Mammalian Cells Contain an Activator of YopJ
Expression of YopJ in cultured mammalian cells results in the acetylation of cellular MEK1/2. We discovered that acetylation of the activation loop of MEK1/2 resulted in the loss of immunodetection of MEK1/2 by the antiserum CST9122 (A
). This antiserum, raised against a peptide comprising the residues of the activation loop of MEK1/2, efficiently recognizes unmodified MEK1/2 but does not recognize MEK1/2 that has been acetylated by YopJ on the serine and threonine residues within the activation loop. Loss of signal on a Western blot thus provided us a stringent readout for the assessment of the extent of YopJ-mediated modification of MEK1/2. The 47E6 antibody, on the other hand, was not sensitive to the modification and allowed visualization of total amounts of MEK1/2. Using the antiserum CST9122 as a readout of MEK modification, we had earlier (6
) concluded that even very small amounts of YopJ expressed in mammalian cells were sufficient to cause the acetylation of most, if not all, of the MEK1/2 in cells, suggesting that YopJ is a very efficient acetyltransferase. A similar inference has also been made by others, wherein it has been reported that as little as 1 ng of transfected YopJ or VopA (the YopJ homologue from V. parahemeolyticus
) results in inhibition of MAPK signaling (12
). Most bacterial toxins are very active in terms of their enzymatic activities; YopH, another type III effector from Yersinia
that manifests a protein-tyrosine phosphatase activity, has been recognized as one of the most potent protein-tyrosine phosphatases isolated to date (13
FIGURE 1. Assays for the acetylation of MEK; eukaryotic HeLa cells contain an activating cofactor for YopJ. A, HeLa cells were transfected with wild-type YopJ (pSFFV-YopJ wt) or with the enzymatically inactive C172A mutant (pSFFV-YopJ C172A). Cell lysates were (more ...)
The acetyltransferase activity of YopJ can also be observed in vitro
using purified recombinant proteins expressed in bacteria (5
). Acetylation of purified MEK2 by YopJ could be monitored by the inclusion of radioactive [14
C]-AcCoA in the reaction mixture (B
However, analysis of the same reactions by Western blotting showed that only a small percentage of the total MEK molecules had been modified when enzymatic concentrations of YopJ were used. Increasing the concentration of AcCoA, raising the temperature or duration of incubation of the reaction mixture did not result in any significant improvement in the extent of acetylation. The poor efficiency of acetylation in vitro, which contrasted with the extremely efficient modification of MEK in mammalian HeLa cells, led us to wonder if there might be a mammalian cell cofactor required for the activation of the acetyltransferase activity of YopJ.
Indeed, inclusion of dialyzed HeLa cytosol resulted in a marked stimulation of the acetyltransferase activity of YopJ (C). Not only was there a significantly higher acetylation of MEK but also an increase in YopJ autoacetylation. These observations suggested that mammalian cell cytosol contains a cofactor that stimulates the acetyltransferase activity of YopJ.
This observation is not without precedent. Another type III effector protein from Yersinia
, YpkA (YopO), requires eukaryotic cell actin as an activator. Binding of G-actin to YpkA stimulates the serine-threonine kinase activity of YpkA (14
). Actin is also reported to be a cofactor required for maximal activity of the adenovirus protease adenain (15
Purification of a Heat-stable Activatory Cofactor Present Only in Eukaryotic Cells
As a first step toward identifying the cofactor required for YopJ we decided to fractionate HeLa cytosol by size-exclusion chromatography. HeLa cytosol corresponding to 30 mg of total protein was resolved over S75 Superdex resin, and 2-ml fractions were collected. 15 μl of each fraction was analyzed for the presence of the cofactor. Cofactor activity was observed (supplemental Fig. S1
) to elute from the gel-filtration column as a relatively broad peak starting at Fraction 52, indicating that the cofactor was likely a low molecular mass molecule (smaller than the 12.4-kDa cytochrome c
YopJ has been suggested to act as a deubiquitinase (16
), although we have not been able to observe any significant deubiquitinase activity associated with YopJ (expressed as recombinant protein in bacteria or in insect cells). We thus examined whether ubiquitin (molecular mass of 8.5 kDa) might be serving as the low molecular weight activating cofactor for YopJ, but this was not the case. Inclusion of various amounts of ubiquitin in YopJ-catalyzed acetyltransferase assays did not result in any stimulation of YopJ activity.
We then tried various treatments on Fraction 52 (obtained from size-exclusion chromatography described above) to better understand the nature of the cofactor. Significantly, heating Fraction 52 at 95 °C for 30 min did not destroy the cofactor activity (A). Protease, nuclease, and phosphatase treatments of Fraction 52 also did not significantly affect cofactor activity (except at very high doses). The results obtained thus far suggested that YopJ required a mammalian cell, low molecular weight, heat-stable cofactor for activation of its acetyltransferase activity. Furthermore, the cofactor did not seem to be proteinaceous or composed of nucleic acids. Consistent with these observations, an acid extract of HeLa cells was found to contain cofactor activity (B). This treatment removes most protein and nucleic acid components and leaves intact only small molecules and metabolites.
FIGURE 2. Analysis of the cofactor activity from HeLa cytosol; absence of cofactor activity in bacteria. A, Fraction 52 obtained from size-exclusion chromatography of HeLa cytosol (supplemental Fig. S1) was used in the in vitro acetyltransferase assay and displayed (more ...)
We next examined whether the cofactor was specific to mammalian cells. Acid extracts were prepared from bacteria, yeast, slime mold, HeLa cells and from rat brain, neutralized, and tested for the presence of cofactor. Interestingly, it was discovered that only bacteria lacked the cofactor (C). The activatory cofactor for YopJ was thus exclusively eukaryotic and not found in bacteria. This observation immediately suggested that the powerful acetyltransferase YopJ is likely kept quiescent in Yersinia by virtue of the lack of a eukaryotic cofactor.
Isolation of the Cofactor and Its Identification as IP6
Having narrowed down the cofactor to an acid extract of eukaryotic cells, we next employed the ion-exchange resin AG1-X8 to further fractionate the extract. Neutralized acid extracts of HeLa cells were passed over acetate-equilibrated AG1-X8 resin, and the resin was subsequently washed with water. Bound material was eluted with hydrochloric acid and tested for cofactor activity in YopJ-catalyzed acetyltransferase assays. A shows that the stimulatory cofactor was, indeed, enriched on AG1-X8 resin.
The eluate from the AG1-X8 resin was then analyzed by mass spectrometry (MS) to identify the molecule(s) contained therein. MS analysis in the negative ion mode revealed the presence of a predominant species with m/z = 658.823 in the elution (B). This moiety was further subjected to fragmentation analysis using MALDI-MS/MS and displayed peaks corresponding to the loss of H3PO4 and HPO3 ions (C). The MS analyses presented in (B and C) suggested that the cofactor present in the elution was likely IP6. The presence of fragments corresponding to breakdown products of IP6, namely IP5 (m/z = 578.9 Da), IP4 (m/z = 498.9 Da), and other fragments resulting from the loss of H2O from these species, were also seen (C). The identification was confirmed by comparing with the MS/MS fragment spectrum of commercially available IP6 acquired under the same conditions (D). These results establish that the activatory cofactor for YopJ purified from mammalian cells using AG1-X8 resin is IP6, also known as phytic acid or phytate.
Efficient analysis of inositol phosphates is difficult, because the compounds do not absorb visible or UV light nor can they easily be identified using specific colorimetric reagents. To verify that IP6
is indeed the activatory cofactor we performed in vitro
acetyltransferase assays using chemically pure IP3
, and inositol hexakissulfate (IS6
has similar structure and charge density compared with IP6
and can be used to examine the specificity of the requirement for IP6
). It is seen in A
could not substitute for IP6
as cofactors of YopJ. This observation indicates that YopJ is selective in its requirement for phosphate groups and that three phosphates, as in IP3
, are insufficient to stimulate the activity of YopJ.
FIGURE 4. Validation of IP6 as the activating cofactor. A, autoradiograph examining acetylation (using [14C]AcCoA) of MEK by YopJ in the presence of increasing doses (1, 10, and 100 nm) of IP3, IP6, and IS6. In A and B, lane 1 depicts the acetylation of MEK by (more ...)
The enzyme phytase (myo-inositol-hexakisphosphate 6-phosphohydrolase), naturally present in many plants and microorganisms, breaks down IP6
(phytate) releasing phosphate (19
). We observed (B
) that phytase destroyed the cofactor activity of both IP6
and of the mammalian cell extract (two dilutions); alkaline phosphatase, on the other hand, did not. This result constitutes further proof of IP6
being the activatory cofactor.
We then examined the extent of MEK acetylation using the discriminatory CST9122 antiserum. As seen in C inclusion of IP6 in acetyltransferase reactions resulted in very high levels of modification of MEK as inferred by the significant loss of immunodetection by the CST9122 antiserum (lanes 1 and 2); also, inclusion of progressively higher amounts of the HeLa cell extract led to progressively higher modification of MEK within the duration of the assay (1 h). This result shows that inclusion of IP6 or HeLa extract can stimulate the acetyltransferase activity of YopJ to achieve near complete acetylation of MEK.
Taken together, these results identify IP6 as the mammalian cell cofactor required by YopJ for activation of its acetyltransferase activity. This is manifested in increased autoacetylation as well as increased substrate acetylation by YopJ.
Our results establish YopJ as the second Yersinia
effector, after YpkA, which requires a host cell molecule for stimulation of its enzymatic activity. Binding of eukaryotic cell actin has been shown to result in increased autophosphorylation of YpkA resulting in elevated kinase activity of YpkA toward substrates (20
). However, we observed that prior autoacetylation of YopJ was not required for acetylation of MEK by YopJ (supplemental Fig. S2
). Thus, unlike the autoacetylation of transcription factor IIB (21
) and of p300/cAMP-responsive element binding protein association factor (22
) that activates their enzymatic activities, the autoacetylation of YopJ has likely no effect on its substrate acetyltransferase activity.
We demonstrated above (A
) that IP3
did not act an activator of YopJ but that the higher phosphorylated inositol polyphosphate IP6
did. The common precursor of all soluble inositol phosphates in most eukaryotic cells is IP3
(which is produced when phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate yielding IP3
and diacylglycerol). IP3
is then processed by a sequence of enzymes to produce a number of more highly phosphorylated inositol species. Inositol pentakisphosphate (IP5
) and inositol hexakisphosphate (IP6
) are the two most abundant inositol polyphosphates in mammalian cells (23
). They are also the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds (24
). We thus examined the absolute requirement for IP6
to be an activator. We have established in C
that an acid extract from wild-type yeast S. cerevisiae
contained cofactor activity. We thus examined extracts from yeast deletion strains that lack one or more enzymes of the inositol phosphate pathway. The inositol polyphosphate content of yeast has been very well characterized (25
The major inositol phosphate present in wild-type yeast is IP6
with modest levels of IP7
also present (11
). In the budding yeast, S. cerevisiae
, the enzyme IPK1 converts IP5
). The ipk1
Δ deletion strain thus lacks IP6
but instead accumulates IP5
and diphosphoinositol tetrakisphosphate (PP-IP4
), a pyrophosphate resulting from the action of the inositol pyrophosphate-forming enzyme, KCS1. Yeast bearing the double deletion ipk1
Δ thus show accumulation of IP4
and do not contain any higher polyphosphates. The enzyme inositol phosphate multikinase, IPMK, metabolizes IP3
Δ yeast thus accumulate only IP2
). The yeast strain kcs1
Δ lacks the inositol pyrophosphate-forming enzyme, KCS1 (which synthesizes the pyrophosphates IP7
). Therefore, kcs1
Δ yeast contain predominantly IP6
but no IP7
We examined extracts from the wild-type yeast strains BY4741 and DDY1810 and from the deletion strains ipk1
Δ (BY4741), ipk1
Δ (BY4741), ipmk
Δ (DDY1810), and kcs1
Δ (DDY1810). The results presented in A
show that of the extracts analyzed only those from ipmk
Δ yeast (lane 6
) were deficient in providing the cofactor for YopJ activity. These yeast contain IP2
as the only inositol phosphates. The extract from ipk1
Δ yeast (lane 4
) that lack IP6
but still contain IP5
was able to activate YopJ. Similarly, the ipk1
Δ yeast extract (lane 5
) that contains IP4
retained activity as well. Inositol pyrophosphates were clearly not required for cofactor activity because kcs1
Δ yeast extract (lane 7
) supported YopJ activation as well. In control experiments it was verified that purified IP5
was indeed able to support the activation of YopJ (supplemental Fig. S3
FIGURE 5. An inositol phosphate-deficient deletion strain of yeast lacks the cofactor; AvrA is stimulated by IP6 undergoing conformational change in response to IP6 addition. A, neutralized acid extracts were prepared from various deletion strains of the yeast (more ...)
We thus conclude that IP5 can also support the activation of YopJ. However, because IP6 is the predominant IPx species present in mammalian cells and was the principal species found in the AG1-X8 purification, it is likely to be the natural activator of the acetyltransferase activity of YopJ. It is interesting to note that, although IP5 is accepted as an efficient activator of YopJ, IS6 was not (A).
IP6 Activates the Acetyltransferase Activity of AvrA and Causes Conformational Change in AvrA
We have noted above that autoacetylation of YopJ provides a convenient readout of its activity. We used this feature as a readout of activation of another member of the YopJ family. We analyzed the activation of AvrA, the YopJ homologue from S. typhimurium. The autoacetylation of AvrA was also found (B) to be activated by IP6. Our results thus establish the generality of the requirement of IP6 as a eukaryotic cell activator for the YopJ family of type III secreted effectors.
We wondered about the nature of the association of IP6
with YopJ and AvrA. The binding of IP6
to TIR1, a receptor for the plant hormone auxin (28
) and to the RNA deaminase ADAR2 (18
), is shown by their crystal structures to occur deep within the core of the protein molecule in a cavity lined by basic residues. In each of these cases IP6
is found to co-purify with the recombinant protein that has been expressed in a eukaryotic expression system. The binding of the deeply embedded IP6
molecule likely happens during the folding of the protein, which is why the IP6
is carried through the various steps of purification (in IP6
-free buffers) to be revealed in the crystal structure. YopJ did not display such an irreversible association with IP6
, because in control experiments it was observed by us that YopJ purified in a single column step from a eukaryotic expression system (baculovirus-mediated expression in insect cells) still required activation by externally added IP6
. Rather, we believe that IP6
binds to basic residues on or close to the surface of YopJ resulting in allosteric activation of the acetyltransferase activity of YopJ (and other YopJ-like molecules). Crystal structures of the cysteine protease domains of the Vibrio cholerae
RTX toxin (29
) and of the Clostridium difficile
Toxin A (30
) show IP6
binding to such basic surface cavities on the proteins distant from the active sites.
Because YopJ was difficult to produce in quantities sufficient for biophysical analyses we chose to examine AvrA by fluorescence and CD spectroscopy. A change in the intrinsic tryptophan fluorescence of a protein upon ligand binding is a very sensitive measure of conformational changes in the environment of the reporter tryptophan residue. AvrA contains a single tryptophan residue (tryptophan 44). We thus examined the effect of IP6 addition on the tryptophan fluorescence of AvrA. As seen in C, addition of IP6 resulted in a decrease in the intensity of emission of intrinsic tryptophan fluorescence of the protein. IP6 binding therefore results in a conformational change in AvrA.
We examined the nature of this IP6
-induced conformational change using CD spectroscopy. The phenomenon of circular dichroism is very sensitive to the secondary structure of polypeptides and proteins and is a particularly powerful technique for monitoring conformational changes. Measurement of CD spectra of AvrA in the far-UV spectral region (190–250 nm) in the presence of increasing amounts of IP6
showed a decrease in ellipticity at 208 nm and 222 nm indicative of an increase in α-helical content of AvrA (supplemental Fig. S4A
We then measured the change in ellipticity of AvrA upon IP6
addition as a function of time (D
). It was observed that addition of IP6
resulted in a saturable decrease of the CD signal at 222 nm. Thus, binding of IP6
resulted in an increase of total helicity of AvrA. In control experiments it was verified that addition of AcCoA to AvrA either before or after the addition of IP6
did not result in any significant conformational change. The most likely interpretation of these observations is that IP6
binds in a basic pocket on the surface of AvrA and induces the formation of a helix leading to allosteric activation of the acetyltransferase activity of AvrA. Furthermore, addition of IP6
to the catalytically inactive C172A mutant of AvrA also resulted in a similar conformational change (supplemental Fig. S4B
). This suggests that the binding site for IP6
on AvrA is likely distant from the active site for catalysis.
Taken together, our results demonstrate that the YopJ family of type III effector proteins, which includes AvrA from S. typhimurium, requires the eukaryotic host cell molecule IP6 for activation of their acetyltransferase activity. This mechanism suggests that YopJ-like molecules are quiescent in the bacterium where they are synthesized, because bacteria do not contain IP6. Upon injection into mammalian cells by the pathogen type III secretion system these molecules bind host cell IP6 and become activated, thereby dampening the host immune response by covalently modifying host cell-signaling proteins.