syringae [9]. Similarly to BRI1, PRR-associated kinases also undergo tyrosine phosphorylation. It is important to note that BAK1 also plays an essential role in PTI by acting as coreceptor in the perception of PAMPs and a positive regulator of subsequent signaling [23–28]. Importantly, phosphorylation of Y610 on BAK1 seems to play a role in the basal expression of immune-related genes beyond its contribution to the initiation of BR-signaling [10,29] (Figure 1; Table 1). The involvement of this and additional BAK1 tyrosine residues in PTI signaling will require further investigation. The cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) interacts with FLS2, EFR, and BAK1, and plays an important role in the activation of specific downstream responses after PAMP perception [30–33]. It was recently
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shown that BIK1 is phosphorylated on several tyrosine residues in vitro, including Y23, Y150, Y168, Y214, Y234, Y243, and Y250 [12,13,34] (Figure 1; Table 1). Notably, several BIK1 tyrosine residues are required for the BIK1mediated phosphorylation of substrates in vitro [13] (Table 1) and for BIK1-dependent PTI responses in planta [12]. Together, these reports indicate that tyrosine phosphorylation is a key regulatory mechanism in the activation of innate immune complexes and subsequent signal transduction. Role of tyrosine phosphorylation in early RK-mediated signaling: a multifunctional mechanism for signaling initiation? In animal RKs, autophosphorylation of specific tyrosine residues is necessary to increase the catalytic efficiency of the RK itself, whereas phosphorylation of additional tyrosine residues creates docking sites for downstream signaling molecules [4,5]. Similarly, a BRI1 tyrosine residue phosphorylated in vivo, Y956, is required for BRI1 kinase activity [8], although this is most likely due to the role of BRI1 Y956 as gatekeeper residue at the back of the ATP binding site, allowing the active conformation of the kinase domain [35]. Consequently, the results obtained by amino acid substitutions require careful interpretation because such substitutions may have direct or indirect structural consequences. Another BRI1 tyrosine residue phosphorylated in vivo, Y831, is not essential for BRI1 kinase activity and plays an inhibitory role in BR-triggered signaling [8]. In addition, two phosphorylated tyrosine residues in BAK1 (Y610) and EFR (Y836) were found to be essential for BRand elf18-triggered responses, respectively, while being non-essential for their respective kinase activities [9,10]. It is therefore possible that, similarly to what is observed in animals, phosphorylation on specific tyrosine residues is required for kinase activation (and subsequent phosphorylation of relevant serine, threonine, or tyrosine residues), while other phosphorylated tyrosine residues are involved in dynamic interactions with downstream kinase substrates or other signaling components. A similar situation was found for phosphorylated tyrosine residues in the cytoplasmic kinase BIK1 in vitro [12,13]. In addition, as seen for BKI1 [11], tyrosine phosphorylation of negative regulators, such as kinase inhibitors or protein phosphatases, could disrupt their inhibition of RK activation. This would provide a prompt and transient switch, contributing to the formation of active receptor complexes in a robust ligand-dependent manner. It is important to note that several central signaling components are present in both BR and PAMP receptor complexes. The most prominent example is BAK1, which acts as coreceptor for BRI1 and several PRRs, and is a positive regulator for both BR- and PAMP-triggered responses. A different case is that of BIK1, which acts as a positive regulator of PTI, but is a negative regulator of BR signaling. How these proteins mediate specific signaling outputs upon perception of different ligands is still unknown [36]. It is tempting to speculate that phosphorylation of particular, distinct tyrosine (as well as serine or threonine) residues could contribute to ligand-dependent 3
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Opinion outputs mediated by these shared components, therefore providing signaling specificity. Concluding remarks Recent work has uncovered tyrosine phosphorylation as an important mechanism for the initiation of signaling mediated by plasma membrane-localized RK complexes in plants. Remarkably, the relevance of tyrosine phosphorylation for BR- and PAMP-triggered signaling goes beyond receptor complexes and extends to downstream signaling cascades because several downstream regulatory components, including glycogen synthase kinase 3 (GSK3)-like kinases, mitogen-activated protein kinases (MAPKs), and calcium-dependent protein kinases (CDPKs), have been shown to phosphorylate tyrosine residues [37–39]. Further work will be necessary to gain insight into the specific hierarchy of phosphorylation of tyrosine, serine, and threonine residues in plant RK complexes and to assess their relative contributions to the recruitment and release of additional regulators and downstream interactors, and hence to the onset of signaling. Acknowledgments Research on this topic in the laboratory of C.Z. is funded by the Gatsby Charitable Foundation and the European Research Council. A.P.M. was funded by a long-term post-doctoral fellowship from the Federation of European Biochemical Societies. R.L-D. was funded by a long-term postdoctoral fellowship from the Fundacio´n Ramo´n Areces. We thank all members of the C.Z. laboratory, as well as Steven Huber, Vardis Ntoukakis, and Benjamin Schwessinger, for helpful discussions. We apologize to colleagues whose work could not be cited due to space limitations.
References 1 Zhu, J.Y. et al. (2013) Brassinosteroid signalling. Development 140, 1615–1620 2 Boller, T. and Felix, G. (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by patternrecognition receptors. Annu. Rev. Plant Biol. 60, 379–406 3 Shiu, S.H. and Bleecker, A.B. (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. U.S.A. 98, 10763–10768 4 Lemmon, M.A. and Schlessinger, J. (2010) Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 5 Lim, W.A. and Pawson, T. (2010) Phosphotyrosine signaling: evolving a new cellular communication system. Cell 142, 661–667 6 Mu, J.H. et al. (1994) Characterization of a pollen-expressed receptorlike kinase gene of Petunia inflata and the activity of its encoded kinase. Plant Cell 6, 709–721 7 Shah, K. et al. (2001) Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation. J. Biol. Chem. 276, 41263–41269 8 Oh, M.H. et al. (2009) Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 106, 658–663 9 Macho, A.P. et al. (2014) A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation. Science 343, 1509–1512 10 Oh, M.H. et al. (2010) Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression. Proc. Natl. Acad. Sci. U.S.A. 107, 17827–17832 11 Jaillais, Y. et al. (2011) Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–237 12 Lin, W. et al. (2014) Tyrosine phosphorylation of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity. Proc. Natl. Acad. Sci. U.S.A. 111, 3632–3637 13 Xu, J. et al. (2013) Identification and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1. Protein Cell 4, 771–781
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14 Dievart, A. and Clark, S.E. (2004) LRR-containing receptors regulating plant development and defense. Development 131, 251–261 15 Shiu, S.H. and Bleecker, A.B. (2003) Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol. 132, 530–543 16 Sun, Y. et al. (2013) Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res. 23, 1326–1329 17 Wang, X. and Chory, J. (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313, 1118–1122 18 Wang, J. et al. (2014) Structural insights into the negative regulation of BRI1 signaling by BRI1-interacting protein BKI1. Cell Res. 24, 1328–1341 19 Macho, A.P. and Zipfel, C. (2014) Plant PRRs and the activation of innate immune signaling. Mol. Cell 24, 263–272 20 Go´mez-Go´mez, L. and Boller, T. (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, 1003–1011 21 Zipfel, C. et al. (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749–760 22 Chen, X. et al. (2010) A conserved threonine residue in the juxtamembrane domain of the XA21 pattern recognition receptor is critical for kinase autophosphorylation and XA21-mediated immunity. J. Biol. Chem. 285, 10454–10463 23 Schulze, B. et al. (2010) Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J. Biol. Chem. 285, 9444–9451 24 Schwessinger, B. et al. (2011) Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet. 7, e1002046 25 Chinchilla, D. et al. (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500 26 Heese, A. et al. (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. U.S.A. 104, 12217–12222 27 Roux, M. et al. (2011) The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23, 2440–2455 28 Sun, Y. et al. (2013) Structural basis for flg22-induced activation of the Arabidopsis FLS2–BAK1 immune complex. Science 342, 624–628 29 Oh, M.H. et al. (2011) Functional importance of BAK1 tyrosine phosphorylation in vivo. Plant Signal. Behav. 6, 400–405 30 Kadota, Y. et al. (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol. Cell 54, 43–55 31 Li, L. et al. (2014) The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15, 329–338 32 Lu, D. et al. (2010) A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl. Acad. Sci. U.S.A. 107, 496–501 33 Zhang, J. et al. (2010) Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7, 290–301 34 Wang, Y. et al. (2014) Assessment of BAK1 activity in different plant receptor-like kinase complexes by quantitative profiling of phosphorylation patterns. J. Proteomics 108, 484–493 35 Bojar, D. et al. (2014) Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation. Plant J. 78, 31–43 36 Lozano-Duran, R. and Zipfel, C. (2014) Trade-off between growth and immunity: role of brassinosteroids. Trends Plant Sci. 20, 12–19 37 Kim, T.W. et al. (2009) Brassinosteroid signal transduction from cellsurface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 11, 1254–1260 38 Mithoe, S.C. et al. (2012) Targeted quantitative phosphoproteomics approach for the detection of phospho-tyrosine signaling in plants. J. Proteome Res. 11, 438–448 39 Oh, M.H. et al. (2012) CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity. FEBS Lett. 586, 4070–4075
Opinion
Importance of tyrosine phosphorylation in receptor kinase complexes Alberto P. Macho*, Rosa Lozano-Dura´n*, and Cyril Zipfel The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK
Tyrosine phosphorylation is an important posttranslational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Receptor kinases RKs are plasma membrane-localized proteins that initiate signaling in a multitude of plant processes. RKs are composed of an ectodomain potentially involved in ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain. Well-studied examples of RK-mediated signaling pathways are those triggered by plant perception of the steroid hormones brassinosteroids (BRs), which promote plant growth [1], or of pathogenassociated molecular patterns (PAMPs), leading to immune responses that constitute the first active layer of plant immunity [2]. Plant RKs have traditionally been classified as serine/ threonine kinases [3]. This is in contrast with most animal RKs where tyrosine phosphorylation is important for their activation and subsequent signaling, leading to the onset of a multitude of cellular processes [4,5]. Phosphorylation on tyrosine residues was originally observed in plant RKs after autophosphorylation in vitro [6–8], although until recently it was not clear whether this also occurred in vivo. Reports over the past 5 years have shown that, Corresponding authors: Macho, A.P. ([email protected]); Zipfel, C. ([email protected]). Keywords: tyrosine phosphorylation; receptor kinases; immunity; brassinosteroids; PAMPs. * Present addresses: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China. 1360-1385/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2015.02.005
in addition to serine/threonine phosphorylation, several RKs in Arabidopsis thaliana (hereafter referred to as Arabidopsis) can undergo tyrosine phosphorylation, and that this modification plays an important role in their activation and in the initiation of subsequent signaling [8–10] (Figure 1, Table 1). In addition, RK-associated proteins are also regulated by tyrosine phosphorylation, uncovering an additional role of this post-translational modification in downstream signal transduction [11–13] (Figure 1; Table 1). In this Opinion article we propose that tyrosine phosphorylation plays a major role in the activation, transduction, and, potentially, specificity of RKmediated signaling in plants. Tyrosine phosphorylation in the BR receptor complex The BR signaling pathway is one of the best studied in plants [1]. In a seminal article published by Oh and coworkers [8] it was observed that the BR receptor, the leucine-rich repeat (LRR)-RK BRASSINOSTEROID-INSENSITIVE 1 (BRI1), is able to autophosphorylate on tyrosine residues, in addition to serine and threonine residues [8]. This was unexpected because BRI1 was classified as a serine/threonine kinase, alongside other plant RKs [14,15]. Strikingly, the authors also reported an unanticipated difficulty in the identification of specific BRI1 phosphorylated tyrosines in vivo when using liquid chromatography–tandem mass spectrometry (LC–MS/ MS). However, through a thorough analysis using sitedirected mutagenesis and modification- and sequence-specific antibodies, the authors identified a single tyrosine residue in the juxtamembrane domain of BRI1 (Y831; Figure 1; Table 1) which plays an important role in BR signaling in vivo, without affecting overall BRI1 kinase activity [8]. Additional tyrosine residues, Y956 and Y1072, were found to be phosphorylated in vivo and in vitro, respectively, although mutations of these residues abolished BRI1 kinase activity [8] (Figure 1; Table 1). Moreover, it was observed that the LRR-RK BRI1ASSOCIATED KINASE 1 (BAK1), which acts as a BR coreceptor with BRI1 [16], was able to autophosphorylate on tyrosine residues, and this prompted a follow-up study on the relevance of different tyrosine residues in BAK1 [10]. Using site-directed mutagenesis and modificationand sequence-specific antibodies, a single tyrosine residue in the C-terminal domain of BAK1 (Y610; Figure 1; Table 1) was identified as a major autophosphorylation site upon Trends in Plant Science xx (2015) 1–4
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(A)
(B)
BRI1
BRI1
EFR
EFR elf18
BAK1
BAK1
BAK1
BR
PM
BAK1
PM P
BKI1 BIK1
P
BIK1
P
BKI1 BIK1
BIK1
P BIK1
BIK1 P
BIK1
BR signaling
PTI signaling Cytoplasm
Cytoplasm TRENDS in Plant Science
Figure 1. Simplified diagram of tyrosine-phosphorylated proteins involved in early brassinosteroid (BR)- and PAMP (here, elf18)-triggered signaling. (A) In the absence of BR, BKI1 and BIK1 negatively regulate the activation of BRI1. BR perception leads to phosphorylation (P) of BKI1 Y211, followed by the release of the protein into the cytosol, which relieves the inhibition of BRI1 kinase activity and increases the affinity between BRI1 and BAK1 kinase domains. BR perception also leads to the release of BIK1. The dissociation of both negative regulators allows the formation of a fully-active BR receptor complex. (B) BIK1 associates with EFR and BAK1 in unelicited conditions. Upon elf18 perception, EFR Y836 is phosphorylated and contributes to the ligand-induced activation of the EFR–BAK1 complex. PAMP perception also leads to the phosphorylation of BIK1, which is released from the receptor complex and activates downstream immune responses. Abbreviations: BAK1, BRI1-ASSOCIATED KINASE 1; BIK1, BOTRYTIS-INDUCED KINASE 1; BKI1, BRI1-KINASE INHIBITOR 1; BRI1, BRASSINOSTEROID-INSENSITIVE 1; EFR, EF-TU RECEPTOR; PAMP, pathogen-associated molecular pattern; PM, plasma membrane.
BR perception that is required for BR-triggered signaling [10]. Together, these two reports unequivocally demonstrated that both BRI1 and BAK1 are dual-specificity kinases, and that tyrosine phosphorylation plays a prominent role
in the activation of the BR receptor complex and subsequent signal transduction. Later work added more complexity to the role of tyrosine phosphorylation in the regulation of BR receptor activation. One of the mechanisms that keeps BRI1 in an inactive state
Table 1. Tyrosine-phosphorylated residues in components of RK complexesa Protein
BRI1
Residue
Y831 Y956
BAK1 BKI1 EFR BIK1
a
Y1072 Y610 Y211 Y836 Y23 Y150 Y168 Y214 Y234 Y243 Y250
Identification
In In In In In In In In In In In In In In In In
vivo vitro vivo vitro vitro vivo vitro vivo vivo vitro vitro vitro vitro vitro vitro vitro
Liganddependency
Auto-/transphosphorylation
Evidence of phosphorylation
Mutation to Phe abolishes kinase activity
Nd
Auto
Yes
Nd
Auto
Nd BL-induced BL-induced elf18-induced Nd Nd Nd Nd Nd Nd Nd
Detection by LC/ MS-MS
Refs
Sitedirected mutagenesis
Yes
Sequence- and phosphorylationspecific antibodies Yes
No
[7]
No
Yes
Yes
Yes
[7]
Auto Auto
No No
Yes Yes
Yes Yes
Yes No
[7] [9]
Trans Nd Auto Nd Auto/trans Auto/trans Auto Auto/trans Auto/trans
No Yes Yes No Yes Yes Yes No Yes
No No No No No No No No No
Yes Yes No Yes No Yes Yes Yes Yes
N/A No No Yes No No No No No
[10] [8] [11] [11] [12,33] [12,33] [11] [11] [11,12,33]
Abbreviations: BAK1, BRI1-ASSOCIATED KINASE 1; BIK1, BOTRYTIS-INDUCED KINASE 1; BKI1, BRI1-KINASE INHIBITOR 1; BRI1, BRASSINOSTEROID-INSENSITIVE 1; EFR, EF-TU RECEPTOR. Nd, not determined.
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Opinion in the absence of the hormone operates via an interaction with the plasma membrane-localized protein BRI1-KINASE INHIBITOR 1 (BKI1) [17]. BR perception leads to phosphorylation of a specific tyrosine residue in BKI1 (Y211), followed by release of the protein into the cytosol [11] (Figure 1; Table 1). BKI1 dissociation relieves the inhibition of BRI1 kinase activity and increases the affinity between BRI1 and BAK1 kinase domains, therefore allowing the formation of a fully-active BR receptor complex [11,18] (Figure 1). Thus, tyrosine phosphorylation appears to be an essential regulatory mechanism for the robustness of the multi-layered regulation of BR signaling initiation in plants. Tyrosine phosphorylation in plasma membrane immune receptor complexes In recent years, several reports have significantly increased our understanding of the initiation of immune signaling triggered by plant pattern-recognition receptors (PRRs), which mediate the perception of PAMPs (reviewed in [19]). Several PRRs are RKs, such as the LRR-RKs FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which perceive bacterial flagellin (or the elicitor peptide flg22) and elongation factor Tu (EF-Tu; or the elicitor peptide elf18), respectively [20,21]. Although protein phosphorylation within PRR complexes was known to be crucial for the initiation of immune signaling [22–24], the exact phosphorylation events occurring within these complexes and their exact biological roles were unknown. Recently, we reported that EFR is activated upon ligand binding by phosphorylation on tyrosine residues [9]. A combination of site-directed mutagenesis and targeted MS analysis identified a single tyrosine residue on EFR (Y836) that is phosphorylated in vivo after elf18 perception and which is required for the activation of EFR and downstream immune responses [9] (Figure 1; Table 1). Interestingly, this residue is not required for the overall kinase activity of EFR, but only for its ligand-induced activation [9]. An additional demonstration of the biological relevance of tyrosine phosphorylation for PRR-triggered immunity (PTI) comes from the finding that the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 injects a tyrosine phosphatase inside the plant cell to target EFR and FLS2. The interaction of this bacterial protein with these PRRs reduces their level of tyrosine phosphorylation, subsequently inhibiting immune signaling and thereby increasing susceptibility to P. syringae [9]. Similarly to BRI1, PRR-associated kinases also undergo tyrosine phosphorylation. It is important to note that BAK1 also plays an essential role in PTI by acting as coreceptor in the perception of PAMPs and a positive regulator of subsequent signaling [23–28]. Importantly, phosphorylation of Y610 on BAK1 seems to play a role in the basal expression of immune-related genes beyond its contribution to the initiation of BR-signaling [10,29] (Figure 1; Table 1). The involvement of this and additional BAK1 tyrosine residues in PTI signaling will require further investigation. The cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) interacts with FLS2, EFR, and BAK1, and plays an important role in the activation of specific downstream responses after PAMP perception [30–33]. It was recently
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shown that BIK1 is phosphorylated on several tyrosine residues in vitro, including Y23, Y150, Y168, Y214, Y234, Y243, and Y250 [12,13,34] (Figure 1; Table 1). Notably, several BIK1 tyrosine residues are required for the BIK1mediated phosphorylation of substrates in vitro [13] (Table 1) and for BIK1-dependent PTI responses in planta [12]. Together, these reports indicate that tyrosine phosphorylation is a key regulatory mechanism in the activation of innate immune complexes and subsequent signal transduction. Role of tyrosine phosphorylation in early RK-mediated signaling: a multifunctional mechanism for signaling initiation? In animal RKs, autophosphorylation of specific tyrosine residues is necessary to increase the catalytic efficiency of the RK itself, whereas phosphorylation of additional tyrosine residues creates docking sites for downstream signaling molecules [4,5]. Similarly, a BRI1 tyrosine residue phosphorylated in vivo, Y956, is required for BRI1 kinase activity [8], although this is most likely due to the role of BRI1 Y956 as gatekeeper residue at the back of the ATP binding site, allowing the active conformation of the kinase domain [35]. Consequently, the results obtained by amino acid substitutions require careful interpretation because such substitutions may have direct or indirect structural consequences. Another BRI1 tyrosine residue phosphorylated in vivo, Y831, is not essential for BRI1 kinase activity and plays an inhibitory role in BR-triggered signaling [8]. In addition, two phosphorylated tyrosine residues in BAK1 (Y610) and EFR (Y836) were found to be essential for BRand elf18-triggered responses, respectively, while being non-essential for their respective kinase activities [9,10]. It is therefore possible that, similarly to what is observed in animals, phosphorylation on specific tyrosine residues is required for kinase activation (and subsequent phosphorylation of relevant serine, threonine, or tyrosine residues), while other phosphorylated tyrosine residues are involved in dynamic interactions with downstream kinase substrates or other signaling components. A similar situation was found for phosphorylated tyrosine residues in the cytoplasmic kinase BIK1 in vitro [12,13]. In addition, as seen for BKI1 [11], tyrosine phosphorylation of negative regulators, such as kinase inhibitors or protein phosphatases, could disrupt their inhibition of RK activation. This would provide a prompt and transient switch, contributing to the formation of active receptor complexes in a robust ligand-dependent manner. It is important to note that several central signaling components are present in both BR and PAMP receptor complexes. The most prominent example is BAK1, which acts as coreceptor for BRI1 and several PRRs, and is a positive regulator for both BR- and PAMP-triggered responses. A different case is that of BIK1, which acts as a positive regulator of PTI, but is a negative regulator of BR signaling. How these proteins mediate specific signaling outputs upon perception of different ligands is still unknown [36]. It is tempting to speculate that phosphorylation of particular, distinct tyrosine (as well as serine or threonine) residues could contribute to ligand-dependent 3
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Opinion outputs mediated by these shared components, therefore providing signaling specificity. Concluding remarks Recent work has uncovered tyrosine phosphorylation as an important mechanism for the initiation of signaling mediated by plasma membrane-localized RK complexes in plants. Remarkably, the relevance of tyrosine phosphorylation for BR- and PAMP-triggered signaling goes beyond receptor complexes and extends to downstream signaling cascades because several downstream regulatory components, including glycogen synthase kinase 3 (GSK3)-like kinases, mitogen-activated protein kinases (MAPKs), and calcium-dependent protein kinases (CDPKs), have been shown to phosphorylate tyrosine residues [37–39]. Further work will be necessary to gain insight into the specific hierarchy of phosphorylation of tyrosine, serine, and threonine residues in plant RK complexes and to assess their relative contributions to the recruitment and release of additional regulators and downstream interactors, and hence to the onset of signaling. Acknowledgments Research on this topic in the laboratory of C.Z. is funded by the Gatsby Charitable Foundation and the European Research Council. A.P.M. was funded by a long-term post-doctoral fellowship from the Federation of European Biochemical Societies. R.L-D. was funded by a long-term postdoctoral fellowship from the Fundacio´n Ramo´n Areces. We thank all members of the C.Z. laboratory, as well as Steven Huber, Vardis Ntoukakis, and Benjamin Schwessinger, for helpful discussions. We apologize to colleagues whose work could not be cited due to space limitations.
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