Delineation of exoenzyme S residues that mediate the
interaction with 14-3-3 and its biological activity
Lubna Yasmin
1,
*, Anna L. Jansson
1,
*, Tooba Panahandeh
1
, Ruth H. Palmer
3
, Matthew S. Francis
2
and Bengt Hallberg
1
1 Department of Medical Biosciences ⁄ Pathology, Umea
˚
University, Sweden
2 Department of Molecular Biology, Umea
˚
University, Sweden
3 Umea
˚
Center for Molecular Pathogenesis, Umea
˚
University, Sweden
14-3-3 proteins are a group of highly conserved intra-
cellular dimeric molecules, expressed in plants, inverte-
brates and higher eukaryotes, with no intrinsic activity.
14-3-3 proteins play an important role in several signa-
ling pathways and 14-3-3 interacts with proteins in a
phospho-specific manner, using a defined consensus-

University, 901 87 Umea
˚
,
Sweden
Fax: + 46 90 785 2829
Tel: + 46 90 785 2523
E-mail:
*Both authors contributed equally to this
work.
(Received 5 October 2005, revised 7
December 2005, accepted 12 December
2005)
doi:10.1111/j.1742-4658.2005.05100.x
14-3-3 proteins belong to a family of conserved molecules expressed in all
eukaryotic cells, which play an important role in a multitude of signaling
pathways. 14-3-3 proteins bind to phosphoserine ⁄ phosphothreonine motifs
in a sequence-specific manner. More than 200 14-3-3 binding partners have
been found that are involved in cell cycle regulation, apoptosis,
stress responses, cell metabolism and malignant transformation. A phos-
phorylation-independent interaction has been reported to occur between
14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial
ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study,
we have investigated the effect of amino acid mutations in this C-terminal
domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interac-
tion. Our results suggest that leucine-428 of ExoS is the most critical resi-
due for ExoS enzymatic activity, as cytotoxicity analysis reveals that
substitution of this leucine significantly weakens the ability of ExoS to
mediate cell death. Leucine-428 is also required for the ability of ExoS to
modify the eukaryotic endogenous target Ras. Finally, single amino acid
substitutions of positions 426–428 reduce the interaction potential of 14-3-3

wanted to define individual residues within the 14-3-3
binding domain of ExoS that are important for the
14-3-3 interaction, as well as the resultant activity
in vivo. We have approached these questions using a
strategy of single amino acid site-directed mutagenesis
of the cofactor interaction domain within ExoS.
Various single mutant ExoS proteins were tested for
their capacity to interact with 14-3-3 and subsequently
for their cytotoxicity and ADP-ribosylation potential
using Ras as a substrate in vivo. We show that the leu-
cine residue at position 428 is necessary for both the
ADP-ribosylation activity and the cytotoxic action of
ExoS in vivo.
Result and discussion
Acidic residues within the 14-3-3 binding
domain of ExoS are not strictly needed for
phosphorylation-independent binding
The interaction between 14-3-3 and ExoS is important
for the ADP ribosylation activity of ExoS and even
more intriguingly, appears to be independent of serine-
phosphorylation [15,26]. The amino acid sequence
between 419 and 428 of ExoS is known to be import-
ant for this interaction [14]. To address exactly
which amino acid residues in the ExoS sequence
S
419
QGLLDALDL
428
are critical for 14-3-3 binding, a
set of single substitution mutants of ExoS were con-

6. GST-ExoS(DALDL424–428AAAAA) S
419
QGLLAAAAA
428
This study
7. GST-ExoS(D424A; D427A) S
419
QGLLAALAL
428
This study
8. GST-ExoS(S419I)
I
419
QGLLDALDL
428
This study
9. GST-ExoS(Q420A) S
419
AGLLDALDL
428
This study
10. GST-ExoS(G421A) S
419
QALLDALDL
428
This study
11. GST-ExoS(L422A) S
419
QGALDALDL
428

This study
18. GST-ExoS(LD426–427AA) S
419
QGLLDAAAL
428
This study
L. Yasmin et al. Delineation of ExoS residues
FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS 639
S(Q420A), GST-ExoS(G421A), GST-ExoS(L422A),
GST-ExoS(L423A), GST-ExoS(D424A), GST-Exo-
S(A425K), GST-ExoS(L426A), GST-ExoS(D427A),
GST-ExoS(L428A) and GST-ExoS(LD426–427AA)
(Table 1). All GST-ExoS derivatives were expressed
and purified, and were then employed in protein pull-
down experiments (Fig. 1). HeLa cells were harvested
and the lysates precleared with GST beads prior to 1-h
incubation with each of the indicated GST-ExoS-
fusion proteins. Samples were subsequently washed
and run on SDS ⁄ PAGE, followed by immunoblotting
with 14-3-3 antibodies. It should be noted that we did
not investigate binding of different 14-3-3 isoforms or
the specificity of different 14-3-3 isoform binding in
this study, as we used a pan-14-3-3 antibody. It is
established that GST-ExoS(wt) interacts with 14-3-3,
but not GST-beads alone or the fusion protein, GST-
ExoS(SD), in which the ExoS residues at positions
419–423 are substituted with alanine and residues
424–428 have been deleted [14] (Fig. 1, compare lane 3
with lanes 2 and 4). We also observed that both
GST-ExoS(DALDL424–428AAAAA) and GST-ExoS-

amino acids, such as glutamic and aspartic acid
residues, are able to mimic the phosphorylated serine
Fig. 1. Interaction of GST-ExoS variants with endogenous 14-3-3 proteins. HeLa cells were harvested and lysates were subjected to ‘pull-
down’ analysis with 5 lg of individual GST-fusion proteins. Samples were separated on a SDS ⁄ PAGE, followed by immunoblotting with
14-3-3antibodies. Upper panel: Lane 1, control HeLa cell lysate, 2 lg; lane 2, GST alone; lane 3, GST-ExoS(wt); lane 4, GST-ExoS(DS); lane 5,
GST-ExoS(LDL426–428AAA); lane 6, GST-ExoS(DALDL424–428AAAAA); lane 7, GST-ExoS(D424A; D427A); lane 8, GST-ExoS(S419I);
lane 9, GST-ExoS(Q420A); lane 10, GST-ExoS(G421A); lane 11, GST-ExoS(L422A); lane 12, GST-ExoS(L423A); lane 13, GST-ExoS(D424A);
lane 14, GST-ExoS(A425K); lane 15, GST-ExoS(L426A); lane 16, GST-ExoS(D427A); lane 17, GST-ExoS(L428A). Lower panel: Coomassie blue
stained SDS ⁄ PAGE showing the purified GST-fusion proteins used in this study. The order corresponds to lanes 2–17 above.
Delineation of ExoS residues L. Yasmin et al.
640 FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS
motif of Raf-1, which would perhaps explain the
binding of 14-3-3 proteins to this motif [28]. To test
the hypothesis put forward by Petosa et al. [28], we
used single or double amino acid substitutions of the
aspartic acid residues at positions 424 and 427 of the
ExoS binding site for 14-3-3. These substitutions did
not alter the ExoS)14-3-3 interaction under the condi-
tions tested (Fig. 1, lanes 7, 13 and 16).
Although from this analysis it is not obvious how
the interaction between 14-3-3 and ExoS occurs,
our pull-down analysis with GST-ExoS(LDL426–
428AAA) still strongly suggests that ExoS must utilize
a strategy for its interaction with 14-3-3 that is similar
to that seen with R18 and serotonin N-acetyltrans-
ferase. This is because R18 is also nonphosphorylated
and serotonin N-acetyltransferase selectively utilizes a
subset of residues both in the conserved basic binding
groove and residues outside the groove [13,28,29]. To
understand the molecular basis for why the triple

amino acid substitution variants into target cells.
Translocation of all ExoS variants resulted in a cyto-
toxic phenotype, e.g., cells rounded up and became
semidetached from the Petri dish. Both loose and
semidetached cytotoxic cells were washed free from
bacteria and transferred to a new Petri dish and incu-
bated overnight with medium containing gentamicin.
Bacterial growth of each strain was assessed by viable
counts, both during initial infection and also after
extended infection, to ensure the same constant bacter-
ial load (data not shown). At the same time, we con-
firmed equivalent levels of ExoS expression and
secretion by each strain (Fig. 3B,C, lanes 2–9). We
then quantitated cell death by a trypan blue exclusion
assay performed 24 h after infection. Infection with
wild-type ExoS mediated a nonreversible cell morphol-
Fig. 2. Effect of using GST-ExoS fusion dilutions during pull-down analysis. Selected GST-ExoS variants were sequentially diluted prior to
analysis of their interaction potential with endogenous 14-3-3 proteins from HeLa cell lysate. Lane 1, 2.5 lg of GST-ExoS(wt); lane 2,
1.25 lg of GST-ExoS(wt); lane 3, 0.75 lg of GST-ExoS(wt); lane 4, 2.5 lg of GST-ExoS(L426A); lane 5, 1.25 lg of GST-ExoS(L426A); lane 6,
0.75 lg of GST-ExoS(L426A); lane 7, 2.5 lg of GST-ExoS(D427A); lane 8, 1.25 lg of GST-ExoS(D427A); lane 9, 0.75 lg of GST-ExoS(D427A);
lane 10, 2.5 lg of GST-ExoS(L428A); lane 11, 1.25 lg of GST-ExoS(L428A); lane 12, 0.75 lg of GST-ExoS(L428A); lane 13, 2.5 lg of GST-
ExoS(LD426–427AA); lane 14, 1.25 lg of GST-ExoS(LD426–427AA); lane 15, 0.75 lg of GST-ExoS(LD426–427AA). Upper panel, 14-3-3 pro-
teins were detected by immunoblotting with anti14-3-3 antibodies. Lower panel, Coomassie blue stained GST-fusion proteins used in the
pull-down experiment.
L. Yasmin et al. Delineation of ExoS residues
FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS 641
ogy, concomitant with a disruption of actin microfila-
ments, and ultimately cell death (compare Fig. 4B with
4F), corroborating with earlier studies [21]. In fact,
only 9% of ExoS(wt) infected cells survived compared

still harbor wild-type GAP activity that enables actin
reorganization through the ability to down regulate
the activity of small GTP binding proteins, such as
Rho and Cdc42 in HeLa cells [21]. However, a reduced
ADP-ribosylation activity permits this phenotype to be
reversed postinfection. This phenotype must be due to
either leucine residues at positions 426 or 428, as a
mutation of aspartic acid at position 427 aggressively
induced cell deaths such as the wild type. Indeed, bac-
teria translocating the ExoS(L428A) mutant poorly
mediated cell death (90% survival) after a 2-h infec-
tion, which is comparable to bacteria expressing the
ExoS(LDL426–428AAA) mutant (Fig. 3A, lane 7, and
Fig. 4, compare D with H). Curiously, this was despite
an interaction between ExoS(L428A) and 14-3-3 in the
pull-down experiment (Figs 1 and 2). In contrast, the
ExoS(L426A) mutant killed all but 8% of infected cells
similar to the wild-type protein (Fig. 3A, lane 5). To
further support this important role for amino acid 428,
a double mutant, ExoS(LD426 : 427AA), was con-
structed. Bacteria translocating ExoS(LD426 : 427AA)
still mediated significant cell death with only 20% sur-
vival (Fig. 3, lane 9). This is similar to the lethal
affects of the single substitution mutants ExoS(L426A)
and ExoS(D427A). This is surprising, as this double
mutant was rather impaired in 14-3-3 binding (Fig. 2,
lanes 13–15). Why this weak interaction between
ExoS(LD426 : 427AA) and 14-3-3 is still enough to
mediate cytotoxicity is currently unclear. We can only
A

conformational change of the ExoS protein that might
be of importance for the activation of the ADP-ribosy-
lation activity. Nevertheless, we define a second resi-
due, leucine at position 428, which is an important
determinant for induced cell death by the ADP-ribosy-
lating domain of ExoS. Whether this serves a similar
function to the critical glutamic acid residue at posi-
tion 381 [32] remains a focus for our future research.
ExoS-dependent in vivo ADP-ribosylation of Ras
requires the Leu-428 residue
Ras is modified by the ADP-ribosylating activity of
ExoS expressed and delivered into the eukaryotic cells
by genetically modified Y. pseudotuberculosis [14]. We
used this assay to further assess the in vivo biological
activity of our ExoS variants. HeLa cells were in-
fected for 2 h with Y. pseudotuberculosis induced by
arabinose to express and translocate ExoS(wt),
ExoS(D424A), ExoS(L426A), ExoS(D427A), Exo-
S(L428A), ExoS(D424A; D427A), ExoS(LDL426–
428AAA) and ExoS(LD426–427AA) into target cells.
The cells were then harvested and the resultant lysate
was separated on a SDS ⁄ PAGE followed by immuno-
blotting with anti-Ras and anti-pan-Erk antibodies as
a loading control (Fig. 3D and E respectively). Ras
was modified in cells infected with bacteria expressing
one of either wild-type ExoS, ExoS(D424A), Exo-
S(L426A), ExoS(D427A), ExoS(D424A; D427A) or
ExoS(LD426–427AA) (Fig. 3D, lanes 2, 4, 5, 6, 8 and
9). This paralleled our analysis of ExoS-induced HeLa
cell death. Significantly, much less modification of Ras

translocating different variants of ExoS, all cells showed a cytotoxic phenotype in that they rounded up and became semidetached from the
Petri dish (A–D). These infections were washed free from bacteria and transferred to new Petri dishes and incubated with medium contain-
ing penicillin, streptomycin and gentamicin to ascertain the reversibility of this cytotoxic response (E–H).
L. Yasmin et al. Delineation of ExoS residues
FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS 643
that the phosphorylation-independent ExoS)14-3-3
interaction is complex, and is likely to involve coordi-
nation of multiple discrete ExoS interaction motifs,
some of which may be acidic in nature, but others not.
It is easy to imagine that these molecular contacts
could generate ExoS conformational changes necessary
for the controlled induction of enzymatic activity or
could even activate a cytosolic targeting mechanism.
Understanding these molecular events will no doubt
require detailed structural analysis, which is not cur-
rently available.
Numerous reports have described the importance of
14-3-3 proteins as a factor involved in the activation
of ExoS [14,26–28,33]. We were therefore very sur-
prised when the single substitution mutant Exo-
S(L428A) lacked ADP-ribosylating activity in vivo,
even though this mutant should still engage 14-3-3
proteins from HeLa cell lysates. This raises the notion
that 14-3-3 binding is not the sole requirement for
ExoS activity. Perhaps Leu-428 is even required for
enzymatic activity per se, such as in directly engaging
the molecular targets of ADP-ribosylation. This
evokes the function of glutamic acid at position 381,
which is a prerequisite for ADP-ribosylating activity.
It has been proposed that E-381 functions in both

It is apparent that more secrets concerning this
intriguingly complex interaction need to be uncovered.
Many of these may be revealed only through compre-
hensive structural analysis. No structural data exists
for the phosphorylation-independent 14-3-3–ExoS
complex, either using native ExoS domains or a syn-
thetic peptide sequence encompassing the 14-3-3 bind-
ing domain (this study) [14,15,26]. An enticing
prospect for future research is to determine how amino
acid Leu-428 of ExoS influences the interaction
dynamics with 14-3-3.
Experimental procedures
Cell cultures, cell lysis
HeLa cells were grown in RPMI 1640 supplemented with
10% (v ⁄ v) fetal bovine serum and 100 units ⁄ mL penicillin.
Following bacterial infection cells were washed in ice-cold
NaCl ⁄ P
i
and lysed on ice in lysis buffer [1%(v ⁄ v) Triton
x-100, 100 mm NaCl, 50 mm Tris ⁄ HCl (pH 7.5), 1 mm
EDTA supplemented with protease inhibitors (Complete,
#1697498, Roche Diagnostics, Basel, Switzerland)]. Lysates
were subsequently cleared by centrifugation at 15 000 g for
10 min at 4 °C. Lysates were precleared with glutathione
S-transferase (GST) for 5 min, before incubation with var-
ious GST-fusion proteins for 1 h prior to the addition of
Glutathione Sepharose (GE Healthcare, Uppsala, Sweden)
for 30 min. After three washes in lysis buffer, samples were
boiled in SDS ⁄ PAGE sample buffer.
Western analysis, peptides and antibodies

nate nonsecreted chaperone of ExoS [30,34]. In all cases,
DNA was amplified by PCR using conditions described pre-
viously [35]. Construction of pMF384 containing arabinose
inducible wild-type exoS has been described in detail previ-
ously [14]. Arabinose inducible exoS variants on the plasmids
pMF493, pMF515, pMF516, pMF518, pMF523, pMF582
and pMF583 were obtained by replacing the C-terminal
ClaI ⁄ KpnI exoS fragment from pMF384 with DNA ampli-
fied and restriction enzyme cut with ClaI ⁄ KpnI from
pGEX-ExoS(D427A), pGEX-ExoS(D424A), pGEX-
ExoS(LDL426–428AAA), pGEX-ExoS(LD426–427AA),
pGEX-ExoS(DD424 : 427AA), pGEX-ExoS(L426A), and
pGEX-ExoS(L428A), respectively (see Supplementary mater-
ial, Table S1), using the exoS-specific primers, pexoSseq3
(position 973991; forward): 5¢-AAGTGATGGCGCTTGG
TCT-3¢ and pexoSd (reverse): 5¢-ATGCATGGTACCTCAG
GCCAGATCAAGGCCGCG-3¢. All constructs were main-
tained in Escherichia coli DH5 and were confirmed by
sequence analysis using the DYEnamic ET terminator
cycle sequencing kit (Amersham Biosciences). Stable induc-
tion of protein expression in strains grown in the presence
of 0.02% l(+)-arabinose was confirmed by western analy-
sis, as described previously [36], using polyclonal rabbit
anti-ExoS [30]. Bacterial infection of cells was performed
in the presence of 0.1% l(+)-arabinose, as described
previously [14].
Acknowledgements
Financial support for this work was from the Swedish
Cancer Society, Carl Tryggers Foundation, and
Riksfo

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acid substitutions corresponding to the ExoS variants
outlined in Table 1.
This material is available as part of the online article
from:
Delineation of ExoS residues L. Yasmin et al.
646 FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS