Apoptin is a nucleocytoplasmic shuttling protein.
The ability of the CAV protein Apoptin to localize to either the nucleus or the cytoplasm depending on cell type suggested that the protein may undergo nucleocytoplasmic shuttling. To determine whether Apoptin shuttles in primary cells, we transiently transfected PFFs with a plasmid expressing Apoptin fused to the C terminus of GFP (GFP-Apwt) and treated the cells with LMB, a compound that specifically blocks Crm1-mediated nuclear export (25
). If Apoptin shuttles in primary cells, then blocking nuclear export should result in the accumulation of Apoptin in the nucleus. Figure shows that LMB treatment resulted in nuclear accumulation of GFP-Apwt in PFFs. To verify this result, PFFs were infected with an adenovirus expressing Flag-tagged Apoptin (42
) and treated in the presence or absence of LMB. Figure shows, as expected, that LMB treatment resulted in nuclear accumulation of Flag-Apwt. Consistent with our previous studies (42
), Flag-tagged Apoptin exhibited a filamentous staining pattern, which was stable after LMB treatment.
FIG. 1. Apoptin shuttles between the nucleus and cytoplasm in a Crm1-dependent manner. (A) PFF cells expressing GFP-Apwt were treated in the presence or absence of LMB. (Top) 3 h later, Apoptin localization was visualized by fluorescence microscopy for GFP, and (more ...)
To test whether Apoptin also shuttles in transformed cells, we developed an assay to monitor the nucleocytoplasmic shuttling of a predominantly nuclear protein. In non-small-cell-lung-carcinoma H1299 cells, we expressed a dominant-negative Ran GTPase mutant (dnRan), which blocks Ran-dependent nuclear import (21
). To validate this approach, we performed a control experiment monitoring the effect of dnRan on localization of HIV Rev, a well-characterized nucleocytoplasmic shuttling protein (20
). Figure shows, as expected, that a dsRed-dnRan fusion-protein localized to the nuclear periphery (13
) and that a Rev-GFP fusion-protein accumulated in the nucleolus (45
). Coexpression of dsRed-dnRan and Rev-GFP resulted in the loss of nuclear GFP signal, confirming that Rev-GFP exited the nucleus and was blocked for subsequent reentry. Similarly, expression of dsRed-dnRan resulted in the loss of nuclear GFP-Apwt, indicating that Apoptin shuttled in transformed cells. Collectively, the data of Fig. indicate that Apoptin localization is regulated by nucleocytoplasmic shuttling in both primary and transformed cells and that nuclear import and export are mediated by the common nuclear trafficking machinery.
Apoptin nuclear localization is mediated by a dominant transformed cell-specific activity.
To determine whether the transformed or primary cell contained a dominant Apoptin localization activity, we performed a heterokaryon experiment. PFFs were transiently transfected with a plasmid expressing GFP-Apwt, and H1299 cells were transiently transfected with a plasmid expressing Apoptin fused to the C terminus of dsRed (dsRed-Apwt). Twelve hours later, the cells were combined, fused with polyethylene glycol, and allowed to recover overnight. Figure shows that when either PFF/GFP-Apwt or H1299/dsRed-Apwt cells were self-fused, the expected localization patterns were observed. However, fusion of PFF/GFP-Apwt cells with H1299/dsRed-Apwt cells resulted in the translocation of PFF-derived GFP-Apwt to the nucleus where it colocalized with dsRed-Apwt. These results indicate that transformed cells contain a dominant activity that confers Apoptin nuclear localization.
We also monitored localization of Apoptin in HA-1 cells, a clonal population of human embryonic kidney cells transformed with SV40 large T antigen that bypass senescence and enter crisis (6
). HA-1 cells were infected with Ad-Apwt and Apoptin localization was monitored both before (passage 44) and after (passage 94) crisis. Figure shows that Apoptin exhibited a filamentous, cytoskeleton-like cytoplasmic staining pattern in precrisis cells, whereas in postcrisis cells Apoptin localized predominantly to the nucleus. Thus, the dominant activity in transformed cells that directs Apoptin to the nucleus appears early during the transformation process.
Biochemical properties of Apoptin differ between primary and transformed cells.
We and others have previously reported that Apoptin forms large cytoplasmic aggregates in primary cells (7
). We hypothesized that this aggregation may reflect a fundamental difference in the biochemical state of Apoptin in primary versus transformed cells. The immunoprecipitation experiment of Fig. shows that Apoptin was highly resistant to extraction with a low ionic strength buffer from PFFs compared to H1299 cells. In fact, in primary cells the majority of Apoptin was not solubilized even under high-ionic-strength-conditions (data not shown). Figure shows that Apoptin derived from precrisis HA-1 cells was also highly resistant to extraction compared to postcrisis cells in which Apoptin was readily immunoprecipitated. These results suggest that in transformed cells, nuclear translocation of Apoptin coincides with a large increase in solubility of the protein. The filamentous, cytoplasmic staining pattern observed in primary cells (see Fig. and references 7
) suggests that Apoptin may be tightly associated with cellular filament networks, which could explain the difference in solubility between primary and transformed cells.
Functional characterization of Apoptin NES and NLS sequences.
We next sought to characterize the specific sequence elements that contribute to nucleocytoplasmic shuttling. Previous studies have noted a putative canonical NES comprising amino acids 37 to 46 in the N terminus and a putative NLS comprising residues 70 to 121 in the C terminus (shown in Fig. ) (9
). To determine whether residues 37 to 46 are in fact a functional NES, we constructed a mutant in which the core residues leucine-44 and leucine-46 were mutated to alanine (GFP-Ap-pmNES). Figure shows that in PFFs GFP-Ap-pmNES mislocalized to the nucleus, indicating that the putative NES is functional and that NES-dependent transport of Apoptin is required for cytoplasmic accumulation in primary cells. The putative bipartite NLS contains two domains highly enriched in lysine and arginine (residues 86 to 88 and residues 116 to 118). We constructed an NLS mutant in which both trios of basic amino acids were mutated to alanine (Ap-pmNLS). This mutant mislocalized to the cytoplasm in H1299 cells, indicating the NLS is required for nuclear localization. Thus, Apoptin contains both a functional NES and NLS, a finding consistent with the nucleocytoplasmic shuttling activity described above.
FIG. 2. Apoptin contains a functional NES and NLS. (A) (Top) Schematic diagrams of N-terminal GFP-tagged Apoptin NES and NLS mutants. (Middle) H1299 and PFF cells expressing GFP-Apoptin mutants were monitored by fluorescence microscopy for GFP (left) and DAPI (more ...)
A recent study has suggested that the Apoptin N-terminal region lacks an NES and that nuclear export is mediated by a noncanonical, Crm1-dependent NES located near the C terminus of the protein (34
). To confirm our conclusion that the Apoptin N-terminal region harbors a functional NES, we sought to determine whether the N terminus was sufficient to mediate nuclear export in a Crm1-dependent manner. H1299 cells were transfected with two GFP-fused Apoptin fragments, GFP-Ap(1-48) or GFP-Ap(1-88), both of which contain the N-terminal NES but lack the C-terminal NLS (see Fig. ) and putative C-terminal NES (34
). Figure shows that both derivatives displayed a predominantly cytoplasmic localization pattern in H1299 cells, indicating the N-terminal NES is sufficient to confer nuclear export. Moreover, treatment with LMB resulted in an increased GFP signal in the nucleus, indicating nuclear export was Crm1 dependent. These results confirm our conclusion that the Apoptin N-terminal region contains a functional, Crm1-dependent NES.
Apoptin fragments containing either the NES or NLS fail to undergo cell type-specific localization.
The simplest explanation for the differential localization of Apoptin in primary and transformed cells is that one of the localization signals is subject to cell type-specific regulation. For example, the NLS might be active in transformed cells and inactive in primary cells. To test this model, we analyzed the localization of GFP-fused Apoptin fragments containing either the NLS or NES in primary and transformed cells. Figure shows that GFP-Ap(42-88), which lacks both the NLS and the NES, displayed a diffuse homogeneous localization pattern in both H1299 and PFF cells similar to that of GFP alone. As expected, the GFP-Ap(1-48) and GFP-Ap(1-88) mutants, which contain the N-terminal NES but lack the C-terminal NLS, displayed a predominantly cytoplasmic localization pattern in PFFs and, as shown above, in H1299 cells, indicating the NES is active in transformed cells. Conversely, the GFP-Ap(42-121) and GFP-Ap(82-121) mutants, which contain the C-terminal NLS but lack the N-terminal NES, localized to the nucleus in H1299 cells, as well as in PFFs, indicating the NLS is active in primary cells. Thus, the NLS and NES are active in both primary and transformed cells and, when uncoupled, both localization signals function constitutively.
FIG. 3. Apoptin fragments containing either the NES or the NLS fail to undergo cell type-specific localization. (Top) Schematic diagrams of GFP-Apoptin deletion mutant derivatives. (Middle) Fluorescence microscopy of H1299 and PFFs expressing GFP-Apoptin derivatives. (more ...) trans restoration of Apoptin cell type-specific localization through protein multimerization.
Previous studies have shown that purified, recombinant Apoptin aggregates in vitro (28
), raising the possibility that Apoptin functions as a multimer in vivo. As a first test of this possibility, we sought to determine whether cell type-specific localization could be restored in trans
by coexpression of two Apoptin fragments, one containing the NLS and the other containing the NES. Figure shows, as expected, that GFP-Ap-pmNLS, which lacks a functional NLS, localized to the cytoplasm of both H1299 cells and PFFs, whereas dsRed-Ap-pmNES, which lacks a functional NES, localized to the nucleus in both H1299 cells and PFFs. However, coexpression of GFP-Ap-pmNLS and dsRed-Ap-pmNES resulted in the localization of both proteins to the nucleus of H1299 cells and the cytoplasm of PFFs. These results suggest that Apoptin is a multimer in vivo and confirm that both localization signals are required for proper cell type-specific localization.
FIG. 4. Apoptin localization signals are modular and can cooperate in trans to confer cell type-specific localization. (A) H1299 (left panels) and PFF (right panels) cells expressing GFP-Ap-pmNLS, dsRed-Ap-pmNES, or both were monitored by fluorescence microscopy. (more ...)
To verify that Apoptin is a multimer in vivo and to map the multimerization domain, we performed a series of coimmunoprecipitation experiments. H1299 cells were transfected with a series of GFP-fused Apoptin derivatives and 12 h later infected with Flag-tagged Ad-Apwt. At 24 h after infection, Flag-Apoptin was immunoprecipitated, and the immunoprecipitate was analyzed by immunoblotting with an α-GFP antibody. Figure shows that only derivatives containing the N-terminal third of Apoptin [GFP-Apwt, GFP-Ap(1-48), GFP-Ap(1-88)] coimmunoprecipitated with the Flag-Apoptin, indicating that this region mediates protein multimerization.
To confirm the results of the coimmunoprecipitation experiments and to verify that multimerization is the basis for restoration of cell type-specific localization in trans, we analyzed three additional Apoptin derivatives in the trans-expression assay. Figure shows that expression of dsRed-Apwt or dsRed-Ap-pmNES restored nuclear localization to the constitutively cytoplasmic GFP-Ap(1-88) mutant in transformed cells and maintained cytoplasmic localization in primary cells. In contrast, dsRed-Ap(82-121) failed to alter cytoplasmic localization of GFP-Ap(1-88) in transformed cells, indicating that the N-terminal multimerization domain is required for trans-association of the localization signals. These results show that the activity of a constitutively cytoplasmic mutant can be restored by expressing an NLS-containing C-terminal fragment in trans.
The specific Apoptin NES is required for cell type-specific localization.
We next sought to determine whether the specific sequence of the Apoptin NES and NLS were required for cell type-specific localization by determining whether they could be functionally substituted with heterologous localization signals. Figure shows that replacement of the Apoptin NLS with that of SV40 large T antigen (Ap-SV40NLS) resulted in nuclear localization in H1299 cells and predominantly cytoplasmic localization in PFFs. Thus, the SV40 large T antigen NLS can functionally substitute for the Apoptin NLS. In contrast, replacement of the Apoptin NES with that of Rev (Ap-RevNES), a well-established, prototypical NES (15
), resulted in a similar diffuse localization pattern in both H1299 and PFFs, indicating that the specific sequence of the Apoptin NES is critical for proper cell type-specific localization.
FIG. 5. The specific Apoptin NES is required for cell type-specific localization and overlaps with the multimerization domain. (A) Fluorescence microscopy of H1299 and PFFs expressing GFP-Apoptin derivatives in which either the NLS was replaced by the SV40 NLS (more ...) Apoptin NES and multimerization domains overlap.
One explanation for the failure of the Rev NES to functionally substitute for the Apoptin NES is that the Apoptin NES provides an activity in addition to nuclear export. Because both the NES and the multimerization domain map to the N terminus, we reasoned that the NES might be an essential part of the multimerization domain. To test this possibility, we analyzed the two NES derivatives, Ap-pmNES and Ap-RevNES, for their ability to multimerize in the coimmunoprecipitation assay. Figure shows that, compared to GFP-Apwt, the GFP-Ap-pmNES mutant showed a somewhat reduced ability to interact with Flag-Apoptin. Significantly, GFP-Ap-RevNES, which contains a functional NES that differs at multiple residues from the Apoptin NES, failed to detectably coimmunoprecipitate with Flag-Apoptin. Interestingly, the intermediate level of multimerization observed with the Ap-pmNES mutant was sufficient to confer function in the trans assay (see Fig. ); however, quantification of Fig. revealed that Ap-pmNES was less effective than wild-type Apoptin in mediating trans localization (the nuclear/cytoplasmic ratios were 1.57 and 2.50, respectively), which presumably reflected the decreased multimerization efficiency. Together, these results suggest that the Apoptin multimerization domain overlaps with the NES.
The NLS and nucleocytoplasmic shuttling are required for APC1 interaction and Apoptin-induced cell death.
The experiments described above address the contribution of the NES and NLS to protein localization but do not indicate whether these sequences are required for the ability of Apoptin to induce apoptosis. To investigate the role of the NLS, we constructed a second NLS mutant in which lysine-116, arginine-117, and arginine-118 were mutated to alanine (Ap-pmNLS2; Fig. ). This mutant protein retained partial nuclear localization in H1299 cells (Fig. ). We also analyzed the Ap-SV40NLS mutant, which localized exclusively in the nucleus in H1299 cells (see Fig. ). To determine the ability of these two mutants to induce cell death in transformed cells, GFP-Apwt, GFP-Ap-pmNLS2, and GFP-Ap-SV40NLS were transiently expressed in H1299 cells and, 3 days after transfection, the cells were fixed, stained with DAPI, and analyzed by microscopy for apoptotic morphology. As expected, cells expressing GFP-Apwt underwent pronounced apoptosis, whereas the ability of the Ap-pmNLS2 and Ap-SV40NLS mutants to induce apoptosis was severely reduced (Fig. , top).
FIG. 6. Functionality of both localization signals is critical for APC1 interaction and Apoptin-induced cell death. (A) Schematic diagrams of Apoptin NLS mutations. (B) Fluorescence microscopy of H1299 cells expressing GFP-Ap-pmNLS2. (C) (Top) Apoptosis assays. (more ...)
To understand why the Ap-pmNLS2 and Ap-SV40NLS mutants failed to induce apoptosis in transformed cells even though they localized to the nucleus, we next monitored their ability to interact with the APC1 subunit of the APC/C, which we have previously shown interacts with the C terminus of Apoptin (42
). A triple Flag-tagged version of each mutant was transiently transfected into H1299 cells and immunoprecipitated with an α-Flag antibody, and the immunoprecipitate was analyzed for APC1 by immunoblotting. As expected, APC1 was present in the immunoprecipitate from wild-type Apoptin but not in that of the Ap-pmNLS2 or Ap-SV40NLS mutants (Fig. , bottom) despite the presence of both of these mutants in the nucleus. These observations are consistent with our previous results (42
) and suggest that the NLS sequence overlaps with the domain required for association with APC1. Furthermore, these results indicate that nuclear localization in the absence of APC1 association is not sufficient to induce apoptosis.
To assess the involvement of the NES in Apoptin-induced cell death, we next investigated the ability of the Ap-pmNES mutant, which contains a wild-type C-terminal domain and localizes to the nucleus in transformed and primary cells (see Fig. ), to interact with APC1 and induce apoptosis. Figure shows that despite its nuclear localization, this mutant failed to induce apoptosis in PFFs, a finding consistent with previous studies showing that nuclear localization of Apoptin is not sufficient to induce apoptosis in primary cells (9
). Surprisingly, however, this mutant also failed to induce apoptosis in transformed cells (Fig. , top) and exhibited greatly reduced ability to interact with APC1 (Fig. , bottom). The dnRan assay of Fig. shows, as expected, that the Ap-pmNES mutant failed to undergo nucleocytoplasmic shuttling. Thus, the shuttling activity of Apoptin is required for effective interaction with APC1 and induction of apoptosis.
Apoptin recruits APC/C to PML bodies in transformed cells.
The fact that Apoptin interacts with APC1 (42
) suggested that in transformed cells Apoptin would colocalize with APC/C complex. Because the APC1 antibody was not suitable for immunofluorescence (data not shown), to test this prediction we performed immunofluorescence for Cdc27 (also known as APC3), a core APC/C subunit routinely used as a marker for APC/C localization (17
). H1299 cells were transiently transfected with a plasmid expressing dsRed-Apwt, incubated for 12 h, fixed, and stained with an α-Cdc27 antibody. Figure shows that dsRed-Apwt colocalized with Cdc27 in the nucleus of transformed cells. However, we failed to detect Cdc27 immunofluorescence in the presence of the Ap-pmNLS2 mutant, which, unlike wild-type Apoptin (42
), does not associate with the APC/C and fails to induce apoptosis (Fig. ) and G2
/M arrest (data not shown). This result is consistent with previous reports showing that several APC/C subunits, including Cdc27, are detected only in mitotic and not in interphase cells (16
FIG. 7. Apoptin colocalizes with APC/C within PML bodies in the nuclei of transformed cells. (A and B) H1299 cells were transiently transfected with a plasmid expressing either dsRed-Apwt or dsRed-Ap-pmNLS2 and, after a 12-h incubation, were fixed and stained (more ...)
It has recently been reported that Apoptin associates with PML nuclear bodies (34
). To verify this result, cells were transfected with either dsRed-Apwt or, as a control, dsRed-Ap-pmNLS2 and stained with an α-PML antibody. Figure confirms that wild-type Apoptin colocalized with PML bodies within the nucleus of transformed cells. Significantly, in cells transfected with dsRed-Ap-pmNLS2, PML immunofluorescence was undetectable, a finding consistent with previous studies showing that in H1299 cells PML nuclear body formation occurs only under conditions of apoptotic stimuli (11
). Thus, Apoptin induces the formation of PML nuclear bodies in transformed cells in a manner that requires APC/C interaction.
The results described above indicate that Apoptin colocalizes with the APC/C (Fig. ) and that Apoptin is present in PML bodies (Fig. ). These results strongly suggested that in cells expressing Apoptin, APC/C would be present in PML bodies. To provide direct evidence for this supposition, plasmids expressing either dsRed-Apwt or dsRed-Ap-pmNLS2 were cotransfected into H1299 cells with a plasmid constitutively expressing PML fused to the C terminus of cyan fluorescent protein (CFP-PML). At 12 h after transfection, cells were fixed and stained with an α-Cdc27 antibody. Figure shows that, as expected, dsRed-Apwt, Cdc27 and CFP-PML colocalized in the nucleus of transformed cells. Thus, in the presence of Apoptin, the APC/C becomes sequestered in PML bodies.