A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2.
ABSTRACT SOS2 (salt overly sensitive 2) is a serine/threonine protein kinase required for salt tolerance in Arabidopsis thaliana. In this study, we identified the protein phosphatase 2C ABI2 (abscisic acid-insensitive 2) as a SOS2-interacting protein. Deletion analysis led to the discovery of a novel protein domain of 37 amino acid residues, designated as the protein phosphatase interaction (PPI) motif, of SOS2 that is necessary and sufficient for interaction with ABI2. The PPI motif is conserved in protein kinases of the SOS2 family (i.e., protein kinase S, PKS) and in the DNA damage repair and replication block checkpoint kinase, Chk1, from various organisms including humans. Mutations in the conserved amino acid residues in the PPI motif abolish the interaction of SOS2 with ABI2. We also identified a protein kinase interaction domain in ABI2 and examined the interaction specificity between PKS and the ABI phosphatases. We found that some PKSs interact strongly with ABI2 whereas others interact preferentially with ABI1. The interaction between SOS2 and ABI2 was disrupted by the abi2-1 mutation, which causes increased tolerance to salt shock and abscisic acid insensitivity in plants. Our results establish the PPI motif and the protein kinase interaction domain as novel protein interaction domains that mediate the binding between the SOS2 family of protein kinases and the ABI1/2 family of protein phosphatases.
- SourceAvailable from: László Szabados[show abstract] [hide abstract]
ABSTRACT: Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of Δ1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3–101.3 contributes 20–40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.The Plant Journal 08/1997; 12(3):557 - 569. · 6.58 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The Arabidopsis ABI1 locus is essential for a wide spectrum of abscisic acid (ABA) responses throughout plant development. Here, ABI1 was shown to regulate stomatal aperture in leaves and mitotic activity in root meristems. The ABI1 gene was cloned and predicted to encode a signaling protein. Although its carboxyl-terminal domain is related to serine-threonine phosphatase 2C, the ABI1 protein has a unique amino-terminal extension containing an EF hand calcium-binding site. These results suggest that the ABI1 protein is a Ca(2+)-modulated phosphatase and functions to integrate ABA and Ca2+ signals with phosphorylation-dependent response pathways.Science 07/1994; 264(5164):1448-52. · 31.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: 1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.European Journal of Biochemistry 09/1991; 199(3):691-7. · 3.58 Impact Factor
A novel domain in the protein kinase SOS2 mediates
interaction with the protein phosphatase 2C ABI2
Masaru Ohta*, Yan Guo, Ursula Halfter, and Jian-Kang Zhu†
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721
Communicated by Andre ´ T. Jagendorf, Cornell University, Ithaca, NY, July 31, 2003 (received for review March 26, 2003)
SOS2 (salt overly sensitive 2) is a serine?threonine protein kinase
identified the protein phosphatase 2C ABI2 (abscisic acid-insensi-
tive 2) as a SOS2-interacting protein. Deletion analysis led to the
discovery of a novel protein domain of 37 amino acid residues,
designated as the protein phosphatase interaction (PPI) motif, of
SOS2 that is necessary and sufficient for interaction with ABI2. The
PPI motif is conserved in protein kinases of the SOS2 family (i.e.,
protein kinase S, PKS) and in the DNA damage repair and replica-
tion block checkpoint kinase, Chk1, from various organisms includ-
ing humans. Mutations in the conserved amino acid residues in the
PPI motif abolish the interaction of SOS2 with ABI2. We also
identified a protein kinase interaction domain in ABI2 and exam-
ined the interaction specificity between PKS and the ABI phospha-
tases. We found that some PKSs interact strongly with ABI2
whereas others interact preferentially with ABI1. The interaction
between SOS2 and ABI2 was disrupted by the abi2-1 mutation,
which causes increased tolerance to salt shock and abscisic acid
insensitivity in plants. Our results establish the PPI motif and the
protein kinase interaction domain as novel protein interaction
domains that mediate the binding between the SOS2 family of
protein kinases and the ABI1?2 family of protein phosphatases.
response to developmental, hormonal, and environmental cues.
The Arabidopsis SOS2 (salt overly sensitive 2) gene is necessary
for sodium and potassium ion homeostasis and salt tolerance (1).
SOS2 encodes a serine?threonine protein kinase with an N-
terminal kinase catalytic domain similar to SNF1?AMPK and a
novel C-terminal regulatory domain (2). SOS2 is normally
inactive, presumably because of an intramolecular interaction
between the catalytic domain and the autoinhibitory regulatory
domain (3). Salt stress elicits a cytosolic calcium signal (4).
Calcium, together with the calcium-binding protein SOS3, acti-
vates SOS2 (5). SOS3 physically interacts with SOS2 in the yeast
two-hybrid system as well as in vitro (5). A 21-aa sequence in the
regulatory domain of SOS2, designated as the FISL motif, is
necessary and sufficient for the interaction with SOS3 (3). The
SOS3–SOS2 kinase complex is required for the phosphorylation
and activation of the plasma membrane Na??H?antiporter
encoded by the SOS1 gene (6–8).
In Arabidopsis, SOS2 is a member of a family of 25 protein
kinases that are known as protein kinase S (PKS) (3). Evidence
suggests that individual PKS proteins interact with specific
calcium-binding proteins in the SOS3 family (known as SCaBPs)
to form distinct protein kinase complexes (3). These protein
kinase complexes may be capable of decoding various calcium
signals elicited by developmental, hormonal, or environmental
cues (3). For example, whereas the SOS3–SOS2 protein kinase
complex mediates salt stress-specific calcium signaling, SCaBP5
and PKS3 form a complex that participates in a regulatory loop
in abscisic acid (ABA) signaling, possibly by decoding an ABA-
elicited calcium signal and by controlling the generation of such
a calcium signal (9).
The phosphorylation status of a protein is determined by the
balance between the activities of protein kinases and protein
eversible protein phosphorylation is a fundamental mecha-
nism by which living organisms regulate cellular processes in
phosphatases. Protein phosphatase 2C is a class of conserved
serine?threonine protein phosphatases involved in stress re-
sponses in plants, yeasts, and animals (10–13). Dominant mu-
tations in two of the homologous protein phosphatase 2Cs,
ABA-insensitive (ABI) 1 and ABI2, render Arabidopsis plants
insensitive to the stress hormone ABA (8, 14–18). The abi1-1
and abi2-1 mutations have been proposed to have dominant
negative effects, and the WT ABI1 and ABI2 proteins are
thought to be negative regulators of ABA signaling (19–21).
Although ABI1 and ABI2 are highly homologous, they may
function at different steps in ABA signaling (22–26).
In this study, we identified ABI2 as a SOS2-interacting
protein. Deletion analysis led to the discovery of a novel protein
domain of 37 amino acid residues, designated as the protein
phosphatase interaction (PPI) motif, of SOS2 that is necessary
and sufficient for interaction with ABI2. The PPI motif is
conserved in Arabidopsis PKS proteins and in the DNA damage
repair and replication block checkpoint kinase, Chk1, from
amino acid residues in the PPI motif abolish the interaction of
SOS2 with ABI2. We also identified a protein kinase interaction
(PKI) domain in ABI2 and examined the interaction specificity
between PKS and the ABI phosphatases. We found that some
PKSs interact strongly with ABI2 whereas others interact pref-
erentially with ABI1. The interaction between SOS2 and ABI2
was disrupted by the abi2-1 mutation, which causes increased
tolerance to salt shock and ABA insensitivity in plants. Our
identification of the PPI motif and the PKI domain that mediate
the interaction between SOS2 and ABI2 contributes substan-
tially to the understanding of the structure and regulation of
these key regulators of stress tolerance.
Materials and Methods
Construction of Plasmids. Plasmids pAS-SOS2 and pAS-SOS2N
were described by Halfter et al. (5) and Guo et al. (3), respec-
tively. Plasmids pAS-SOS1, pACT2-ABI1, pact-ABI2, and pAS-
PKS3 were constructed previously (9). Plasmids containing the
C-terminal portions of SOS2 cDNA (pAS-SOS2-T1 and pAS-
SOS2-T3) were constructed by moving the NcoI and BamHI
fragments from pACT-SOS2-T1 and pACT-SOS2-T3 (3) into
the pAS2 vector. Full-length cDNAs for PKS11, PKS18, and
PKS24 were amplified by PCR using specific primers and
inserted into the pAS2 vector to make pAS-PKS11, pAS-PKS18,
and pAS-PKS24. The pACT2-SOS3 plasmid was made by mov-
ing a SmaI and an EcoRI fragment from pGEX-SOS3 (5) to
To make pAS-SOS2 and pAS-PKS3 deletion constructs [pAS-
SOS2 (333?385), pAS-SOS2 (333?369), pAS-SOS2 (386?446),
and pAS-PKS3 (327?371)], PCR was performed with pairs of
Abbreviations: SOS2, salt overly sensitive 2; ABA, abscisic acid; ABI, ABA-insensitive; PPI,
protein phosphatase interaction; PKS, protein kinase S; PKI, protein kinase interaction; SC,
synthetic complete; ?-gal, ?-galactosidase; MS, Murashige and Skoog.
*Present address: Institute of Agricultural and Forest Engineering, University of Tsukuba,
Tsukuba, Ibaraki 305-0572, Japan.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
September 30, 2003 ?
vol. 100 ?
no. 20 ?
forward primers (containing an NcoI site) and reverse primers
(containing a BamHI site). The resulting PCR products were
digested with NcoI and BamHI and inserted between the NcoI
and BamHI sites of pAS2. Deletion constructs of pACT-ABI2
(1?224, 1?111, 112?224, 225?423, 112?147, and 148?193) were
made by PCR amplification using specific primers and inserting
the DNA fragments into the SmaI and EcoRI sites of pACT2.
Inverse PCR-based site-directed mutagenesis was carried out on
double-stranded plasmid DNA with high-fidelity Taq polymerase
by using the following primers: 5?-AGAAACAGCAGCGGTTT-
GCCTTTTAAC-3? and 5?- CGAAGGGAACCTAGTGAG-3?
for pAS-SOS2 (R340A?F341A); 5?-TTGTGATAAGGTA-
ATCATC-3? and 5?-GGCTTAAATGACTCTGCAC-3? for
pAS-SOS2 (L324D); 5?-GCCATCGTAAACATCAAAG-3?
and 5?-CATGGCGGTTCTCAGG-3? for pACT-ABI2 (G162D);
5?-GCCATCGTAAACACCAAAG-3? and 5?-CATGACGGT-
TCTCAGG-3? for pACT-ABI2 (G168D); 5?-CTCCTTCGT-
CAAAGCCAG-3? and 5?-ATAGTGAAGGAGAAACCG-3? for
pACT-ABI2 (E186K); 5?-CTCCGTCAAAGCCAAATG-3? and
5?-GAGATAGCTAAGGAGAAAC-3? for pACT-ABI1
GAGATAGTTAAGGAGAAAC-3? for pACT-ABI1 (A201V);
and 5?-CTCCGTCAAAGCCAAATG-3? and 5?-GAGATAGT-
TAAGGAGAAAC-3? for pACT-ABI1 (A197TA201V). The
resulting PCR products were digested with DpnI and purified by
gel electrophoresis, followed by self-ligation. The ligated prod-
ucts were transformed into Escherichia coli DH5a cells. All
plasmid constructs were completely sequenced to ensure that
there were no PCR or cloning errors.
Yeast Two-Hybrid Screen and Interaction Assays. The SOS2 coding
region was amplified by PCR with primers containing restriction
sites and cloned in-frame between the NcoI and BamHI sites of
pAS2 to make the bait plasmid pAS-SOS2. The screening of
pACT plasmid library (27) was performed as described (5).
Plasmid DNA of bait and prey constructs were transformed
were grown overnight at 30°C in synthetic complete (SC) media
cell suspension containing ?4 ? 104cells was dropped onto SC
agar plates lacking tryptophan, uracil, and leucine, and the cells
were grown for 2 days at 30°C. After colonies were transferred
onto a nitrocellulose transfer membrane (NT, BioTrace, 0.45
mm; Gelman), ?-galactosidase (?-gal) filter assays were carried
out as described (5).
In Vitro Protein Binding Assays. To produce E. coli-expressed
GST-ABI2, the coding region of ABI2 was cloned in-frame into
the BamHI and EcoRI sites of pGEX-2TK. The resulting
construct was transformed into E. coli BL21 DE3 cells to obtain
the GST-ABI2 fusion protein. The 37-aa PPI sequence of SOS2
was cloned into the BamHI and EcoRI sites of pGEX-2TK to
produce the GST-PPI fusion protein. Radiolabeled ABI2 and
SOS2 were produced from pET14b-ABI2 and pET14b-SOS2,
respectively, using an in vitro transcription and translation assay
kit (TNT Coupled Reticulocyte Lysate System, Promega) with
[35S]methionine as the sole source of methionine, according to
the manufacturer’s instructions. In vitro protein affinity pull-
down assays were carried out as described (3).
Plant Growth and Salt Treatments. Seeds of WT and mutants were
sown on Murashige and Skoog (MS) plates containing 1.2%
(wt?vol) agar and 3% sucrose. The seeds were stratified at 4°C
for 3 days and then transferred to 22°C under continuous light
for germination and growth. Five days after germination, seed-
lings of WT and mutants were transferred onto either MS agar
plates or MS agar plates containing 150 mM NaCl for salt shock
Identification of ABI2 as a SOS2-Interacting Protein. Using a yeast
two-hybrid approach, we screened for proteins that interact with
the protein kinase SOS2. The entire SOS2 cDNA was fused
in-frame with the DNA binding domain of GAL4 to generate a
bait construct in the plasmid pAS2, and the construct was
transformed into the yeast strain Y190 (28). We screened a
Arabidopsis seedlings (27). Sequence analysis revealed that 7 of
the 101 putative interacting clones that we isolated encode ABI2
(see refs. 17 and 18).
A combination of the bait plasmid pAS-SOS2 with the empty
prey vector pACT2 did not activate transcription of the ?-gal
reporter gene (Fig. 1A). Strong ?-gal activity was observed when
the SOS2 bait was combined with ABI2 in the prey vector (Fig.
1A). To confirm whether SOS2 can bind to ABI2 in vitro, an
affinity pull-down assay was carried out. [35S]Methionine-
labeled SOS2 protein was incubated with the GST-ABI2 or
GST-RB (maize retinoblastoma protein; see ref. 5) fusion pro-
tein on Sepharose beads. The beads were pelleted and washed,
and the bound proteins were resolved by SDS?PAGE. The
labeled SOS2 protein was detected from the GST-ABI2 beads
but not from the control GST-RB beads, demonstrating that
SOS2 can bind to ABI2 in vitro (Fig. 1B).
Although ABI1 is very similar in sequence to ABI2 (15, 16),
we did not find ABI1 among the putative interacting clones from
the yeast two-hybrid screen (data not shown). A combination of
the SOS2 bait and ABI1 in the prey vector led only to a slight
activation of the ?-gal reporter gene expression (Fig. 1A). As a
negative control, a combination of the C-terminal tail of SOS1
(6) as a bait with either ABI1 or ABI2 in the prey vector failed
to activate the reporter gene (Fig. 1A). Together, these results
show that SOS2 preferentially binds to ABI2.
Identification of an ABI2-Binding Motif in SOS2. To identify a
minimal region of SOS2 that is sufficient for interaction with
ABI2, we made serial deletions of SOS2 in the bait vector pAS2
(Fig. 2A). When combined with the empty prey plasmid pACT2,
none of the deletion constructs of SOS2 baits activated the ?-gal
reporter gene (Fig. 2B). The bait construct SOS2-T1, which
contains the C-terminal regulatory domain of SOS2, activated
transcription of the ?-gal reporter gene when combined with the
ABI2 prey plasmid (Fig. 2B). In contrast, the N-terminal region
only weakly with ABI1 in the yeast two-hybrid assay. The pAS-SOS2?pACT2,
pAS-SOS1?pACT-ABI1, and pAS-SOS1?pACT-ABI2 combinations were used as
grown on SC plates (Left) and the corresponding ?-gal filter assays (Right) are
shown. (B) SOS2 binds to ABI2 in vitro. [35S]Methionine-labeled SOS2 was
pulled down by GST-ABI2 but not by GST-RB. GST-RB was used as a negative
SOS2 interacts with ABI2. (A) SOS2 interacts strongly with ABI2 but
www.pnas.org?cgi?doi?10.1073?pnas.2034853100Ohta et al.
of SOS2 (SOS2-N), which corresponds to the kinase catalytic
domain (2, 3, 5), did not activate the reporter gene. These results
indicate that the C-terminal, but not the N-terminal, region of
SOS2 interacts with ABI2.
The FISL motif (amino acids 309–329) of SOS2 mediates the
interaction with SOS3 (3). SOS2-T3, which does not contain the
FISL motif, also interacted with ABI2 (Fig. 2B), showing that
the FISL motif is not important for SOS2 interaction with ABI2.
We found that the SOS2 sequence between amino acid residues
333 and 385 but not 386 and 446 interacted with ABI2 (Fig. 2B).
Further analysis showed that the SOS2 sequence between amino
acids 333 and 369 is sufficient for interaction with ABI2 in the
yeast two-hybrid system (Fig. 2B). We designated this 37-aa
sequence of SOS2 the PPI motif.
To determine whether the 37-aa PPI motif can bind ABI2 in
vitro, we carried out a pull-down assay with35S-labeled ABI2 and
was found to bind to the GST-PPI but not GST control beads
(Fig. 2C). The result shows that the PPI peptide is sufficient for
binding to ABI2 in vitro.
The PPI Motif Is Conserved in PKS. Previously, we showed that a
SOS2-like protein kinase, PKS3, interacts strongly with ABI2
and weakly with ABI1 (9). To examine whether other PKSs can
assays with PKS11 (29), PKS18 (30), and PKS24 (3) as baits.
PKS11 and PKS24 interacted preferentially with ABI2 but not
with ABI1 (Fig. 3A). In contrast, PKS18 interacted strongly with
ABI1 but only weakly with ABI2 (Fig. 3A).
Sequence alignment shows that the PPI motif of SOS2 is
conserved in these protein kinases (Fig. 3B). Interestingly, the
Chk1 from humans (Fig. 3B). To determine whether the putative
PPI motif of PKS3 can mediate its interaction with ABI2, we
shown in Fig. 3C, this putative PPI sequence interacted with
In the PPI motif of SOS2, Arg-340 and Phe-341 are highly
conserved among the protein kinases (Fig. 3B). To further
Schematic representation of the constructs used in the yeast two-hybrid
interaction assay. The indicated regions of SOS2 were cloned into the bait
vector pAS2. (B) Interaction in the yeast two-hybrid assay. The SOS2 bait
plasmids were transformed with either pACT2 or pACT-ABI2 into the yeast
shown. (C) In vitro binding assay. [35S]Methionine-labeled ABI2 protein was
pulled down by GST-PPI but not by GST.
Identification of a PPI motif in SOS2 by deletion mapping. (A)
the PPI motif. (A) Interaction of PKS11, PKS18, and PKS24 with ABI1 and ABI2.
Yeast strains containing the pAS-PKS11, pAS-PKS18, and pAS-PKS24 baits and
pACT-ABI1 and pACT-ABI2 prey were assayed for ?-gal activity. Combinations
with the empty pACT2 prey vector were used as negative controls. Yeast
grown on SC plates (Left) and ?-gal filter assay (Right) are shown. (B) Align-
ment of the PPI sequences of SOS2 and PKS proteins. A putative regulatory
region of the human protein kinase Chk1 is shown under the alignment.
Identical and similar amino acid residues are shaded in black and gray,
respectively. Asterisks indicate amino acid residues identical between Chk1
interaction with ABI2. Full-length PKS3 or its PPI motif (amino acids 327–371)
in bait vector was transformed with either pACT2 or pACT-ABI2 into the yeast
strain Y190 for two-hybrid assay. Yeast grown on SC plates (Left) and ?-gal
filter assay (Right) are shown.
Differential interaction between PKS and ABI proteins mediated by
Ohta et al.PNAS ?
September 30, 2003 ?
vol. 100 ?
no. 20 ?
elucidate the amino acids important for the kinases to interact
with protein phosphatases, we mutated these two residues of
SOS2 and tested the impact of the mutations on the protein
interaction. Interaction of the SOS2 bait with the ABI2 prey was
abolished when Arg-340 and Phe-341 were both substituted with
alanine (R340AF341A) (Fig. 4), suggesting that these two
conserved residues in the protein kinase are important for
interaction with the ABI phosphatases. In comparison, substi-
tution of Leu-324 with aspartic acid (L324D), which is located
ABI2 (Fig. 4B). As expected, the L324D mutation abolished the
interaction with SOS3 (Fig. 4B). The R340AF341A mutations
also weakened the interaction with SOS3 (Fig. 4B), which may
be caused by a steric hindrance effect of the mutations on the
neighboring FISL motif.
Identification of a SOS2-Interacting Domain in ABI2. To determine
which region of ABI2 is involved in the interaction with SOS2,
we made deletion constructs of ABI2 in the prey plasmid pACT2
(Fig. 5A), and their interaction with SOS2 was evaluated by using
the yeast two-hybrid assay. The N-terminal region (amino acids
1–224, 1?224) of ABI2 retained the interaction with SOS2,
whereas the C-terminal region (amino acids 225–423, 225?423)
did not interact with SOS2 (Fig. 5B). This finding suggests that
the N-terminal half of ABI2 is sufficient for the interaction with
SOS2, although the interaction is not as strong as for the
full-length ABI2. Weak but significant ?-gal activity was still
observed when the prey plasmid harbored only amino acid
residues 112–224 (112?224) of ABI2 (Fig. 5B). Further deletions
(amino acids 148–193) (Fig. 5B).
The abi2-1 Mutant Protein Cannot Interact with SOS2. The PKI
domain of ABI2 is conserved among ABI1, ABI2, and other
protein phosphatase 2Cs from Arabidopsis (Fig. 6A). Several
amino acid residues in this region are known to be important for
the function of ABI1 and ABI2. In the dominant abi2-1 mutant
of Arabidopsis, Gly-168 is substituted by an aspartic acid residue
(17, 18). Substitution of amino acid Gly-174 of ABI1 by aspartic
acid, which corresponds to Gly-162 of ABI2, abolishes the ability
of ABI1 to block ABA-inducible transcription in maize proto-
plasts (19). A recessive mutation (R1) in the abi2-1 mutant
background, which converts Glu-186 to lysine, suppresses the
ABI phenotype of the abi2-1 mutant (21). To determine whether
these mutations might affect the interaction of ABI2 with SOS2,
these mutations (G162D, G168D, and E186K) were introduced
into the ABI2 prey construct, and the interaction of the resultant
ABI2 mutant proteins with SOS2 was evaluated by the yeast
two-hybrid assay. Either of the G162D and G168D mutations in
ABI2 abolished the interaction with SOS2 (Fig. 6B). However,
SOS2 (Fig. 6B). These results further confirmed that some of the
148–193) are important for ABI2 to interact with SOS2.
Amino Acid Substitutions in ABI1 That Increase Its Interaction with
SOS2. The minimal SOS2 interaction domain of ABI2 is highly
conserved in ABI1. In particular, amino acid residues 158–193
of ABI2 are almost identical to those of ABI1 except that the
Thr-197 and Val-201 in ABI2 are replaced by Ala in ABI1 (Fig.
SOS2 spanning the FISL motif and the PPI motif is shown. Arrows indicate
introduced amino acid substitutions. (B) Interaction in the yeast two-hybrid
system. WT and the mutated SOS2-bait plasmids were transformed with
pACT2, pACT-ABI2, or pACT-SOS3 into the yeast strain Y190. Yeast grown on
SC plates (Left) and ?-gal filter assay (Right) are shown.
Mutational analysis of the PPI motif in SOS2. (A) The WT sequence of
representation of the constructs used in the yeast two-hybrid interaction
assay. The indicated regions of ABI2 were cloned into the prey vector pACT2.
(B) Interaction in the yeast two-hybrid assay. The pAS-SOS2 bait was trans-
SC plates (Left) and ?-gal filter assay (Right) are shown.
Identification of a SOS2 interaction domain in ABI2. (A) Schematic
Alignment of several Arabidopsis protein phosphatase 2Cs in the PKI domain.
Identical and conserved amino acid residues are shaded in black and gray,
respectively. Arrows indicate positions of amino acid substitutions. (B) Muta-
tional analysis of the PKI domain in ABI2. Yeast strains containing the pAS-
SOS2 bait combined with WT or the mutated ABI2 in the pACT2 prey vector
assay (Right) are shown. (C) Mutational analysis of the PKI domain in ABI1.
ABI1 in the pACT2 prey vector were assayed for ?-gal activity. pACT2 and
pACT-ABI2 were used as negative and positive controls, respectively. Yeast
grown on SC plates (Left) and ?-gal filter assay (Right) are shown.
Mutational analysis of the PKI domains of ABI1 and ABI2. (A)
www.pnas.org?cgi?doi?10.1073?pnas.2034853100 Ohta et al.
weakly with SOS2 (Figs. 1A and 6C). To test whether the two
divergent amino acid residues (Thr-197 and Val-201 in ABI2)
contribute to the specificity of the interaction with SOS2, we
introduced targeted mutations that convert the amino acid
residues of ABI1 into those of ABI2. Single amino acid substi-
tutions in ABI1 (A197T and A201V) did not result in stronger
interactions with SOS2 (Fig. 6C). However, a double amino acid
substitution in ABI1 (A197TA201V) conferred a stronger in-
teraction with SOS2 (Fig. 6C). These results suggest that the two
divergent amino acid residues are important for SOS2 to dis-
tinguish ABI2 from ABI1.
The abi2-1 Mutant Is Tolerant to Salt Shock. The protein kinase
SOS2 is a positive regulator of salt tolerance, and sos2 mutants
are hypersensitive to salt stress (1). To begin to determine the in
vivo role of ABI2 in the SOS regulatory pathway for salt
tolerance, we examined the salt tolerance of the abi1-1 and
abi2-1 mutants (Fig. 7). WT, abi1-1, and abi2-1 plants were
germinated and grown on normal MS agar medium without
NaCl for 5 days and then transferred onto MS agar medium
either with or without supplementation of 150 mM NaCl.
Without NaCl supplementation, the abi1-1 and abi2-1 mutants
grew as well as the WT. However, in the presence of 150 mM
mutants are more tolerant to salt shock than the WT plants. This
result suggests that ABI2 is involved in salt tolerance in vivo.
Protein–protein interactions are fundamental to our under-
standing of protein regulation and signal transfer mechanisms of
signal transduction. The interactions between proteins are often
Well-known protein interaction domains include those desig-
nated SH3, SH2, WW, EH, and PDZ (31, 32). Undoubtedly, the
identification of protein interaction domains is a critical part of
signal transduction studies and contributes to a fundamental
knowledge base of cell biology (31, 32). However, very few
protein interaction domains have been defined in plant proteins.
SOS2 is a serine?threonine protein kinase that is required for
plant salt tolerance (2). We have shown that SOS2 interacts with
and is activated by SOS3 (5). SOS3 binds to the FISL motif, a
21-aa sequence close to the kinase catalytic domain of SOS2 (3).
In this study, we show that SOS2 also interacts with the protein
phosphatase 2C ABI2 and identify a minimal ABI2 binding
sequence in SOS2. This sequence is designated as the PPI motif
and is adjacent to the FISL motif. The identification of this PPI
motif represents an important contribution to our understanding
of the structure and regulation of SOS2.
The PPI motif is conserved in the SOS2 class of protein
found to interact with ABI2 and?or ABI1. The PPI motif in
PKS3 is also sufficient for interaction with ABI2. PKS represent
a large family of 25 protein kinases (3), and there are eight
2C-type protein phosphatases in the ABI2?ABI1 subclass (33).
Sequence variations in the PPI motif of PKS may determine
whether a particular PKS interacts with ABI2, ABI1, or another
related protein phosphatase. For example, residues R340 and
S343 of SOS2 are conserved in PKS3, PKS11, and PKS18, all of
which interact strongly with ABI2 but not ABI1 (Fig. 3B). In
contrast, these two residues are changed to K and T in PKS18,
only weakly with ABI2. On the protein phosphatase side,
variations in the PKI domain likely determine which protein
phosphatase interacts with which PKS. Indeed, although the WT
ABI1 protein interacts only weakly with SOS2, substitutions of
two amino acid residues within the PKI domain with corre-
sponding residues in ABI2 led to an interaction with SOS2 that
was nearly as strong as that of ABI2.
The G162D and G168D mutations in the PKI domain disrupt
the interaction between ABI2 and SOS2. These mutations also
have important functional consequences on ABA signaling and
salt tolerance. In fact, the G168D mutation is responsible for the
dominant abi2-1 mutant phenotypes in ABA insensitivity (17)
and tolerance to salt shock. The G162D mutation in ABI2
corresponds to the G174D mutation in ABI1, which has been
shown to abolish the ability of ABI1 to block ABA-inducible
the PKI domain is functionally important for the ABI protein
The PPI sequence is not only conserved in the PKS proteins
but is also found in the cell cycle checkpoint kinase Chk1 from
various organisms. In response to DNA damage or replication
block, two upstream protein kinases, ATM and ATR, phosphor-
ylate and activate Chk1, which in turn phosphorylates and
inhibits the activities or enhances the degradation of the Cdc25
family of protein phosphatases required for cell cycle progres-
sion (34). The presence of a putative PPI sequence motif in Chk1
suggests that it may interact with Cdc25 or an unknown protein
ABI2 and ABI1 are well-known regulators of ABA signaling
(35, 36), which is important for plant tolerance to several abiotic
stresses including salt, drought, and freezing (37). ABA may
exert its effect on salt tolerance through the interaction between
ABI2 and SOS2. In this scenario, ABI2 would be a point of cross
talk between the ABA pathway and the SOS pathway for ion
homeostasis. Alternatively, ABI2 may have a role in the SOS
pathway that is independent of ABA. In this model, SOS2 can
be considered as a scaffold protein that holds SOS3 and ABI2
in one complex, which functions specifically in salt tolerance by
responding to a distinct calcium signal triggered by sodium stress
(Fig. 8). Similarly, PKS3 would serve as a scaffold for SCaBP5
and ABI2, and this complex would function specifically in ABA
signaling (9) by responding to a specific calcium signal elicited by
ABA. Consistent with this hypothesis, sos3 and sos2 mutants are
specifically defective in salt tolerance but not in ABA responses
(1, 38), whereas scabp5 and pks3 mutants are altered only in
ABA sensitivity but not significantly in salt tolerance (9).
The functional relationship between SOS2 and ABI2 needs
further investigation. Theoretically, SOS2 and ABI2 may control
the phosphorylation status of each other, or they may regulate
the phosphorylation status of common protein substrates. SOS2
did not phosphorylate ABI2 in vitro, nor did ABI2 dephosphor-
ylate autophosphorylated SOS2 (data not shown). However,
WT and the abi1-1 and abi2-1 mutant seedlings were grown on vertical MS
plates containing 1.2% (wt?vol) agar and 3% sucrose. After 5 days of germi-
nation, the seedlings were transferred onto either the control MS agar plates
(Left) or MS agar plates containing 150 mM NaCl (Right). The pictures were
taken 6 days after the seedling transfer.
abi1-1 and abi2-1 mutant seedlings are more tolerant to salt shock.
Ohta et al. PNAS ?
September 30, 2003 ?
vol. 100 ?
no. 20 ?