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Interactions between the S -Domain Receptor Kinases
and AtPUB-ARM E3 Ubiquitin Ligases Suggest a
Conserved Signaling Pathway in Arabidopsis
1[W][OA]
Marcus A. Samuel
2
, Yashwanti Mudgil
2,3
,JenniferN.Salt,Fre
´
de
´
ric Delmas, Shaliny Ramachandran,
Andrea Chilelli, and Daphne R. Goring*
Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2
The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular
domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been
deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI)
response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase,
ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in
Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative
Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were
found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein
kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation
assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum)BY-2
cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these
interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the
plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well,
T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or
upstream of ABI3, indicating potential involvement of these proteins in ABA responses.
The process of ubiquitin-mediated protein degrada-
tion is activated in many biological processes during
the plant life cycle and is an equally important step in
the regulation of protein activities (Moon et al., 2004;
Smalle and Vierstra, 2004). Disruptions to the process
can lead to prolonged activity of a target protein and
clearly have effects on the plant growth and develop-
ment. Three enzymes are involved in the ubiquitina-
tion of a target protein, the E1 ubiquitin-activating
enzyme, the E2 ubiquitin-conjugating enzyme, and the
E3 ubiquitin ligase. By far, the E3 ubiquitin ligase is the
largest group of these enzymes that is related to its role
in defining the substrate specificity in this pathway
(Devoto et al., 2002; Dill et al., 2004). For example, there
are two E1 enzymes and 41 E2 enzymes annotated in
the Arabidopsis (Arabidopsis thaliana) genome (Kraft
et al., 2005). The E3 ligase group is a far more diverse
group and, based on known E3 ligase motifs, there are
at least 1,30 0 predicted E3 ligase genes in the Arabi-
dopsis genome (Smalle and Vierstra, 2004). The larger
known Arabidopsis families include the RING family
with approximately 469 predicted proteins and the
F-box family with approximately 700 predicted proteins
(Gagne et al., 2004; Stone et al., 2005). The Arabidopsis
U-box family is a smaller predicted family with 62
members (Azevedo et al., 2001; Andersen et al., 2004).
The U-box is an E3 ligase motif conserved in all
eukaryotes (Aravind and Koonin, 2000) and is a mod-
ified ring finger shown to ubiquitinate substrates in the
presence of the appropriate E1 and E2 (Hatakeyama
et al., 2001; Mudgil et al., 2004). The plant U-box (PUB)
family can be divided into five groups based on the
presence of other distinguishing domains, such as the
UFD2, ARM repeats, UND, Ser/Thr kinase, WD40
repeats (Azevedo et al., 2001; Mudgil et al., 2004;
Wiborg et al., 2008). The PUB-ARM family comprises
the largest group with 41 predicted members in the
Arabidopsis genome and 43 members in the rice (Oryza
sativa) genome (Mudgil et al., 2004; Samuel et al., 2006).
Despite the limited knowledge about the biological
functions for these predicted PUB-ARM proteins, they
have been shown to function as E3 ubiquitin ligases
(Andersen et al., 2004; Mudgil et al., 2004). In various
plant species, diverse biological functions have emerged
for related PUB-ARM proteins. A strong connection to
1
This work was supported by grants from the Natural Sciences
and Engineering Research Council of Canada and a Canada Re-
search Chair to D.R.G.
2
These authors contributed equally to the article.
3
Present address: Department of Biology, The University of
North Carolina, Coker Hall, Chapel Hill, NC 27599.
* Corresponding author; e-mail d.goring@utoronto.ca.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Daphne R. Goring (d.goring@utoronto.ca).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a sub-
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.123380
2084 Plant Physiology, August 20 08, Vol. 147, pp. 2084–2095, www.plantphysiol.org Ó 2008 Ameri can Society of Plant B iologists
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plant defense responses is emerging for several PUB-
ARM proteins. The rice SPL11 gene was identified in
a genetic screen for lesion mimic mutants (Yin et al.,
2000), and t he spl11 mutant disp lays spontaneous
lesions and enhance resistance to fungal and bacterial
pathogens implicating SPL11 as a negative regula-
tor of c ell death (Zeng et al., 2004). In contrast, the
Arabidopsis PUB17 protein and the orthologous to-
bacco (Nicotia na tab ac um) ACRE276 protein appear
to be positi ve regulators of cell death and defense
responses because RNAi and knockout plants are com-
promised in theseresponses(Yang et al., 2006). Similarly,
the Arabidopsis PUB21 and the orthologous tobacco
CMPG1 are required for hypersensitive response devel-
opment and disease resistance (Gonza
´
lez-Lamothe
et al., 2006). A role in plant hormone responses has
also been reported with the potato (Solanum tuberosum)
PHOR1 protein being identified as a positive regulator
of GA signaling (Amador et al., 2001). Finally, connec-
tions are emerging between PUB-ARM proteins and
receptor kinases. The Brassica ARC1 protein has been
found to bind to the S-receptor kinase (SRK) and is
required for the Brassica self-incompatibility (SI) re-
sponse where it functions downstream of the SRK to
cause self-pollen rejection (Gu et al., 1998; Stone et al.,
1999). Interestingly, a related member, Arabidopsis
PUB8, has been implicated in the regulation of mRNA
levels of Arabidopsis lyrata SRK genes (P. Liu et al., 2007).
Last, the tobacco PUB4 protein was identified as an
interacting protein for the CHRK1 receptor kinase (Kim
et al., 2003).
The Brassica and tobacco studies suggest a role for the
PUB-ARM proteins as potential signaling proteins for
receptor kinases. In Arabidopsis, there are a large
number of receptor kinases with a range of extracellular
domains (Morris and Walker, 2003; Haffani et al., 2004).
The Brassica SRK, which interacts with ARC1, is very
closely related to the Arabidopsis S-domain-1 (SD1)
receptor kinase subfamily. The Arabidopsis S-domain
receptor kinases fall into three classes with more than
40 members (Shiu and Bleecker, 2003) and the functions
of this family of kinases have remained largely unde-
fined thus far. Overexpression of ARK1 was shown to
result in severe developmental abnormalities (Tobias
and Nasrallah, 1996), whereas promoter analysis and
expression studies indicated that RLK4 was one of the
targets of pathogen and wound-induced WRKY tran-
scription factor targets (Du and Chen, 2000). The tobacco
CHRK-1 receptor kinase possesses a chitinase-like
extracellular domain that is not found in Arabidopsis;
however, the intracellular kinase domain, which is
required for the interaction with NtPUB4, is most
closely related to members of the Arabidopsis SD1
receptor kinase subfamily (Kim et al., 2000). Cosup-
pression of the endogenous tobacco CHRK1 gene was
found to have a range of phenotypes, including callus
formation following seed germination, increased shoot
formation, reduced apical dominance, and abnormal
flowers. This was also accompanied by increased cyto-
kinin levels in the transgenic plants (Lee et al., 2003).
The observed interaction between Brassica SRK-
ARC1 and tobacco CHRK1-NtPUB4 and the conserva-
tion of signaling components across Brassica and
Arabidopsis suggested to us that the Arabidopsis
S-domain receptor kinase family could potentially utilize
the numerous AtPUB-ARM family members as their
downstream signaling components. To investigate
this, we have performed a selected interaction screen
between the SD1 receptor kinases and AtPUB-ARM
family proteins and identified either common or spe-
cific interactors. Further analyses of these interactions
were carried out using in vitro phosphorylation assays
and transient expression assays. In addition, potential
links to the plant hormone abscisic acid (ABA) were
further investigated by functiona l analyses with se-
lected SD1 receptor kinase and AtPUB-ARM proteins.
RESULTS
AtPUB-ARM Proteins Interact with Arabidopsis
and Brassica S-Domain Receptor Kinases
A directed yeast two-hybrid interaction screen was
conducted with ARM-repeat domains from multiple
AtPUB-ARM proteins (Fig. 1A) against kinase domains
from selected receptor kinases. AtPUB-ARM proteins
were chosen to represent the different modular combi-
nations found in the AtPUB-ARM family (Fig. 1A; Mudgil
et al., 2004). AtPUB13, 14, and 45 represented the Brassica
ARC1-like domain organization (UND, U-box, and
ARM domains) with AtPUB13 and 14 being more closely
related to ARC1 and AtPUB45 being more distantly re-
lated to ARC1. AtPUB9, 29, and 38 were selected to rep-
resent AtPUB-ARM proteins that lack the UND domain
(U-box and ARM only), and AtPUB9 and 38 are more
closely related to ARC1 relative to AtPUB29. Finally,
AtPUB44 was chosen to represent the dual ARM-repeat
clade (U-bo x:ARM:ARM) and is the most distantly re-
lated AtPUB relative to ARC1 (Mudgil et al., 2004; Samuel
et al., 2006). All six of these AtPUB-ARM proteins have
been shown to have in vitro E3 ubiquitin ligase activity
(Andersen et al., 2004; Mudgil et al., 2004; J. Salt, M.A.
Samuel, and D.R. Goring, unpublished data).
The various kinases included the Arabidopsis
S-domain receptor kinases representing vario us sub-
groups: SD1-7 (ARK1), SD1-6 (ARK2), SD1-8 (ARK3),
SD1-29, SD1-13 (RKS2), SD1-1, SD2-5, SD2-2 (RLK4),
SD3-1, DUF26-21 (RKC1), and DUF26-4 (RLK3), along
with two Arabidopsis Leu-rich repeat (LRR) receptor
kinases, LRR XI-16 (HAESA) and LRR XI-23. In addi-
tion, related Brassica SD1 receptor kinases, SFR1, SFR2,
and SRK
910
, which were previously shown to interact
with Brassica ARC1, were included in the screen
(Mazzurco et al., 2001). Selected kinase domains were
tested for kinase activity by using an in vitro autophos-
phorylation assay with purified glutathione S-transferase
(GST):kinase fusion proteins. The kinase domains were
found to have strong autophosphorylation activity as
shown in Figure. 1B or as previously shown (Mazzurco
et al., 2001). Protein expression of all the constructs in
AtPUB Proteins and S-Domain Receptor Kinase Interactions
Plant Physiol. Vol. 147, 2008 2085
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the transformed yeast (Saccharomyces cerevisiae) was
confirmed using immunoblot analysis with either the
VP16 or LexA antibodies.
From the yeast two-hybrid analyses, it was found
that the kinase domains from selected SD1 receptor
kinases generally interacted well with the AtPUB-ARM
proteins while very low-level or no interactions were
observed with non-SD1 receptor kinase family mem-
bers (Fig. 1C). For exampl e, AtPUB13, 14, and 9 in-
teracted with all the Arabidopsis and Brassica SD1
receptor kinases as well as SD2-5, but no interactions
were observed with the remaining receptor kinases.
AtPUB38 also interacted with a number of the Arabi-
dopsis and Brassica SD1 receptor kinases as wel l as
Figure 1. Yeast two-hybrid interactions between selected AtPUB-ARM proteins and receptor kinases. A, Domain organization of
AtPUB-ARM proteins tested in the yeast two-hybrid screen. The motif arrangements were previously identified in Mudgil et al.
(2004). The predicted domain organizations for the full-length AtPUB-ARM proteins are shown on the left, while the ARM
domains used in the yeast two-hybrid interaction studies for the respective AtPUBs are shown on the right. B, In vitro
autophosphorylation assay. Selected kinases were expressed as GST-fusion proteins in Escherichia coli and subjected to an in
vitro [g
32
P]-labeled autophosphorylation assay followed by autoradiography. C, Yeast two-hybrid interactions between selected
kinase domains and ARM domains. For all AtPUB-ARMs, the entire ARM region following the U-box was used. The exception is
AtPUB44a and AtPUB44b, where the longer ARM repeat region was split in half, with each half being tested. The Arabidopsis
receptor kinase nomenclature used is according to Shiu and Bleecker (2003). The AtPUB nomenclature is according to Azevedo
et al. (2001) and Mudgil et al. (2004) and can be found at http://www.arabidopsis.org/info/genefamily/pub.html. Interactions
were detected by the activation of the lacZ reporter gene leading to b-galactosidase activity, which, in the presence of X-gal,
produced a blue color on filter lifts of transformed yeast. The time required for the formation of the blue color was monitored and
roughly documented with (1111) indicating a very rapid blue color development to (1) indicating a weak, but reproducible,
blue color development after several hours. No detection of any b-galactosidase activity (blue color) was interpreted as no
interaction and indicated as (2). C, Control; nd, not determined.
Samuel et al.
2086 Plant Physiol. Vol. 147, 2008
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