Articles pubs.acs.org/acschemicalbiology
Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR’s Phosphatase Activity Karolina Pavic,† Pablo Rios,† Kristina Dzeyk,‡ Christine Koehler,§ Edward A. Lemke,§ and Maja Köhn*,† †
Genome Biology Unit, ‡Proteomics Core Facility and §Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany S Supporting Information *
ABSTRACT: Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR’s biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique “variable insert” region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.
Protein tyrosine phosphorylation is a crucial process in cellular signaling. Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of the phosphate from the substrate. Classical PTPs dephosphorylate preferentially phosphotyrosine residues. Dualspecificity phosphatases (DSPs or DUSPs) constitute a heterogeneous group within the superfamily of PTPs. DSPs show diverse substrate specificities ranging from phosphotyrosine and phosphoserine/threonine residues to nonpeptidic substrates such as phosphoinositides.1,2 The classical PTPs and DSPs share a similar catalytic mechanism which involves the catalytically active cysteine of the consensus active site (“Ploop”) signature motif HC(X)5R(S/T) (single letter amino acid code), and, in most cases, a general acid/base aspartate in the so-called WPD loop named after the conserved amino acids of this loop.3 Vaccinia H1-related (VHR) phosphatase (also known as DUSP3) is a constitutively expressed 20.5 kDa phosphatase with predominantly nuclear localization.4 The level of VHR was found to fluctuate during cell cycle progression, and downregulation of VHR by RNA interference showed that cells lacking VHR exhibit signs of senescence.5 VHR was found to be overexpressed in prostate cancer6 and in several cervix cancer cell lines, and it was postulated that the increased protein level of VHR in cervix cancer cells would enable the cells to proliferate.7 The active conformation of VHR is defined by the low pKa (5.52) of the active site cysteine (Cys124),8 and VHR exhibits a preference for dephosphorylating phosphotyrosine.9,10 VHR dephosphorylates the Extracellular signal regulated kinases (Erk) 1/2 and c-Jun N-terminal kinase (JNK) in vivo.4,11 In addition, signal transducer and activator of © 2014 American Chemical Society
transcription (STAT) 5 and the receptor tyrosine kinase ErbB2 have also been reported to be dephosphorylated by VHR.12,13 There are only few reports addressing the regulatory principles of VHR. Phosphorylation of Y138 of VHR by ZAP-70 tyrosine kinase is required for down-regulation of the Erk1/2-Elk pathway in activated T-cells14 and STAT5 dephosphorylation following cytokine stimulation. Moreover, VHR Y138 phosphorylation by tyrosine kinase (Tyk) 2 was observed only in the catalytically deficient forms of VHR.12 Lastly, Vacciniarelated kinase (VRK) 3 was linked to the enhancement of VHR-mediated down-regulation of Erk-activity.15 Although sharing substrates with mitogen-activated protein kinase phosphatases (MKPs), VHR is smaller in size, and it lacks the N-terminal MAPK-binding (MKB) domain and additional adapter/regulatory domains that may regulate substrate specificity.16 The absence of regulatory domains in this small protein makes the prediction of regulatory protein−protein interactions difficult, and oligomerization as potential regulatory mechanism has not yet been reported for VHR. A unique structural region in VHR, which could potentially be involved in specific interactions, resides between the chains β3−β7 and is termed “variable insert”.9,17,18 When compared to structures of homologous PTPs, Phe68 and Met69 of the protruding β3−α4 loop in VHR cannot be aligned in the structure-based sequence alignment.17,18 With the exception of Met69, which Received: April 1, 2014 Accepted: May 5, 2014 Published: May 5, 2014 1451
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Figure 1. Selected residues in VHR. (a) Overall crystal structure of VHR (PDB entry number 1VHR) presented as a cartoon (gray). The “variable insert” region in VHR (residues 61−83) is pointed out in orange. Phe68 is colored green, Asp92 red, Cys124 cyan, and Met69 violet. (b) Surface representation of the zoomed-in active site and Phe68 Met69 of the “variable insert” of VHR. The structure is left-tilted with respect to the one in part a to make the Cys124 visible in this representation. The residues are colored as indicated in part a. (c) Chemical structure of pBPA.
was also generated (amino acids 1−67 of VHR), although to a much lesser extent than the full length variant. This product remained in the purified samples due to the N-terminal Histagging. As this truncated product contained neither the pBPA nor the active site, we did not expect it to interfere negatively with the following studies (see below). After removal of the His-tag, all recombinant proteins were left N-terminally Flag3tagged, which was required for some of the following experiments. Characterization of VHR Variants. To test whether introducing pBPA at the position of Phe68 affected the secondary structure, we analyzed it by far UV−circular dichroism spectroscopy (CD).25 When comparing the spectrum acquired for the F68pBPA variant with the spectrum obtained for the wild type VHR (wtVHR), we observed only minor modifications, suggesting that structural perturbations were imposed neither by pBPA in this VHR variant nor by its truncated form (Supporting Information, Figure S2). Next, we sought to determine kinetic constants of the enzymes by examining their catalytic efficiency in dephosphorylating pNPP (Supporting Information, Table S2). In addition to the pBPAcontaining variant, we included a systematic mutational analysis of Phe68 and a M69A mutation in order to elucidate a possible functional impact of the region bearing Phe68 and Met69 on catalytic activity. The kinetic constants that we obtained for wtVHR were similar to those noted previously.26,27 For all VHR variants generated by site-directed replacement at position 68 with Ala, Asp, Trp, Cys, or pBPA, turnover numbers were within 1.6 fold of the value obtained for the wtVHR. KM values were not substantially affected. We concluded that the overall catalytic performance of these variants was not altered much. With respect to the pBPA variant, this meant that neither the incorporation of pBPA nor the minor amount of truncated form present in the sample had an effect on the catalytic performance. In contrast, M69A exhibited a stronger shift in the values of KM and kcat/KM compared to the wtVHR. Since Met69 anchors Arg130 of the active site cleft,9 we reasoned that substitution with a residue that lacks hydrogen bonding capacity could disrupt the proper geometry of the active site cleft. Thus, this residue outlining the
anchors Arg130 of the active site cleft, the function of the other residues of the unique “variable insert” region is still unknown.9 To study if a potential role of the distinct Phe68 and Met69 containing patch of the “variable insert” region could be to mediate specific protein−protein interactions, we exploited here the nonsense suppression technology utilizing the amber STOP codon (nucleotides TAG) for site-directed incorporation of the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA).19,20 This approach was previously shown to be effective in capturing not only stably associated protein interactions by covalent cross-linking, but also weak, transient, pH- or subcellular localization-dependent complexes.21−23 pBPA is reversibly photoexcited upon exposure to 345−365 nm light, minimizing photodamage to other biomolecules, while providing a covalent snapshot of interactions.24 Using this approach and complementary methods, we demonstrate that VHR dimerizes in vitro and within cells, and that the “variable insert” region is involved in this specific VHR self-association. We also show that the dimeric association negatively affects the enzymatic activity against para-nitrophenylphosphate (pNPP) and, physiologically more relevant, Erk1/2. Thus, our findings indicate that self-association is a mechanism for the regulation of VHR phosphatase.
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RESULTS AND DISCUSSION Incorporation of pBPA into VHR by Amber Suppression in E. coli. In the VHR protein sequence, Phe68 of the “variable insert” region, which contains residues 61−83, was selected to be replaced by pBPA (Figure 1). Phe68 was chosen (i) because of its localization in the loop region of the “variable insert”, which might minimize a potential disruptive effect that the substitution to pBPA could have on the structural integrity, (ii) because of its close proximity to the active site (Figure 1b), which could imply an influence on catalytic activity, and (iii) because it and its neighboring residue Met69 protrude out of the structure and could thus be involved in potential protein− protein interactions. The VHR F68pBPA variant exhibited good incorporation efficiency and its identity was confirmed using mass spectrometry (MS) (Supporting Information, Table S1 and Figure S1). The truncated variant with the protein translation aborted at the position of the amber STOP codon 1452
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Figure 2. Photoinduced cross-linking of purified recombinant VHR. Dimer formation of the Flag3-tagged F68pBPA VHR upon exposure to UV light. The Flag3-wtVHR, with or without UV exposure, and its F68pBPA variant without UV exposure, were used as negative controls. Bands: Monomeric full length proteins ≈ 24 kDa; covalently cross-linked dimers of the Flag3-F68pBPA variant ≈ 50 kDa; Flag3-VHR 1−67 ≈ 14 kDa; cross-linked monomeric form to Flag3-VHR 1−67 ≈ 40 kDa. The analyses were done by Western blotting using an anti-VHR antibody. The results are representative of two independent experiments.
Figure 3. Detection of VHR self-association by chemical cross-linking. (a) Evaluating the intrinsic ability of VHR to self-associate by examining the oligomeric state of the purified recombinant Flag3-tagged wtVHR and its C124S and F68pBPA variants after cross-linking with glutaraldehyde. The control samples were incubated without glutaraldehyde. (b) Cross-linking of Flag3-tagged wtVHR with DSS. The control sample was treated without the presence of DSS. (c) Profiling the effect of the structural and electrostatic properties of the residues incorporated at position 68 on the ability of VHR to self-associate, by cross-linking with glutaraldehyde. For panels a−c, the cross-linked species were detected by Western blotting using an antiVHR antibody. The results reflect the minimum of two independent experiments.
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propensity of wtVHR using complementary methods to the genetic encoding of pBPA. To this end, we applied the crosslinking reagents glutaraldehyde29 and disuccinimidyl suberate (DSS).30 The control samples that were treated without a cross-linker regularly exhibited no dimer formation. The monomeric forms of Flag3-tagged wtVHR and F68pBPA VHR ran as a band at apparent molecular weight (MW) of approximately 20 kDa (Figure 3a). For both proteins, a second population at about 40 kDa clearly corresponded to the size of the dimer. The faint band at about 30 kDa, present only in the glutaraldehyde-treated sample of the Flag3-tagged F68pBPA variant, matched the MW of the cross-linked complex between the full length and the truncated form. These results confirm the self-association of VHR, including the involvement of the sequence of the truncated product (VHR1−67) in the interaction. Furthermore, they also demonstrate that the truncated product does not have an effect on the homodimerization of the full length VHR, as wtVHR, not containing a truncated form, also dimerizes. We also observed faint bands of higher order oligomers (Figure 3a), which could be a result of the applied method, as we did not detect these when using the site-selective pBPA-mediated cross-linking. Moreover, VHR dimerization was independent of the availability of the active site cysteine because incubation with glutaraldehyde also led to the detection of dimers and higher order oligomers for the C124S variant of VHR, in which the catalytically active cysteine was replaced by serine. The self-association of VHR was crossconfirmed using DSS, showing an increase in the cross-linking yield concomitant with the increase of the protein concentration (Figure 3b), demonstrating once more the potential of VHR to self-associate. To investigate the impact of steric/electrostatic features of the amino acid at position 68 on the formation of homodimers, we next profiled the series of F68-variants. After treating the samples with glutaraldehyde, we observed that all F68-variants were able to form dimers and higher order oligomers (Figure 3c). The abundance of the detected oligomers in case of the F68W and F68D variants did not differ much to that observed with wtVHR, indicating that electrostatic features and more bulk do not play a significant role here. Exchanging the hydrophobic aromatic residue in the native protein (Phe) for a small nonpolar one (Ala) led to a stronger self-association (Figure 3c) than was observed with the wtVHR and the other variants (Figure 3a and c) except F68C and F68pBPA, possibly by enabling closer association of the monomers. Coomassie staining of the resolved samples on an SDS gel (Supporting Information, Figure S4) showed that equal amounts of VHR variants were used in the cross-linking experiments (Figure 3a and c). The F68C variant demonstrated cross-linking efficiency similar to what we observed for the F68A variant, which was stronger than that of the wtVHR (Figure 3a and c). Disulfide formation is recognized as a mechanism of protein selfassociation31 and could enhance the dimerization also in this case. In addition to the UV-induced cross-linking of the full length F68pBPA to the fragment containing Flag3-VHR 1−67, this finding supports that the “variable insert” is involved in the self-association of VHR. To verify this, we investigated in more detail if the F68C mutation renders the protein more susceptible to intermolecular disulfide bond formation. We checked the oligomerization state of the variants by resolving the samples, dialyzed with or without the reducing agent βmercaptoethanol, by SDS-PAGE under nondenaturing or
rim of the substrate-binding cleft (Figure 1b) could be indirectly engaged in promoting efficient turnover. F68pBPA VHR Dimerizes upon UV Exposure In Vitro. In order to determine if the “variable insert” region of VHR could be involved in homomeric protein−protein interactions, we exposed the Flag3-pBPA variant of VHR in buffer to UV light. We indeed detected a band on the Western blot, which in size corresponded to the homodimer (48 kDa). Analogous experiments without UV exposure showed no dimer formation (Figure 2). Also, the truncated fragment that did not contain pBPA (Flag3-VHR 1−67, band around 14 kDa) did not show any homodimerization, irrespective of UV irradiation. However, the full length Flag3-F68pBPA VHR was found to cross-link to the truncated product (together 35 kDa). This led us to hypothesize that the region of the protein corresponding to the truncated product (which also contains residues 61−67 of the flexible loop of the “variable insert”) bears sufficient sequential requirements to drive intermolecular association between VHR monomers. Since photo-cross-linking using pBPA was previously shown to enable the detection of weak and transient protein interactions,21−23 we investigated in the following the potential of VHR to self-associate. To this end, we evaluated the dependence of the dimer formation on the protein concentration. Purified recombinant Flag3-tagged F68pBPA and wtVHR were exposed to UV light for 30 min, spanning protein concentrations from 50 to 500 μg/mL. Subsequent Western blot analysis detected bands corresponding to the cross-linked complexes of the F68pBPA variant already at 50 μg/mL, with increase in detected complexes at higher protein concentrations demonstrating the concentration-dependence of the interaction (Figure 2). Finally, we sought to prove covalent dimerization by MS. After UV-irradiation of F68pBPA, we detected in the SDS gel of the sample the monomeric fraction of full length VHR, traces of the truncated form, the covalently cross-linked homodimer and, to a much lesser extent, the cross-linked heterodimer of full length and truncated version (Figure 2). To increase the likelihood for detecting the full mass of the dimer by MS, we attempted to isolate the covalent homodimer by size exclusion chromatography. Only a small amount of the monomeric fraction (M) could be isolated (Supporting Information, Figure S3a). We were not able to obtain the pure fraction of the covalent homodimer. Nevertheless, the mass corresponding to the covalent dimer was identified by LC-MS/MS analysis in the mixture of the four components enriched in the covalent homodimer (D) (Supporting Information, Figure S3a and b). In this enriched mixture (D), the mass of the cross-linked heterodimer of full length and truncated version was also detected (Supporting Information, Figure S3b). Furthermore, LC-MS/MS analysis of the band corresponding to the monomeric fraction (M) identified intramolecular cross-linking (Supporting Information, Methods). When the monomeric sample (M) isolated by size exclusion was resubjected to UV light, no further dimers were formed (Supporting Information, Figure S3c). These results indicated that UV-exposure of F68pBPA triggers dimer formation, while simultaneously attenuating cross-linking efficiency due to intramolecular cross-linking, explaining the limitations in the yield of dimer formation (Figure 2). Photoinduced destruction of pBPA or side reactions with buffer components could also be also potential causes for the observed limited yield.28 Confirmation of the VHR Dimerization by Alternative Methods. We next sought to confirm the dimerization 1454
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Figure 4. Self-association of VHR in COS-1 cells. (a) COS-1 cells were transiently cotransfected with myc- and Flag3-tagged wtVHR. The analysis was conducted 24 h post-transfection. The cells were stimulated with 10 mM H2O2 for 1 h at 37 °C prior to lysis. The lysate was immunoprecipitated using antibody against the Flag-tag and immunoblots were probed by using an antimyc antibody. Afterward, the membrane was stripped and reblotted using an anti-Flag antibody. (b) COS-1 cells were transfected with pCMV/Flag3-wtVHR and probed for dimeric association inside the cell. Twenty-four hours post-transfection, the cells were treated with 0.8% (w/v) PFA in PBS for 150 min and then lysed. The control samples were incubated in growth medium only. The samples were heated at 60 °C for 20 min or at 95 °C for 5 min. The pellets (P) that remained from clearing the lysates were resuspended and incubated at 95 °C for 5 min. The samples were resolved by 4−12% SDS-PAGE and probed with an anti-Flag antibody. The images in panels a and b represent the results of two independent experiments.
denaturing conditions (Supporting Information, Figure S5). For wtVHR, we observed traces of dimers under nondenaturing compared to denaturing conditions, and no difference was observed in the extent of dimer formation with respect to βmercaptoethanol treatment. The F68C variant showed a strongly increased potential for self-association compared to the wtVHR under nondenaturing conditions. In general, under denaturing SDS-PAGE conditions hardly any homodimers could be detected, suggesting together that the self-association is reversible and that, under the in vitro conditions, the interaction is rather weak and can be destroyed during gel electrophoresis if not stabilized by Cys68-disulfide formation. Thus, these observations demonstrate that the Cys68 is involved in the self-association. Strikingly, the F68pBPA variant exhibited a pattern more resembling that of the F68C variant than that of the wtVHR (Figure 3a and c). The “variable insert” contains a small hydrophobic and protruding patch of nonconserved amino acid composition.9 Phe68 is a part of this island. Exchanging Phe68 for the more hydrophobic pBPA is likely to further increase the extent of hydrophobic interactions, which could further stabilize the self-association of VHR, again confirming that the position 68 of VHR is important for the self-association. In addition, since the catalytic cysteine was excluded from being involved in the dimer formation (Figure 3a), and the weak dimer formation of wtVHR under nondenaturing electrophoretic conditions was not reduced by β-mercaptoethanol treatment (Supporting
Information, Figure S5), our results indicate that the dimer formation is likely based on structural factors and involves noncovalent interactions. VHR Forms Dimers in Cells. To investigate homodimeric association of VHR in cells, COS-1 cells were cotransfected with myc- and Flag3-tagged VHR constructs. We intended to demonstrate coprecipitation of the two differently tagged VHR variants but, under nonstimulated conditions, were not able to observe cosedimentation. Because previous reports showed that dimerization of PTPs can be enhanced by oxidative conditions,32 we pretreated the cells with 10 mM H2O2. Under these conditions, we indeed observed the self-association of VHR (Figure 4a and Supporting Information, Figure S6a). Repetition of this experiment in vitro confirmed that H2O2 treatment stabilizes the VHR dimers (Supporting Information, Figure S6b), suggesting a direct effect of oxidative species on VHR dimerization but not excluding that other indirect effectors inside cells could also play a role. We also applied paraformaldehyde (PFA) at 0.8% in PBS (w/v) in Flag3-tagged VHR overexpressing COS-1 cells to probe for VHR dimerization in intact cells, in the absence of H2O2. We captured the VHR self-association, and the cross-link was irreversible as shown by denaturing the samples through heating (Figure 4b). The untreated control cells showed no complexes of higher molecular masses. Capturing two VHR proteins covalently inside cells is a persuasive indication of their spatial proximity.33 The dimers, nevertheless, need to be 1455
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Figure 5. Functional implications of VHR dimerization. (a) The proteins were subjected to 345 nm light (1000 W) for 30 min at 250 μg/mL concentration, followed by dilution to 500 nM before subjecting to the activity assay with pNPP at 10 mM. F68pBPA Monomer (M) = isolated by size-exclusion after UV-cross-linking (Supporting Information, Figure S3a). The nonirradiated wtVHR was used as a control. A discrete time-point (30 min) is represented as percentage of activity with respect to the wtVHR and corresponds to the average of at least two experiments with triplicate measurements. See Supporting Information, Figure S7, for the kinetic profiles. The GraphPad 5.0 software (GraphPad, San Diego, CA, U.S.A.) was used to plot the data as mean ± SEM and to perform the statistical analysis. A Student’s unpaired t test was used to calculate the statistical significance and the asterisks indicate ***p < 0.001. (b) Dephosphorylation of Erk1/2 in COS-1 cell extracts by the wtVHR and its variants, with or without exposure to 345 nm light. Lysates were incubated with 500 nM of recombinant VHR, or an equal volume of buffer, for 0, 30, and 90 min at 30 °C. Time-dependent reduction of pErk1/2 in COS-1 extracts was analyzed with an anti-pErk1/2 antibody. An anti-Erk1/2 and an anti-VHR antibodies were used to assess equivalent amounts of lysates and bait used, respectively. The result is representative of three independent experiments.
on VHR’s enzymatic activity, we first subjected the wtVHR and the F68pBPA variant to UV light, followed by activity measurement employing pNPP. We observed that the catalytic activity of the wtVHR was not affected by UV-exposure. In addition, the activity of the UV-irradiated F68pBPA was about 60% lower than that of the nonirradiated sample, which equaled the activity of the wtVHR (Figure 5a). These results could indicate that (i) pBPA-mediated cross-linking randomly lowers the activity or (ii) the topology of the dimeric association might curtail efficient catalytic performance, potentially by providing
stabilized to enable their detection. Stabilization by covalent cross-linking showed that VHR is present as dimers inside cells. The fact that oxidative conditions enabled the detection without requiring a chemical cross-linker suggests a strengthening of the interaction as response to this stimulus. Together, these results indicate that VHR exists inside cells in an equilibrium of monomers and dimers, which is shifted in response to oxidative stimulation. Assessment of Functional Consequences of VHR Dimerization. To assess the effect of the dimer formation 1456
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Conclusions. Taken together, the results demonstrate that VHR dimerizes through Phe68 and Met69 of the “variable insert” region, and indicate that this could be a novel regulatory mechanism for VHR activity. In a nonstimulated state VHR might be present as a monomer or loosely packed dimer as indicated by cross-linking inside cells using PFA (Figure 4b), with the overall catalytic activity dependent on the mutual orientation of the self-associating units. Under stimulated conditions, such as oxidative stress as presented here, the dimerization of VHR appears to be stabilized. While the active site cysteine was observed to not be essential for dimerization, it cannot be excluded at this point that other cysteines in VHR may be involved in the mechanism of dimerization, although none appears to be structurally very close to the observed dimerization interface.9 The intermolecular self-association of VHR might impose steric hindrance to the active site cleft, therefore rendering it inaccessible to substrates. Oligomer formation, also in response to H2O2, has been recognized as a regulatory principle within the PTP superfamily, serving as an important determinant of substrate specificity and catalytic activity.31,32 These reported mechanisms render our hypothesis of oxidative stress-induced VHR dimerization feasible. It is known that the active site cysteine residue of VHR is sensitive to H2O2-induced oxidative inhibition.35 However, VHR showed a lack of excessive, irreversible oxidation observed for other PTPs, and the authors speculated that this might be due to the active site Cys124 being protected from solvent exposure. Dimerization could therefore also provide a mechanism to protect the catalytic cysteine from irreversible oxidation. Selfassociation of VHR would also result in a high local concentration of the enzymatically active form after release of the stimulus, which could act rapidly to elicit a specific cellular response after the dimeric complex is disassembled into monomers. Finally, there is an interest to discover VHR inhibitors due to its role in different cancers.6,7 In this regard, stabilization of VHR dimerization could be a new means to interfere with VHR activity. Our findings were enabled by selective incorporation of the photo-cross-linkable pBPA. This strategy allowed us to investigate how dimerization affects the enzymatic activity of VHR, whose self-association is not stable enough to purify the dimer and assay its activity. When applying traditional crosslinkers, the random and multiple cross-linking exerted by them would usually prevent such a study. Based on the work presented here, it is reasonable to assume that the selective incorporation of photo-cross-linkable amino acids will be a useful and feasible tool for investigating interactions of PTPs in general, and for those in particular where dimerization was suggested by one experiment but could not be confirmed by another, such as for DUSP26.36
sterical hindrance to the active site pocket. As the UV-irradiated F68pBPA sample was a mixture containing the monomeric forms (full length and truncated form), the cross-linked dimer of the full length and the truncated version (length 1−67 of VHR) and the homodimer (Figure 2), the reduced activity is exerted by the combination of these species. However, as stated above, the pure photo-cross-linked F68pBPA dimer could not be obtained by size exclusion chromatography and was therefore not available for activity assays. Other chemical cross-linkers bind randomly, including potential binding to the active site, and can consequently not be applied for activity studies. Therefore, to investigate the contribution of different species, the monomeric fraction of the UV-irradiated F68pBPA variant, isolated through size exclusion chromatography, was tested for its activity and showed to be about 40% less active compared to the wtVHR (Figure 5a). This indicated that intramolecular cross-linking, which we had observed by MS, affected catalytic efficiency, possibly by limiting access to the active site or by disrupting the most optimal configuration of catalytically important residues. Moreover, the reduction in activity of the UV-irradiated F68pBPA previous to size exclusion chromatography can therefore be in part attributed to intramolecular cross-linking. Nevertheless, this sample showed even further reduced activity (Figure 5a and Supporting Information, Figure S7), suggesting that the selfassociated VHR also has reduced activity. In order to obtain a more physiologically relevant model, we assessed levels of dephosphorylated Erk1/2 in COS-1 cell extracts by adding the recombinant proteins. The wtVHR and the F68pBPA variant were pretreated as described above. In the buffer control samples no significant dephosphorylation of Erk1/2 was observed over 90 min (Figure 5b). The wtVHR samples showed a strong reduction in the pErk1/2 level after 90 min irrespective of UV exposure, which is in accord with the experiments using pNPP as substrate (Figure 5a and Supporting Information, Figure S7). The level of pErk1/2 in the sample incubated with irradiated F68pBPA was almost at the level of the buffer control after 90 min, which is in stark contrast to the nonirradiated sample. Taken together, the results from two activity assays indicate that dimerization is likely a negative regulator of VHR enzymatic activity. VHR Dimerization Occludes the Active Site. To investigate further if dimerization could lead to occlusion of the active site, we mutated the catalytically active Asp92 to pBPA. This residue is part of the WPD-loop of VHR. It is in close proximity to both the active site and Phe68 (Figure 1b) and could thus also be able to cross-link with another VHR molecule if the active site were occluded by the dimerization. Like the F68pBPA mutant, the D92pBPA variant showed good incorporation efficiency (Supporting Information, Figure S8a and b). We detected less truncated product (containing Flag3VHR 1−91) for the D92pBPA than for the F68pBPA variant, which can be explained by the dependence of the efficiency of the amber codon suppression on its sequence context.34 When testing its catalytic activity, we found about 25-fold reduction in the kcat of VHR D92pBPA compared to the wtVHR (Supporting Information, Figure S8c and Table S2), which was expected due to the involvement of D92 in the catalytic mechanism.27 Finally, we also observed the formation of a D92pBPA-dimer after UV-irradiation (Supporting Information, Figure S8d), indicating that the active site could indeed be occluded by the dimerization.
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ASSOCIATED CONTENT
Materials and methods are described in detail in Supporting Information.
S Supporting Information *
Materials and methods section. Additional tables and figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 1457
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(14) Alonso, A., Rahmouni, S., Williams, S., van Stipdonk, M., Jaroszewski, L., Godzik, A., Abraham, R. T., Schoenberger, S. P., and Mustelin, T. (2003) Tyrosine phosphorylation of VHR phosphatase by ZAP-70. Nat. Immunol. 4, 44−48. (15) Kang, T.-H., and Kim, K.-T. (2006) Negative regulation of ERK activity by VRK3-mediated activation of VHR phosphatase. Nat. Cell Biol. 8, 863−869. (16) Tonks, N. K. (2013) Protein tyrosine phosphatasesfrom housekeeping enzymes to master regulators of signal transduction. FEBS J. 280, 346−378. (17) Lountos, G. T., Tropea, J. E., and Waugh, D. S. (2011) Structure of human dual-specificity phosphatase 27 at 2.38 Å resolution. Acta crystallog. 67, 471−479. (18) Kim, S. J., Jeong, D.-G., Yoon, T.-S., Son, J.-H., Cho, S. K., Ryu, S. E., and Kim, J.-H. (2007) Crystal structure of human TMDP, a testis-specific dual specificity protein phosphatase: Implications for substrate specificity. Proteins 245, 239−245. (19) Ryu, Y., and Schultz, P. G. (2006) Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat. Methods 3, 263−265. (20) Liu, W., Brock, A., Chen, S., Chen, S., and Schultz, P. G. (2007) Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat. Methods 4, 239−244. (21) Krishnamurthy, M., Dugan, A., Nwokoye, A., Fung, Y.-H., Lancia, J. K., Majmudar, C. Y., and Mapp, A. K. (2011) Caught in the act: Covalent crosslinking captures activator−coactivator interactions in vivo. ACS Chem. Biol. 6, 1321−1326. (22) Davis, L., and Chin, J. W. (2012) Designer proteins: Applications of genetic code expansion in cell biology. Nat. Rev. Mol. Cell. Biol. 13, 168−182. (23) Pham, N. D., Parker, R. B., and Kohler, J. J. (2012) Photocrosslinking approaches to interactome mapping. Curr. Opin. Chem. Biol. 17, 90−101. (24) Tanaka, Y., Bond, M. R., and Kohler, J. J. (2008) Photocrosslinkers illuminate interactions in living cells. Mol. Biosyst. 4, 473− 480. (25) Ranjbar, B., and Gill, P. (2009) Circular dichroism techniques: Biomolecular and nanostructural analyses. A review. Chem. Biol. Drug. Des. 74, 101−120. (26) Denu, J. M., Lohse, D. L., Vijayalakshmi, J., Saper, M., and Dixon, J. E. (1996) Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis. Proc. Natl. Acad. Sci. U.S.A. 93, 2493−2498. (27) Denu, J. M., Zhou, G., Guo, Y., and Dixon, J. E. (1995) The catalytic role of aspartic acid-92 in a human dual-specific proteintyrosine-phosphatase. Biochemistry 34, 3396−3403. (28) Zhang, M., Lin, S., Song, X., Liu, J., Fu, J., Ge, X., Fu, X., Chang, Z., and Chen, P. R. (2011) A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat. Chem. Biol. 7, 671−677. (29) Sun, J.-P., Luo, Y., Yu, X., Wang, W.-Q., Zhou, B., Liang, F., and Zhang, Z.-J. (2007) Phosphatase activity, trimerization, and the Cterminal polybasic region are all required for PRL1-mediated cell growth and migration. J. Biol. Chem. 282, 29043−29051. (30) Cheng, D., Meegalla, R. L., He, B., Cromley, D. A., Billheimer, J. T., and Young, P. R. (2001) Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein. Biochem. J. 359, 707−714. (31) Deb, I., Poddar, R., and Paul, S. (2011) Oxidative stress-induced oligomerization inhibits the activity of the non-receptor tyrosine phosphatase STEP61. J. Neurochem. 116, 1097−1111. (32) Nardozza, A. P., D’Orazio, M., Trapannone, R., Corallino, S., Filomeni, G., Tartaglia, M., Battistoni, A., Cesareni, G., and Castagnoli, L. (2012) Reactive oxygen species and epidermal growth factor are antagonistic cues controlling SHP-2 dimerization. Mol. Cell. Biol. 32, 1998−2009. (33) Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M., and Aebersold, R. (2010) Probing native protein structures by chemical crosslinking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics 9, 1634−1649.
AUTHOR INFORMATION
Corresponding Author
*Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS K.P. and M.K. thank the German Science Foundation (DFG) for support within the Emmy Noether program (KO 4013/11). E.A.L. acknowledges funding from the Emmy Noether program of the DFG. We thank S. Keyse for discussions and suggestions. We thank the Protein Expression and Purification Core Facility and the Proteomics Core Facility at EMBL for support.
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REFERENCES
(1) Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman, A., Godzik, A., Hunter, T., Dixon, J., and Mustelin, T. (2004) Protein tyrosine phosphatases in the human genome. Cell 117, 699−711. (2) Li, X., Wilmanns, M., Thornton, J., and Köhn, M. (2013) Elucidating phosphatase−substrate networks. Sci. Signal. 6, rs10. (3) Jackson, M. D., and Denu, J. M. (2001) Molecular reactions of protein phosphatasesInsights from structure and chemistry. Chem. Rev. 101, 2313−2340. (4) Todd, J. L., Tanner, K. G., and Denu, J. M. (1999) Extracellular signal regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. J. Biol. Chem. 274, 13271−13280. (5) Rahmouni, S., Cerignoli, F., Alonso, A., Tsutji, T., Henkens, R., Zhu, C., Louis-dit-Sully, C., Moutschen, M., Jiang, W., and Mustelin, T. (2006) Loss of the VHR dual-specific phosphatase causes cell-cycle arrest and senescence. Nat. Cell Biol. 8, 524−531. (6) Arnoldussen, Y. J., Lorenzo, P. I., Pretorius, M. E., Waehre, H., Risberg, B., Maelandsmo, G. M., Danielsen, H. E., and Saatciouglu, F. (2008) The mitogen-activated protein kinase phosphatase Vaccinia H1-related protein inhibits apoptosis in prostate cancer cells and is overexpressed in prostate cancer. Cancer Res. 68, 9255−9264. (7) Henkens, R., Delvenne, P., Arafa, M., Moutschen, M., Zeddou, M., Tautz, L., Boniver, J., Mustelin, T., and Rahmouni, S. (2008) Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR. BMC Cancer 8, 147−155. (8) Kim, J.-H., Shin, D. Y., Han, M.-H., and Choi, M.-U. (2001) Mutational and kinetic evaluation of conserved His-123 in dual specificity protein-tyrosine phosphatase vaccinia H1-related phosphatase: Participation of Tyr-78 and Thr-73 residues in tuning the orientation of His-123. J. Biol. Chem. 276, 27568−27574. (9) Yuvaniyama, J., Denu, J. M., Dixon, J. E., and Saper, M. A. (1996) Crystal structure of the dual specificity protein phosphatase VHR. Science 272, 1328−1331. (10) Schumacher, M. A., Todd, J. L., Rice, A. E., Tanner, K. G., and Denu, J. M. (2002) Structural basis for the recognition of a bisphosphorylated MAP kinase peptide. Biochemistry 41, 3009−3017. (11) Todd, J. L., Rigas, J. D., Rafty, L. A., and Denu, J. M. (2002) Dual-specificity protein tyrosine phosphatase VHR down-regulates cJun N-terminal kinase (JNK). Oncogene 21, 2573−2583. (12) Hoyt, R., Zhu, W., Cerignoli, F., Alonso, A., Mustelin, T., and David, M. (2007) Cutting edge: Selective tyrosine dephosphorylation of interferon-activated nuclear STAT5 by the VHR. J. Immunol. 179, 3402−3406. (13) Wang, J.-Y., Yeh, C.-L., Chou, H.-C., Yang, C.-H., Fu, Y.-N., Chen, Y.-T., Cheng, H.-W., Huang, C.-Y. F., Liu, H.-P., Huang, S.-F., and Chen, Y.-R. (2011) Vaccinia H1-related Phosphatase is a phosphatase of ErbB receptors and is down-regulated in non-small cell lung cancer. J. Biol. Chem. 286, 10177−10184. 1458
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(34) Miller, J. H., and Albertini, A. M. (1983) Effects of surrounding sequence on the suppression of nonsense codons. J. Mol. Biol. 164, 59−71. (35) Denu, J. M., and Tanner, K. G. (1998) Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: Evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 37, 5633−5642. (36) Lokareddy, R. K., Bhardwaj, A., and Cingolani, G. (2013) Atomic structure of dual-specificity phosphatase 26, a novel p53 phosphatase. Biochemistry 52, 938−948.
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Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR’s Phosphatase Activity Karolina Pavic,† Pablo Rios,† Kristina Dzeyk,‡ Christine Koehler,§ Edward A. Lemke,§ and Maja Köhn*,† †
Genome Biology Unit, ‡Proteomics Core Facility and §Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany S Supporting Information *
ABSTRACT: Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR’s biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique “variable insert” region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.
Protein tyrosine phosphorylation is a crucial process in cellular signaling. Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of the phosphate from the substrate. Classical PTPs dephosphorylate preferentially phosphotyrosine residues. Dualspecificity phosphatases (DSPs or DUSPs) constitute a heterogeneous group within the superfamily of PTPs. DSPs show diverse substrate specificities ranging from phosphotyrosine and phosphoserine/threonine residues to nonpeptidic substrates such as phosphoinositides.1,2 The classical PTPs and DSPs share a similar catalytic mechanism which involves the catalytically active cysteine of the consensus active site (“Ploop”) signature motif HC(X)5R(S/T) (single letter amino acid code), and, in most cases, a general acid/base aspartate in the so-called WPD loop named after the conserved amino acids of this loop.3 Vaccinia H1-related (VHR) phosphatase (also known as DUSP3) is a constitutively expressed 20.5 kDa phosphatase with predominantly nuclear localization.4 The level of VHR was found to fluctuate during cell cycle progression, and downregulation of VHR by RNA interference showed that cells lacking VHR exhibit signs of senescence.5 VHR was found to be overexpressed in prostate cancer6 and in several cervix cancer cell lines, and it was postulated that the increased protein level of VHR in cervix cancer cells would enable the cells to proliferate.7 The active conformation of VHR is defined by the low pKa (5.52) of the active site cysteine (Cys124),8 and VHR exhibits a preference for dephosphorylating phosphotyrosine.9,10 VHR dephosphorylates the Extracellular signal regulated kinases (Erk) 1/2 and c-Jun N-terminal kinase (JNK) in vivo.4,11 In addition, signal transducer and activator of © 2014 American Chemical Society
transcription (STAT) 5 and the receptor tyrosine kinase ErbB2 have also been reported to be dephosphorylated by VHR.12,13 There are only few reports addressing the regulatory principles of VHR. Phosphorylation of Y138 of VHR by ZAP-70 tyrosine kinase is required for down-regulation of the Erk1/2-Elk pathway in activated T-cells14 and STAT5 dephosphorylation following cytokine stimulation. Moreover, VHR Y138 phosphorylation by tyrosine kinase (Tyk) 2 was observed only in the catalytically deficient forms of VHR.12 Lastly, Vacciniarelated kinase (VRK) 3 was linked to the enhancement of VHR-mediated down-regulation of Erk-activity.15 Although sharing substrates with mitogen-activated protein kinase phosphatases (MKPs), VHR is smaller in size, and it lacks the N-terminal MAPK-binding (MKB) domain and additional adapter/regulatory domains that may regulate substrate specificity.16 The absence of regulatory domains in this small protein makes the prediction of regulatory protein−protein interactions difficult, and oligomerization as potential regulatory mechanism has not yet been reported for VHR. A unique structural region in VHR, which could potentially be involved in specific interactions, resides between the chains β3−β7 and is termed “variable insert”.9,17,18 When compared to structures of homologous PTPs, Phe68 and Met69 of the protruding β3−α4 loop in VHR cannot be aligned in the structure-based sequence alignment.17,18 With the exception of Met69, which Received: April 1, 2014 Accepted: May 5, 2014 Published: May 5, 2014 1451
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Figure 1. Selected residues in VHR. (a) Overall crystal structure of VHR (PDB entry number 1VHR) presented as a cartoon (gray). The “variable insert” region in VHR (residues 61−83) is pointed out in orange. Phe68 is colored green, Asp92 red, Cys124 cyan, and Met69 violet. (b) Surface representation of the zoomed-in active site and Phe68 Met69 of the “variable insert” of VHR. The structure is left-tilted with respect to the one in part a to make the Cys124 visible in this representation. The residues are colored as indicated in part a. (c) Chemical structure of pBPA.
was also generated (amino acids 1−67 of VHR), although to a much lesser extent than the full length variant. This product remained in the purified samples due to the N-terminal Histagging. As this truncated product contained neither the pBPA nor the active site, we did not expect it to interfere negatively with the following studies (see below). After removal of the His-tag, all recombinant proteins were left N-terminally Flag3tagged, which was required for some of the following experiments. Characterization of VHR Variants. To test whether introducing pBPA at the position of Phe68 affected the secondary structure, we analyzed it by far UV−circular dichroism spectroscopy (CD).25 When comparing the spectrum acquired for the F68pBPA variant with the spectrum obtained for the wild type VHR (wtVHR), we observed only minor modifications, suggesting that structural perturbations were imposed neither by pBPA in this VHR variant nor by its truncated form (Supporting Information, Figure S2). Next, we sought to determine kinetic constants of the enzymes by examining their catalytic efficiency in dephosphorylating pNPP (Supporting Information, Table S2). In addition to the pBPAcontaining variant, we included a systematic mutational analysis of Phe68 and a M69A mutation in order to elucidate a possible functional impact of the region bearing Phe68 and Met69 on catalytic activity. The kinetic constants that we obtained for wtVHR were similar to those noted previously.26,27 For all VHR variants generated by site-directed replacement at position 68 with Ala, Asp, Trp, Cys, or pBPA, turnover numbers were within 1.6 fold of the value obtained for the wtVHR. KM values were not substantially affected. We concluded that the overall catalytic performance of these variants was not altered much. With respect to the pBPA variant, this meant that neither the incorporation of pBPA nor the minor amount of truncated form present in the sample had an effect on the catalytic performance. In contrast, M69A exhibited a stronger shift in the values of KM and kcat/KM compared to the wtVHR. Since Met69 anchors Arg130 of the active site cleft,9 we reasoned that substitution with a residue that lacks hydrogen bonding capacity could disrupt the proper geometry of the active site cleft. Thus, this residue outlining the
anchors Arg130 of the active site cleft, the function of the other residues of the unique “variable insert” region is still unknown.9 To study if a potential role of the distinct Phe68 and Met69 containing patch of the “variable insert” region could be to mediate specific protein−protein interactions, we exploited here the nonsense suppression technology utilizing the amber STOP codon (nucleotides TAG) for site-directed incorporation of the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA).19,20 This approach was previously shown to be effective in capturing not only stably associated protein interactions by covalent cross-linking, but also weak, transient, pH- or subcellular localization-dependent complexes.21−23 pBPA is reversibly photoexcited upon exposure to 345−365 nm light, minimizing photodamage to other biomolecules, while providing a covalent snapshot of interactions.24 Using this approach and complementary methods, we demonstrate that VHR dimerizes in vitro and within cells, and that the “variable insert” region is involved in this specific VHR self-association. We also show that the dimeric association negatively affects the enzymatic activity against para-nitrophenylphosphate (pNPP) and, physiologically more relevant, Erk1/2. Thus, our findings indicate that self-association is a mechanism for the regulation of VHR phosphatase.
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RESULTS AND DISCUSSION Incorporation of pBPA into VHR by Amber Suppression in E. coli. In the VHR protein sequence, Phe68 of the “variable insert” region, which contains residues 61−83, was selected to be replaced by pBPA (Figure 1). Phe68 was chosen (i) because of its localization in the loop region of the “variable insert”, which might minimize a potential disruptive effect that the substitution to pBPA could have on the structural integrity, (ii) because of its close proximity to the active site (Figure 1b), which could imply an influence on catalytic activity, and (iii) because it and its neighboring residue Met69 protrude out of the structure and could thus be involved in potential protein− protein interactions. The VHR F68pBPA variant exhibited good incorporation efficiency and its identity was confirmed using mass spectrometry (MS) (Supporting Information, Table S1 and Figure S1). The truncated variant with the protein translation aborted at the position of the amber STOP codon 1452
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Figure 2. Photoinduced cross-linking of purified recombinant VHR. Dimer formation of the Flag3-tagged F68pBPA VHR upon exposure to UV light. The Flag3-wtVHR, with or without UV exposure, and its F68pBPA variant without UV exposure, were used as negative controls. Bands: Monomeric full length proteins ≈ 24 kDa; covalently cross-linked dimers of the Flag3-F68pBPA variant ≈ 50 kDa; Flag3-VHR 1−67 ≈ 14 kDa; cross-linked monomeric form to Flag3-VHR 1−67 ≈ 40 kDa. The analyses were done by Western blotting using an anti-VHR antibody. The results are representative of two independent experiments.
Figure 3. Detection of VHR self-association by chemical cross-linking. (a) Evaluating the intrinsic ability of VHR to self-associate by examining the oligomeric state of the purified recombinant Flag3-tagged wtVHR and its C124S and F68pBPA variants after cross-linking with glutaraldehyde. The control samples were incubated without glutaraldehyde. (b) Cross-linking of Flag3-tagged wtVHR with DSS. The control sample was treated without the presence of DSS. (c) Profiling the effect of the structural and electrostatic properties of the residues incorporated at position 68 on the ability of VHR to self-associate, by cross-linking with glutaraldehyde. For panels a−c, the cross-linked species were detected by Western blotting using an antiVHR antibody. The results reflect the minimum of two independent experiments.
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propensity of wtVHR using complementary methods to the genetic encoding of pBPA. To this end, we applied the crosslinking reagents glutaraldehyde29 and disuccinimidyl suberate (DSS).30 The control samples that were treated without a cross-linker regularly exhibited no dimer formation. The monomeric forms of Flag3-tagged wtVHR and F68pBPA VHR ran as a band at apparent molecular weight (MW) of approximately 20 kDa (Figure 3a). For both proteins, a second population at about 40 kDa clearly corresponded to the size of the dimer. The faint band at about 30 kDa, present only in the glutaraldehyde-treated sample of the Flag3-tagged F68pBPA variant, matched the MW of the cross-linked complex between the full length and the truncated form. These results confirm the self-association of VHR, including the involvement of the sequence of the truncated product (VHR1−67) in the interaction. Furthermore, they also demonstrate that the truncated product does not have an effect on the homodimerization of the full length VHR, as wtVHR, not containing a truncated form, also dimerizes. We also observed faint bands of higher order oligomers (Figure 3a), which could be a result of the applied method, as we did not detect these when using the site-selective pBPA-mediated cross-linking. Moreover, VHR dimerization was independent of the availability of the active site cysteine because incubation with glutaraldehyde also led to the detection of dimers and higher order oligomers for the C124S variant of VHR, in which the catalytically active cysteine was replaced by serine. The self-association of VHR was crossconfirmed using DSS, showing an increase in the cross-linking yield concomitant with the increase of the protein concentration (Figure 3b), demonstrating once more the potential of VHR to self-associate. To investigate the impact of steric/electrostatic features of the amino acid at position 68 on the formation of homodimers, we next profiled the series of F68-variants. After treating the samples with glutaraldehyde, we observed that all F68-variants were able to form dimers and higher order oligomers (Figure 3c). The abundance of the detected oligomers in case of the F68W and F68D variants did not differ much to that observed with wtVHR, indicating that electrostatic features and more bulk do not play a significant role here. Exchanging the hydrophobic aromatic residue in the native protein (Phe) for a small nonpolar one (Ala) led to a stronger self-association (Figure 3c) than was observed with the wtVHR and the other variants (Figure 3a and c) except F68C and F68pBPA, possibly by enabling closer association of the monomers. Coomassie staining of the resolved samples on an SDS gel (Supporting Information, Figure S4) showed that equal amounts of VHR variants were used in the cross-linking experiments (Figure 3a and c). The F68C variant demonstrated cross-linking efficiency similar to what we observed for the F68A variant, which was stronger than that of the wtVHR (Figure 3a and c). Disulfide formation is recognized as a mechanism of protein selfassociation31 and could enhance the dimerization also in this case. In addition to the UV-induced cross-linking of the full length F68pBPA to the fragment containing Flag3-VHR 1−67, this finding supports that the “variable insert” is involved in the self-association of VHR. To verify this, we investigated in more detail if the F68C mutation renders the protein more susceptible to intermolecular disulfide bond formation. We checked the oligomerization state of the variants by resolving the samples, dialyzed with or without the reducing agent βmercaptoethanol, by SDS-PAGE under nondenaturing or
rim of the substrate-binding cleft (Figure 1b) could be indirectly engaged in promoting efficient turnover. F68pBPA VHR Dimerizes upon UV Exposure In Vitro. In order to determine if the “variable insert” region of VHR could be involved in homomeric protein−protein interactions, we exposed the Flag3-pBPA variant of VHR in buffer to UV light. We indeed detected a band on the Western blot, which in size corresponded to the homodimer (48 kDa). Analogous experiments without UV exposure showed no dimer formation (Figure 2). Also, the truncated fragment that did not contain pBPA (Flag3-VHR 1−67, band around 14 kDa) did not show any homodimerization, irrespective of UV irradiation. However, the full length Flag3-F68pBPA VHR was found to cross-link to the truncated product (together 35 kDa). This led us to hypothesize that the region of the protein corresponding to the truncated product (which also contains residues 61−67 of the flexible loop of the “variable insert”) bears sufficient sequential requirements to drive intermolecular association between VHR monomers. Since photo-cross-linking using pBPA was previously shown to enable the detection of weak and transient protein interactions,21−23 we investigated in the following the potential of VHR to self-associate. To this end, we evaluated the dependence of the dimer formation on the protein concentration. Purified recombinant Flag3-tagged F68pBPA and wtVHR were exposed to UV light for 30 min, spanning protein concentrations from 50 to 500 μg/mL. Subsequent Western blot analysis detected bands corresponding to the cross-linked complexes of the F68pBPA variant already at 50 μg/mL, with increase in detected complexes at higher protein concentrations demonstrating the concentration-dependence of the interaction (Figure 2). Finally, we sought to prove covalent dimerization by MS. After UV-irradiation of F68pBPA, we detected in the SDS gel of the sample the monomeric fraction of full length VHR, traces of the truncated form, the covalently cross-linked homodimer and, to a much lesser extent, the cross-linked heterodimer of full length and truncated version (Figure 2). To increase the likelihood for detecting the full mass of the dimer by MS, we attempted to isolate the covalent homodimer by size exclusion chromatography. Only a small amount of the monomeric fraction (M) could be isolated (Supporting Information, Figure S3a). We were not able to obtain the pure fraction of the covalent homodimer. Nevertheless, the mass corresponding to the covalent dimer was identified by LC-MS/MS analysis in the mixture of the four components enriched in the covalent homodimer (D) (Supporting Information, Figure S3a and b). In this enriched mixture (D), the mass of the cross-linked heterodimer of full length and truncated version was also detected (Supporting Information, Figure S3b). Furthermore, LC-MS/MS analysis of the band corresponding to the monomeric fraction (M) identified intramolecular cross-linking (Supporting Information, Methods). When the monomeric sample (M) isolated by size exclusion was resubjected to UV light, no further dimers were formed (Supporting Information, Figure S3c). These results indicated that UV-exposure of F68pBPA triggers dimer formation, while simultaneously attenuating cross-linking efficiency due to intramolecular cross-linking, explaining the limitations in the yield of dimer formation (Figure 2). Photoinduced destruction of pBPA or side reactions with buffer components could also be also potential causes for the observed limited yield.28 Confirmation of the VHR Dimerization by Alternative Methods. We next sought to confirm the dimerization 1454
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Figure 4. Self-association of VHR in COS-1 cells. (a) COS-1 cells were transiently cotransfected with myc- and Flag3-tagged wtVHR. The analysis was conducted 24 h post-transfection. The cells were stimulated with 10 mM H2O2 for 1 h at 37 °C prior to lysis. The lysate was immunoprecipitated using antibody against the Flag-tag and immunoblots were probed by using an antimyc antibody. Afterward, the membrane was stripped and reblotted using an anti-Flag antibody. (b) COS-1 cells were transfected with pCMV/Flag3-wtVHR and probed for dimeric association inside the cell. Twenty-four hours post-transfection, the cells were treated with 0.8% (w/v) PFA in PBS for 150 min and then lysed. The control samples were incubated in growth medium only. The samples were heated at 60 °C for 20 min or at 95 °C for 5 min. The pellets (P) that remained from clearing the lysates were resuspended and incubated at 95 °C for 5 min. The samples were resolved by 4−12% SDS-PAGE and probed with an anti-Flag antibody. The images in panels a and b represent the results of two independent experiments.
denaturing conditions (Supporting Information, Figure S5). For wtVHR, we observed traces of dimers under nondenaturing compared to denaturing conditions, and no difference was observed in the extent of dimer formation with respect to βmercaptoethanol treatment. The F68C variant showed a strongly increased potential for self-association compared to the wtVHR under nondenaturing conditions. In general, under denaturing SDS-PAGE conditions hardly any homodimers could be detected, suggesting together that the self-association is reversible and that, under the in vitro conditions, the interaction is rather weak and can be destroyed during gel electrophoresis if not stabilized by Cys68-disulfide formation. Thus, these observations demonstrate that the Cys68 is involved in the self-association. Strikingly, the F68pBPA variant exhibited a pattern more resembling that of the F68C variant than that of the wtVHR (Figure 3a and c). The “variable insert” contains a small hydrophobic and protruding patch of nonconserved amino acid composition.9 Phe68 is a part of this island. Exchanging Phe68 for the more hydrophobic pBPA is likely to further increase the extent of hydrophobic interactions, which could further stabilize the self-association of VHR, again confirming that the position 68 of VHR is important for the self-association. In addition, since the catalytic cysteine was excluded from being involved in the dimer formation (Figure 3a), and the weak dimer formation of wtVHR under nondenaturing electrophoretic conditions was not reduced by β-mercaptoethanol treatment (Supporting
Information, Figure S5), our results indicate that the dimer formation is likely based on structural factors and involves noncovalent interactions. VHR Forms Dimers in Cells. To investigate homodimeric association of VHR in cells, COS-1 cells were cotransfected with myc- and Flag3-tagged VHR constructs. We intended to demonstrate coprecipitation of the two differently tagged VHR variants but, under nonstimulated conditions, were not able to observe cosedimentation. Because previous reports showed that dimerization of PTPs can be enhanced by oxidative conditions,32 we pretreated the cells with 10 mM H2O2. Under these conditions, we indeed observed the self-association of VHR (Figure 4a and Supporting Information, Figure S6a). Repetition of this experiment in vitro confirmed that H2O2 treatment stabilizes the VHR dimers (Supporting Information, Figure S6b), suggesting a direct effect of oxidative species on VHR dimerization but not excluding that other indirect effectors inside cells could also play a role. We also applied paraformaldehyde (PFA) at 0.8% in PBS (w/v) in Flag3-tagged VHR overexpressing COS-1 cells to probe for VHR dimerization in intact cells, in the absence of H2O2. We captured the VHR self-association, and the cross-link was irreversible as shown by denaturing the samples through heating (Figure 4b). The untreated control cells showed no complexes of higher molecular masses. Capturing two VHR proteins covalently inside cells is a persuasive indication of their spatial proximity.33 The dimers, nevertheless, need to be 1455
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Figure 5. Functional implications of VHR dimerization. (a) The proteins were subjected to 345 nm light (1000 W) for 30 min at 250 μg/mL concentration, followed by dilution to 500 nM before subjecting to the activity assay with pNPP at 10 mM. F68pBPA Monomer (M) = isolated by size-exclusion after UV-cross-linking (Supporting Information, Figure S3a). The nonirradiated wtVHR was used as a control. A discrete time-point (30 min) is represented as percentage of activity with respect to the wtVHR and corresponds to the average of at least two experiments with triplicate measurements. See Supporting Information, Figure S7, for the kinetic profiles. The GraphPad 5.0 software (GraphPad, San Diego, CA, U.S.A.) was used to plot the data as mean ± SEM and to perform the statistical analysis. A Student’s unpaired t test was used to calculate the statistical significance and the asterisks indicate ***p < 0.001. (b) Dephosphorylation of Erk1/2 in COS-1 cell extracts by the wtVHR and its variants, with or without exposure to 345 nm light. Lysates were incubated with 500 nM of recombinant VHR, or an equal volume of buffer, for 0, 30, and 90 min at 30 °C. Time-dependent reduction of pErk1/2 in COS-1 extracts was analyzed with an anti-pErk1/2 antibody. An anti-Erk1/2 and an anti-VHR antibodies were used to assess equivalent amounts of lysates and bait used, respectively. The result is representative of three independent experiments.
on VHR’s enzymatic activity, we first subjected the wtVHR and the F68pBPA variant to UV light, followed by activity measurement employing pNPP. We observed that the catalytic activity of the wtVHR was not affected by UV-exposure. In addition, the activity of the UV-irradiated F68pBPA was about 60% lower than that of the nonirradiated sample, which equaled the activity of the wtVHR (Figure 5a). These results could indicate that (i) pBPA-mediated cross-linking randomly lowers the activity or (ii) the topology of the dimeric association might curtail efficient catalytic performance, potentially by providing
stabilized to enable their detection. Stabilization by covalent cross-linking showed that VHR is present as dimers inside cells. The fact that oxidative conditions enabled the detection without requiring a chemical cross-linker suggests a strengthening of the interaction as response to this stimulus. Together, these results indicate that VHR exists inside cells in an equilibrium of monomers and dimers, which is shifted in response to oxidative stimulation. Assessment of Functional Consequences of VHR Dimerization. To assess the effect of the dimer formation 1456
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Conclusions. Taken together, the results demonstrate that VHR dimerizes through Phe68 and Met69 of the “variable insert” region, and indicate that this could be a novel regulatory mechanism for VHR activity. In a nonstimulated state VHR might be present as a monomer or loosely packed dimer as indicated by cross-linking inside cells using PFA (Figure 4b), with the overall catalytic activity dependent on the mutual orientation of the self-associating units. Under stimulated conditions, such as oxidative stress as presented here, the dimerization of VHR appears to be stabilized. While the active site cysteine was observed to not be essential for dimerization, it cannot be excluded at this point that other cysteines in VHR may be involved in the mechanism of dimerization, although none appears to be structurally very close to the observed dimerization interface.9 The intermolecular self-association of VHR might impose steric hindrance to the active site cleft, therefore rendering it inaccessible to substrates. Oligomer formation, also in response to H2O2, has been recognized as a regulatory principle within the PTP superfamily, serving as an important determinant of substrate specificity and catalytic activity.31,32 These reported mechanisms render our hypothesis of oxidative stress-induced VHR dimerization feasible. It is known that the active site cysteine residue of VHR is sensitive to H2O2-induced oxidative inhibition.35 However, VHR showed a lack of excessive, irreversible oxidation observed for other PTPs, and the authors speculated that this might be due to the active site Cys124 being protected from solvent exposure. Dimerization could therefore also provide a mechanism to protect the catalytic cysteine from irreversible oxidation. Selfassociation of VHR would also result in a high local concentration of the enzymatically active form after release of the stimulus, which could act rapidly to elicit a specific cellular response after the dimeric complex is disassembled into monomers. Finally, there is an interest to discover VHR inhibitors due to its role in different cancers.6,7 In this regard, stabilization of VHR dimerization could be a new means to interfere with VHR activity. Our findings were enabled by selective incorporation of the photo-cross-linkable pBPA. This strategy allowed us to investigate how dimerization affects the enzymatic activity of VHR, whose self-association is not stable enough to purify the dimer and assay its activity. When applying traditional crosslinkers, the random and multiple cross-linking exerted by them would usually prevent such a study. Based on the work presented here, it is reasonable to assume that the selective incorporation of photo-cross-linkable amino acids will be a useful and feasible tool for investigating interactions of PTPs in general, and for those in particular where dimerization was suggested by one experiment but could not be confirmed by another, such as for DUSP26.36
sterical hindrance to the active site pocket. As the UV-irradiated F68pBPA sample was a mixture containing the monomeric forms (full length and truncated form), the cross-linked dimer of the full length and the truncated version (length 1−67 of VHR) and the homodimer (Figure 2), the reduced activity is exerted by the combination of these species. However, as stated above, the pure photo-cross-linked F68pBPA dimer could not be obtained by size exclusion chromatography and was therefore not available for activity assays. Other chemical cross-linkers bind randomly, including potential binding to the active site, and can consequently not be applied for activity studies. Therefore, to investigate the contribution of different species, the monomeric fraction of the UV-irradiated F68pBPA variant, isolated through size exclusion chromatography, was tested for its activity and showed to be about 40% less active compared to the wtVHR (Figure 5a). This indicated that intramolecular cross-linking, which we had observed by MS, affected catalytic efficiency, possibly by limiting access to the active site or by disrupting the most optimal configuration of catalytically important residues. Moreover, the reduction in activity of the UV-irradiated F68pBPA previous to size exclusion chromatography can therefore be in part attributed to intramolecular cross-linking. Nevertheless, this sample showed even further reduced activity (Figure 5a and Supporting Information, Figure S7), suggesting that the selfassociated VHR also has reduced activity. In order to obtain a more physiologically relevant model, we assessed levels of dephosphorylated Erk1/2 in COS-1 cell extracts by adding the recombinant proteins. The wtVHR and the F68pBPA variant were pretreated as described above. In the buffer control samples no significant dephosphorylation of Erk1/2 was observed over 90 min (Figure 5b). The wtVHR samples showed a strong reduction in the pErk1/2 level after 90 min irrespective of UV exposure, which is in accord with the experiments using pNPP as substrate (Figure 5a and Supporting Information, Figure S7). The level of pErk1/2 in the sample incubated with irradiated F68pBPA was almost at the level of the buffer control after 90 min, which is in stark contrast to the nonirradiated sample. Taken together, the results from two activity assays indicate that dimerization is likely a negative regulator of VHR enzymatic activity. VHR Dimerization Occludes the Active Site. To investigate further if dimerization could lead to occlusion of the active site, we mutated the catalytically active Asp92 to pBPA. This residue is part of the WPD-loop of VHR. It is in close proximity to both the active site and Phe68 (Figure 1b) and could thus also be able to cross-link with another VHR molecule if the active site were occluded by the dimerization. Like the F68pBPA mutant, the D92pBPA variant showed good incorporation efficiency (Supporting Information, Figure S8a and b). We detected less truncated product (containing Flag3VHR 1−91) for the D92pBPA than for the F68pBPA variant, which can be explained by the dependence of the efficiency of the amber codon suppression on its sequence context.34 When testing its catalytic activity, we found about 25-fold reduction in the kcat of VHR D92pBPA compared to the wtVHR (Supporting Information, Figure S8c and Table S2), which was expected due to the involvement of D92 in the catalytic mechanism.27 Finally, we also observed the formation of a D92pBPA-dimer after UV-irradiation (Supporting Information, Figure S8d), indicating that the active site could indeed be occluded by the dimerization.
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METHODS
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ASSOCIATED CONTENT
Materials and methods are described in detail in Supporting Information.
S Supporting Information *
Materials and methods section. Additional tables and figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 1457
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AUTHOR INFORMATION
Corresponding Author
*Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS K.P. and M.K. thank the German Science Foundation (DFG) for support within the Emmy Noether program (KO 4013/11). E.A.L. acknowledges funding from the Emmy Noether program of the DFG. We thank S. Keyse for discussions and suggestions. We thank the Protein Expression and Purification Core Facility and the Proteomics Core Facility at EMBL for support.
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