As this interaction is necessary for the ADP-ribosyla-tion activity of ExoS, and more intriguingly appears to be independent of phosphorylation [14,15,26], we wanted to define individual
Trang 1interaction with 14-3-3 and its biological activity
Lubna Yasmin1,*, Anna L Jansson1,*, Tooba Panahandeh1, Ruth H Palmer3, Matthew S Francis2 and Bengt Hallberg1
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-binding motif [1–3] Several of these interacting
part-ners have recognized functions, which include enzymes
in biosynthetic metabolism, ion channels and
regula-tors of growth in plants [4–6] It has been shown that
many human proteins can also bind directly to 14-3-3
in a phosphorylation-dependent manner, placing
14-3-3 as a central regulatory molecule in several
physiological processes such as biosynthetic metabo-lism, cell proliferation, and survival in human cells [3,7,8]
Crystal structure analyses of the 14-3-3 dimer alone
or in complex with peptides or native binding partners has revealed the presence of a basic cluster in the amphipathic groove of each monomer which mediates the interaction of 14-3-3 with the phospho-amino acid residues in its interaction partners Therefore it is likely that each dimer contains two binding pockets and can interact with a single target or with multiple binding partners Further, it has been observed that interaction between 14-3-3 proteins and its target partner(s) can
Keywords
ADP-ribosylation; coenzyme binding site;
cytotoxicity; NAD-dependent; cystic fibrosis;
Pseudomonas aeruginosa
Correspondence
B Hallberg, Department of Medical
Biosciences ⁄ Pathology, Building 6 M ,
2nd floor, Umea˚ University, 901 87 Umea˚,
Sweden
Fax: + 46 90 785 2829
Tel: + 46 90 785 2523
E-mail: Bengt.Hallberg@medbio.umu.se
*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 with ExoS in vitro
Abbreviations
ADPRT, ADP-ribosyltransferase; BD, binding domain; ExoS, exoenzyme S; FAS, factor activating exoenzyme S; GAP, GTPase-activating protein; GEF, guanine exchange factor; GTPase, GTP binding protein; Ras, rat sarcoma.
Trang 2occur outside the amphipathic groove, which probably
contributes to a stable three-dimensional configuration
with an opportunity for conformational modulation of
the target [2,9–13] 14-3-3 also interacts in a
phos-phorylation independent manner with some proteins
and peptides, such as exoenzyme (Exo) S of
Pseudo-monas aeruginosa, p190RhoGEF and the R18 peptide
inhibitor [14–17]
P aeruginosais an opportunistic pathogen that
cau-ses acute infections mainly in immunocompromised
individuals, such as children and patients with cystic
fibrosis, burn wounds or leukemia [18] The virulence
toxin ExoS from P aeruginosa is first secreted and
then translocated from the bacteria into the eukaryotic
cell via a bacterial encoded type III secretion system
[19] ExoS is a bifunctional toxin with an N-terminal
Rho GTPase activating protein (GAP) activity [20,21]
and a highly promiscuous C-terminally encoded
ADP-ribosylation activity towards small GTPases [21–23]
Its function is dependent on interactions with 14-3-3
and factor activating ExoS (FAS) protein cofactors
[24–26]
As this interaction is necessary for the
ADP-ribosyla-tion activity of ExoS, and more intriguingly appears
to be independent of phosphorylation [14,15,26], we
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
S419QGLLDALDL428are critical for 14-3-3 binding, a set of single substitution mutants of ExoS were con-structed together with some additional variants (Table 1) These variant alleles were then fused to GST giving rise to the following fusion proteins: GST-ExoS(wt), GST-ExoS(SD), GST-ExoS(LDL426– 428AAA), ExoS(DALDL424–428AAAAA), GST-ExoS(D424A; D427A), GST-ExoS(S419I),
GST-Exo-Table 1 Summary of the various GST-fusion protein constructs of ExoS used in the present study Substituted amino acid(s) are underlined GST-ExoS(88–453) is the parental allele (‘wild-type’), such that all other alleles listed differ only by the amino acid substitution indicated in parentheses The number in front of plasmid indicates lane numbering in Fig 1.
Plasmid
Substituted amino acid(s)
Reference or source
Trang 3S(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 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-(LDL426–428AAA) lack the ability to interact with
14-3-3 proteins from whole cell lysates of HeLa cells
(Fig 1, lane 5 and 6) At first glance, none of the
single amino acid substitutions of GST-ExoS between amino acid 419–428 showed any obvious inability to interact with endogenous 14-3-3 proteins (Fig 1, lanes 8–17) The same was true for a series of double substi-tution mutants: GST-ExoS(D424A; D427A) (Fig 1, lane 7), GST-ExoS(LD426–427AA) (Fig 2, lane 13), GST-ExoS(DL427-428AA) and GST-ExoS(LL426 : 428AA) (data not shown)
The basic cluster of amino acids in the binding groove of 14-3-3, including amino acids Lys-49,
Arg-56, Lys-120 and Arg-127, in an otherwise acidic mole-cule, are important for the interaction with ExoS, while residues on the hydrophobic surface of the groove are dispensable [27] Moreover, an artificial nonphosphorylated peptide ‘R18’ from a phage display library, binds within the same amphipathic groove of 14-3-3 [28] In this case the negatively charged aspartic (Asp-12) and glutamic acid (Glu-14) residues in the R18 peptide were found to interact in the 14-3-3 pocket Furthermore, a peptide sequence from ExoS including the motif D424ALDL428 has the same poten-tial as R18 to inhibit ExoS ADP-ribosylating activity [14] One suggestion was that the negatively charged 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.
Trang 4motif 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
substitution mutant ExoS(LDL426–428AAA) bound
cellular 14-3-3 proteins poorly, we tested whether
decreasing amounts of single amino acid substitution
mutant of GST-fusion proteins containing
Exo-S(L426A), ExoS(D427A) or ExoS(L428A) altered the
outcome of our pull-down assay A dilution series (2.5,
1.25 or 0.75 lg) of ExoS(wt) or of
GST-ExoS(D427A) gave similar pull-down equivalent
amounts of 14-3-3 proteins (Fig 2, lanes 1–3 and 7–9)
In contrast, diluted GST-ExoS(L426A) and
GST-Exo-S(L428A) precipitated fewer 14-3-3 proteins (Fig 2,
lanes 4–6 and 10–12) Thus, the two leucine amino
acids at positions 426 and 428 might still play a role in
the interaction between ExoS and 14-3-3
Leucine 428 is an important determinant for induced cell death by the ADP-ribosylating domain of ExoS
Having shed some light on the residues more import-ant for the interaction between ExoS and 14-3-3, we wanted to investigate how they affected the biological function of ExoS in vivo We first employed a live⁄ dead assay, capitalizing on the fact that before the ADP-ribosylation activity of translocated ExoS causes cell death, the infected cells undergo a morphology change whereby they round up due to disruption of actin microfilaments [21,30] HeLa cells were infected for 2 h with the surrogate bacterium Yersinia pseudotu-berculosis [21], which was engineered to express and translocate, under the control of arabinose [31], ExoS wild type as well as several single, double and 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 ExoS(L428A); lane 11, 1.25 lg of ExoS(L428A); lane 12, 0.75 lg of ExoS(L428A); lane 13, 2.5 lg of GST-ExoS(LD426–427AA); lane 14, 1.25 lg of GST-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 propro-teins used in the pull-down experiment.
Trang 5ogy, 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
with noninfected cells (Fig 3A, compare lane 2 with
lane 1) We also observed that single substitutions of
aspartic acid residues at position 424 or 427 of ExoS
[ExoS(D424A) or ExoS(D427A)] and the double
mutant ExoS(DD424 : 427AA) were as aggressive as
wild-type ExoS in their ability to induce cell death, as
infected cells were unable to recover from the initial infection (Fig 3A, lanes 4, 6 and 8) Together with the results from the GST-pull-down assay (Figs 1 and 2) using ExoS mutants with the same amino acid substi-tution, it is noticeable that negatively charged amino acids at positions 424 and 427 do not mimic phosphor-ylated serine motifs important for the interaction between 14-3-3 and ExoS Therefore, this interaction is more complex and must occur in another way, whereby amino acids 426 and 428 have a more prom-inent role
Significantly, the translocated triple mutant ExoS(LDL426–428AAA), which is unable to interact with 14-3-3 proteins in pull-down experiments, was sig-nificantly impaired in its ability to induce cell death, with the majority of cells (92%) surviving the infection with ExoS(LDL426–428AAA) toxin (Fig 4, compare
C with G and Fig 3A, lane 3) Therefore, mutant ExoS(LDL426–428AAA) has a reduced ADP-ribosyla-tion activity, the main cause of cell death This pheno-type is reminiscent of cells transiently infected with the ADP-ribosylation mutant ExoS(E381A), which recover their original cell structure and morphology overnight [21,30] By analogy, ExoS(LDL426–428AAA) must 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
B
C
D
E
Fig 3 Phenotypic analysis of ExoS during infection of HeLa cells
in vivo (A) Viability of HeLa cells are expressed as percentage
survi-val rate HeLa cells in the presence of 0.1% arabinose, were
infec-ted for 2 h with Yersinia (YPIII) expressing different variants of
ExoS, lane 1, noninfected cells; lane 2, YPIII(pMF384) expressing
ExoS(wt); lane 3, YPIII(pMF516) expressing ExoS(LDL426–428AAA);
lane 4, YPIII(pMF515) expressing ExoS(D424A); lane 5,
YP-III(pMF582) expressing ExoS(L426A); lane 6, YPIII(pMF493)
expres-sing ExoS(D427A); lane 7, YPIII(pMF583) expresexpres-sing ExoS(L428A);
lane 8, YPIII(pMF523) expressing ExoS(DD424 : 427AA); lane 9,
YP-III(pMF518) expressing ExoS(LD426–427AA) Both loose and
semi-detached cytotoxic cells were washed free from bacteria and
transferred to a new Petri dish and incubated overnight with
med-ium containing gentamicin A trypan blue exclusion assay was
per-formed 24 h after infection to quantitated the percentage of dead
cells Each bar represents the mean values of five independent
experiments (B) and (C) ExoS expression (B) and secretion (C) after
each Y pseudotuberculosis strain was induced in calcium-depleted
medium in the presence of arabinose Proteins were analyzed on
SDS ⁄ PAGE followed by western blot using anti-ExoS antibodies (D)
and (E) Cells were lysed and samples were separated by
SDS ⁄ PAGE Western blot analysis was performed on
immunoblot-ted filters with anti-Ras (D) and with anti-Erk 2 (E) antibodies.
Trang 6speculate that the weak interaction is able to induce 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 was observed in bacteria translocating either Exo-S(L428A) or ExoS(LDL426–428AAA) into infected cells (Fig 3D, lanes 3 and 7), which again correlated
to the extent of cell survival in these infections While, for the most part, our results herein reflect the established principle that 14-3-3 proteins act as cofactors in activating ExoS located in the cytosol [14,26–28,33], a notable exception was revealed The ExoS(LD426–427AA) double mutant and, to a lesser extent, the single mutant ExoS(L426A), showed weakened 14-3-3 binding potential However, like ExoS(D427A), these toxin variants were still biologic-ally active This suggests that the limited binding was still productive, in the sense that an initial contact of ExoS by 14-3-3 proteins or a fast on-off ratio is suffi-cient for ADP-ribosylation activation of ExoS
One goal of this study was to identify single ExoS amino acids residues, which are important for the phosphorylation independent interaction with 14-3-3 This is important considering that most interactions between 14-3-3 and cellular proteins require a phosphorylation-dependent event At least for ExoS, however, earlier predictions that phosphorylation–inde-pendent interactions were mediated by acidic residues are not the whole truth Interestingly, residues Leu-426 and Leu-428 were found to be most important for ini-tial binding in our assay We interpret this to mean
Fig 4 Morphological analysis of HeLa cells infected with variants of ExoS HeLa cells in the presence of 0.1% arabinose, were infected with: (B) and (F) YPIII (pMF384) expressing arabinose induced ExoS(wt); (C) and (G) YPIII(pMF516) expressing ExoS(LDL426–428AAA); (D) and (H) YPIII(pMF583) expressing ExoS(L428A) (A) and (E) Uninfected cells were used as a control After infection for 2 h with bacteria 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).
Trang 7that 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
catalysis and in contributing to the structural integrity
of the active site [32] Could it be that Leu-428
exhib-its similar properties? Another possibility is the
Exo-S(L428A))14-3-3 interaction is not productive While
14-3-3 can still bind to this mutant, perhaps it is
unable to induce a putative conformational change
that may be necessary for ExoS activation If this
were true, it would not appear to be due to a
differ-ent fold in ExoS(L428A) compared to any other ExoS
variant used in this study, because we did not detect
any difference in protein production or stability
(Fig 3, and data not shown)
In summary, we propose that ExoS of P aeruginosa
has evolved to recruit 14-3-3 to regulate its enzymatic
activity, which is similar to many other signal-induced
interactions between 14-3-3 and its targets ([3,7,8]
and refs therein) It is noteworthy that 14-3-3 proteins
are only expressed in eukaryotic cells, including plants,
yeast and protozoa No clear prokaryotic ancestor has
been identified Thus, it would be interesting to
deter-mine if bacteria expressing a 14-3-3 isoform in the
presence of ExoS can survive, as it may be the absence
of 14-3-3 homologues in prokaryotes that safeguard
them against the deleterious effects of their own toxins
This suggests that prokaryotic evolution has created a
new way to take advantage of an evolutionary ‘novel’
eukaryotic 14-3-3 protein family, using them as a
necessary cofactor to activate lethal bacterial toxins,
but only after they have been safely transported from the bacteria into the eukaryotic cell
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⁄ Pi 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 4C 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
Anti-14–3-3b (SC-629) was purchased from Santa Cruz (New York, NY, USA); monoclonal Ras (cat 610002) was obtained from BD Biosciences (Stockholm, Sweden) Anti-ExoS was from Agrisera AB, Sweden Immunoblotting was performed according to the manufacturer’s instructions using secondary antibodies conjugated to horseradish peroxidase sheep antimouse or rabbit antibodies (Pierce, Rockford, IL, USA, and ECL Plus, Amersham-Biosciences)
Plasmids
pGEX-ExoS(SD) is derivative of pGEX-ExoS(88–453], both
of which have been described previously [14] The substi-tution mutants [Table 1], pGEX-ExoS(S419I), pGEX-ExoS(Q420A), pGEX-ExoS(G421A), pGEX-ExoS(L422A), pGEX-ExoS(L423A), pGEX-ExoS(D424A), pGEX-Exo-S(A425K), pGEX-ExoS(L426A), pGEX-ExoS(D427A), ExoS(L428A), ExoS(D424A:D427A), pGEX-ExoS(LD426–427AA), pGEX-ExoS(LDL426–428AAA),
Trang 8and pGEX-ExoS (DALDL424–428AAAAA) were
construc-ted by digesting pGEX-ExoS (SD) with NdeI⁄ NheI and
inserting oligomers (supplementary material, Table S1)
cor-responding to the appropriate amino acid substitutions, as
outlined in Table 1 All constructs were confirmed by
sequencing with DYEnamic ET terminal cycle sequencing
kit (Amersham-Biosciences)
Construction of arabinose inducible ExoS
derivatives and infection of cells
To ensure protein stability of full-length ExoS derivatives,
mutant alleles were coexpressed with orf1, encoding the
cog-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¨rbundet Cystisk Fibros Forskningsfond
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Supplementary material
The following supplementary material is available online:
Table S1 Construction of plasmids used in this study
pGEX-2TK-ExoS(Sn) [14] was digested with NdeI
and NheI, followed by insertion of the annealed oligo-mers listed, which contained the appropriate amino acid substitutions corresponding to the ExoS variants outlined in Table 1
This material is available as part of the online article from: http://www.blackwell-synergy.com