Download Importance of tyrosine phosphorylation in receptor kinase

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
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5
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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