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: firstname.lastname@example.org.
© 2003 by The National Academy of Sciences of the USA
September 30, 2003 ?
vol. 100 ?
no. 20 ?
evidence suggests that SOS2 may be phosphorylated by an
unknown upstream kinase (39), so it is still possible that ABI2
might dephosphorylate SOS2 that is phosphorylated by the
upstream kinase. The protein phosphatase KAPP from Arabi-
dopsis binds to the catalytic domain of receptor-like protein
kinases (40, 41). It has been suggested that KAPP may dephos-
phorylate the receptor-like kinases (42).
It is also possible that ABI2 dephosphorylates proteins that
are phosphorylated by SOS2 (Fig. 8). For example, SOS2
activates SOS1 by phosphorylation (8), and this may be coun-
tered by ABI2-catalyzed dephosphorylation. Consistent with a
role of ABI2 in salt tolerance, we found that abi2 mutant
seedlings are more tolerant to salt shock. The abi1 mutant plants
are also more tolerant to salt shock, possibly because the abi1
mutation is dominant and thus masks the effect of the WT ABI2.
The opposite activities of SOS2 and ABI2 are expected to be
under tight regulation so that they do not occur simultaneously.
Because SOS3 and ABI2 bind to adjacent sequences in SOS2,
SOS3 and ABI2 are also in proximity. The close proximity of
SOS3 to both SOS2 and ABI2 suggests that this calcium sensor
may simultaneously regulate the opposing enzymes.
We thank Ms. Becky Stevenson for excellent technical assistance and Dr.
Andre ´ Jagendorf for critical reading of the manuscript. This work was
supported by National Institutes of Health Grant R01 GM59138 and
United States–Israel Binational Agricultural Research and Develop-
ment Fund Grant IS-2971-98 (to J.-K.Z.).
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SOS3 and ABI2 in salt tolerance. Salt stress elicits a calcium signal that is
perceived by SOS3. SOS3 activates SOS2, which in turn phosphorylates ion
transporters important for ion homeostasis and salt tolerance. ABI2 may
and SOS3 are shown to bind to SOS2 via the FISL and PPI motifs, respectively.
Model showing the involvement of SOS2 and its binding proteins
www.pnas.org?cgi?doi?10.1073?pnas.2034853100 Ohta et al.