PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA
2001; Springer Nature; Volume: 20; Issue: 16 Linguagem: Inglês
10.1093/emboj/20.16.4547
ISSN1460-2075
Autores Tópico(s)Virus-based gene therapy research
ResumoArticle15 August 2001free access PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA Natalie Cohen Natalie Cohen Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Madhulika Sharma Madhulika Sharma Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Alex Kentsis Alex Kentsis Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Jacqueline M. Perez Jacqueline M. Perez Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Stephen Strudwick Stephen Strudwick Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Katherine L.B. Borden Corresponding Author Katherine L.B. Borden Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Natalie Cohen Natalie Cohen Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Madhulika Sharma Madhulika Sharma Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Alex Kentsis Alex Kentsis Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Jacqueline M. Perez Jacqueline M. Perez Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Stephen Strudwick Stephen Strudwick Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Katherine L.B. Borden Corresponding Author Katherine L.B. Borden Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA Search for more papers by this author Author Information Natalie Cohen1, Madhulika Sharma1, Alex Kentsis1, Jacqueline M. Perez1, Stephen Strudwick1 and Katherine L.B. Borden 1 1Structural Biology Program, Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY, 10029 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4547-4559https://doi.org/10.1093/emboj/20.16.4547 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The promyelocytic leukemia protein PML is organized into nuclear bodies which mediate suppression of oncogenic transformation and of growth. The biochemical functions of PML bodies are unknown, despite their involvement in several human disorders. We demonstrate that eukaryotic initiation factor 4E (eIF4E) directly binds the PML RING, a domain required for association with bodies and for suppression of transformation. Nuclear eIF4E functions in nucleocytoplasmic transport of a subset of transcripts including Cyclin D1. Present studies indicate that some PML requires the evolutionarily older eIF4E protein for association with nuclear bodies. Further more, PML RING modulates eIF4E activity by drastically reducing its affinity for its substrate, 5′ m7G cap of mRNA. We demonstrate that eIF4E requires cap binding for transport of Cyclin D1 mRNA and subsequent transformation activity. Additionally, PML reduces the affinity of eIF4E for m7G mRNA cap, causing a reduction in Cyclin D1 protein levels and consequent transformation inhibition. PML is the first factor shown to modulate nuclear eIF4E function. These findings provide the first biochemical framework for understanding the transformation suppression activity of PML. Introduction The promyelocytic leukemia protein (PML) is an important regulator of mammalian cell growth and apoptosis, and when overexpressed it suppresses oncogenic transformation (Melnick and Licht, 1999). These physiological functions of PML are correlated with its organization into nuclear matrix-associated multiprotein complexes (Melnick and Licht, 1999). These complexes are known as PML nuclear bodies, nuclear domain 10 (ND10) or PML oncogenic domains (POD). PML nuclear bodies are characteristically disrupted in acute promyelocytic leukemia (APL), spinocerebellar ataxia and by a variety of viruses (Melnick and Licht, 1999). In 98% of APL cases, a single PML allele is fused to the retinoic acid receptor α (RARα). The resulting PML–RARα fusion protein disrupts PML bodies in a dominant-negative fashion (Grimwade and Solomon, 1997). Remission in these patients is correlated with re-formation of bodies after treatment with all-trans retinoic acid (ATRA; Melnick and Licht, 1999). Despite their clear biomedical relevance, the molecular function of PML nuclear bodies has remained an enigma. The PML gene is absent from organisms such as the budding yeast Saccharomyces cerevisae (http://genome-www.stanford.edu/Saccharomyces) and the fruit fly Drosophila melanogaster (http://www.fruitfly.org). This apparent lack of phylogenetic conservation of PML bodies is intriguing, since they are thought to underlie basic cellular processes in mammals. The integrity of PML and its nuclear bodies is essential for its physiological functions (Melnick and Licht, 1999). Its N-terminal RING, double B-box, coiled-coil (RBCC) motif (Goddard et al., 1991) is required for association of PML with nuclear bodies (Borden et al., 1995, 1996). The RING and B-box are cysteine-rich zinc-binding domains that mediate protein–protein interactions (Borden, 2000). The three-dimensional structure of the PML RING reveals two zinc binding sites (I and II), and zinc binding is required for its native fold (Figure 1C; Borden et al., 1995). The transformation suppressive and pro-apoptotic functions of PML require intact RING and B-box domains. For example, single Cys-Ala substitutions that ablate zinc-binding in either domain disrupt body formation and lead to aberrant cell growth and loss of transformation suppression (Mu et al., 1994; Borden et al., 1995, 1996). Figure 1.Purified PML and eIF4E directly interact. (A and D) GST pull-down analysis of PML and eIF4E. Wild-type eIF4E (A) or mutant eIF4E (D) were incubated with GST fusion proteins as indicated. (B and E) Coomassie Blue-stained SDS–PAGE demonstrating production and purity of the mutant proteins used in (A) and (D). In all cases, proteins of the predicted molecular weight were produced. Further, identical quantities of each protein were used in the pull-down assays shown in (A) and (D). For each pull-down, fusion proteins attached to glutathione beads were resolved in the absence of bait proteins to demonstrate that there was no cross-reaction of these proteins with the antibodies used (data not shown). W.B., western blot; C.B., Coomassie Blue-stained SDS–PAGE. RBB indicates that constructs contained the RING and double B-box of PML; I and II indicate targeted mutation of either zinc site I or zinc site II. 10, 1 and 0.1% represent eIF4E input for (A). The lower of the two bands present in lanes 5, 9 and 10 of (A) and in (D) represents a minor degradation product of eIF4E, which is present in some eIF4E protein preparations and is still able to bind PML RING. (C) The three-dimensional structures of eIF4E and PML are shown (1ej1 and 1bor; Borden et al., 1995; Marcotrigiano et al., 1997). Arrows indicate β-sheets; coils, α-helix; balls, zinc atoms. The m7GpppG dinucleotide substrate of eIF4E is shown in yellow. Structures were rendered in PREPI (S.Islam and M.Sternberg). Download figure Download PowerPoint Despite the progress in defining the physiological roles of PML in transformation suppression and growth, the biochemical basis for these actions remains unclear. Efforts to understand PML body function have centered on identifying its components and using this information to infer the function of the nuclear body itself. This type of analysis has limitations, since PML bodies are heterogeneous in composition and have constituents possessing a variety of seemingly unconnected functions. PML bodies contain a number of proteins involved in transcriptional regulation such as Sp100 (Sternsdorf et al., 1999). However, neither DNA nor RNA polymerase II associates with PML bodies under normal conditions (Maul et al., 2000; von Mikecz et al., 2000), suggesting that they are not sites of active transcription. Moreover, PML nuclear bodies contain proteins involved in nuclear RNA metabolism, translation and ribosome assembly such as eIF3/int-6 (Asano et al., 1997), the ribosomal P-proteins (Borden et al., 1998a) and especially interesting, the eukaryotic translation initiation factor eIF4E (Lai and Borden, 2000). In contrast to PML, eIF4E promotes growth and oncogenic transformation (Sonenberg and Gingras, 1998). In the cytoplasm, eIF4E plays well defined biochemical roles acting as the limiting component in translation initiation (Sonenberg and Gingras, 1998). eIF4E, also known as the cap-binding protein, binds the 5′ m7G cap of mRNA where the cap intercalates between two tryptophans of eIF4E, W56 and W102 (Figure 1C; Marcotrigiano et al., 1997). Cap-binding by eIF4E is essential for initiation of cap-dependent translation (Keiper and Rhoads, 1997) and for regulation of mRNA stability (Dehlin et al., 2000). eIF4E directly binds several regulatory proteins through its dorsal surface. Mutation of W73 to alanine on this surface leads to loss of association of eIF4E with the eIF4G, eIF4E-BP1 and BP2 regulatory proteins (Ptushkina et al., 1998, 1999). In the nucleus the majority of eIF4E is localized to bodies, and appears to function in nucleocytoplasmic mRNA transport of a subset of specific mRNAs (Lejbkowicz et al., 1992; Rosenwald et al., 1995; Rousseau et al., 1996; Lai and Borden, 2000). Overexpression of eIF4E is correlated with increased nucleocytoplasmic transport of Cyclin D1 mRNA, without affecting transport of other mRNAs such as GAPDH and actin (Rousseau et al., 1996; Lai and Borden, 2000). eIF4E overexpression does not lead to increased transcription of Cyclin D1 or to more efficient loading of these mRNAs onto polysomes, but to an increase in the cytoplasmic levels of Cyclin D1 mRNA (Rosenwald et al., 1995; Rousseau et al., 1996). Thus, the effective increase in Cyclin D1 protein results largely from increased mRNA transport. If serine 53 is mutated to alanine (Figure 1C), the resulting mutant eIF4E is unable to transport Cyclin D1 mRNA to the cytoplasm, unable to increase Cyclin D1 protein levels and subsequently unable to form foci in NIH 3T3 cells (Lazaris-Karatzas et al., 1990; Rousseau et al., 1996). The S53A mutant does not affect translation (Rousseau et al., 1996). Thus, eIF4E-dependent transport of Cyclin D1 mRNA is linked to its transformation activity. Previous studies have demonstrated that endogenous PML localizes with and can be immunoprecipitated by eIF4E (Lai and Borden, 2000). This interaction is functional, since PML antagonizes eIF4E-dependent mRNA transport and the RING domain of PML is required for this activity (Lai and Borden, 2000). However, it was not clear whether the antagonism of eIF4E function by PML was a direct consequence of a PML–eIF4E interaction. In addition, the biochemical basis for the antagonism and the physiological effects of this antagonism were unknown. In order to determine biochemical functions for the PML protein and subsequently the nuclear body, we sought to identify proteins that interact directly with its functionally important RING domain. A biochemical analysis of these interactions should reveal molecular functions of the PML protein and a biochemical basis for its physiological effects. This report demonstrates that eIF4E is a RING-binding protein and that eIF4E itself forms both a functional and structural basis for some PML nuclear bodies. We show that the inhibition of eIF4E function by PML is a direct consequence of the interaction between these proteins. These findings have allowed us to develop a biochemical model for PML function as it pertains to eIF4E and suppression of transformation. Results The RING of PML directly binds eIF4E In order to determine whether PML could directly alter eIF4E function, we first examined whether PML interacts directly with eIF4E and whether this interaction is through the RING domain of PML. We used bacterially expressed proteins purified to homogeneity. Constructs contained either the RING double B-box (PML RBB) or only the RING (PML RING). As can be seen from Figure 1A, both PML RING–GST (lane 2) and PML RBB–GST (lane 1) bind directly to eIF4E, demonstrating that the RING domain is sufficient for this interaction. These constructs bound ∼10% of the eIF4E input (lane 5) as indicated by western analysis. eIF4E does not bind GST alone (lane 3). Importantly, the PML RING–eIF4E interaction is specific, since the RING domain from an unrelated protein, Cbl, does not bind eIF4E (lane 4). The corresponding Coomassie Blue-stained gel demonstrates similar loading of all the GST constructs (Figure 1B). Thus, eIF4E interacts directly and specifically with the functionally important RING domain of PML. Previous studies have indicated that other RING proteins, e.g. Msl2 (Copps et al., 1998), bind distinct partner proteins near zinc site I or II. We examined whether this would be the case for PML and eIF4E. Cysteines in either site I or II were mutated to alanine in PML RING or PML RBB and their ability to bind eIF4E was tested. As shown in Figure 1A, mutation of site I reduced eIF4E binding (lanes 8 and 11), whereas mutation of site II in either PML RBB (lane 9) or PML RING (data not shown) constructs was less detrimental to eIF4E binding (compare lanes 8 and 9). The faster migrating band is a degradation product observed in some protein preparations of eIF4E. Thus, our conclusions are based on the full-length eIF4E (the slower migrating band). Notably, although the site I mutation reduces binding, it does not abolish association with eIF4E. These data suggest that eIF4E binds to the RING near site I, but that unfolding of site I (by the cysteine to alanine mutation) does not completely disrupt the conformation of the eIF4E-binding site, suggesting that a short stretch of amino acids could be responsible for eIF4E recognition. In other eIF4E-binding proteins, the YXXXLΦ motif is used for recognition (Sonenberg and Gingras, 1998). PML, however, does not contain this motif, suggesting that another short motif in the RING, involving site I, is responsible for the PML–eIF4E association. The precise region is being defined currently by limited proteolysis and mass spectrometry (our unpublished observations). To gain an insight into the molecular interaction, we examined the effects of m7G cap-binding by eIF4E and of specific mutations in eIF4E on its affinity for the PML RING. Mutation of tryptophan 56 to alanine did not alter the affinity for PML RING, relative to wild-type eIF4E (Figure 1D, compare lanes 1 and 2). However, this mutation substantially reduced retention of eIF4E on m7GTP-Sepharose beads (Figure 6C). Thus, the ability of eIF4E to bind the cap is not required for association with PML, and nor is the presence of W56. Conversely, we investigated whether the presence of m7G cap could inhibit the PML eIF4E interaction. Bacteria do not synthesize an m7G cap, thus all eIF4E produced in these assays is 'cap free'. Therefore, the effect of adding the cap analog m7GpppG was examined. In agreement with the W56 mutation data above, pre-incubation of more than a 10-fold excess of m7GpppG with eIF4E did not alter the affinity of eIF4E for either PML RBB or PML RING (data not shown). W73 is located on the dorsal surface of eIF4E (Figure 1C) and is required for association with several regulatory proteins. We show that the W73A mutant protein has a substantially lower affinity for PML RING (Figure 1D, compare lanes 1 and 3). Thus, PML and eIF4E interact through functionally important parts of each protein. PML and eIF4E co-localize in several leukemic cell lines Previous studies have demonstrated that endogenous eIF4E co-localizes and co-immunoprecipitates with endogenous PML in NIH 3T3 cells (Lai and Borden, 2000). Using immunofluorescence methods in conjunction with confocal microscopy, we examined the localization of endogenous eIF4E and endogenous PML in some leukemic cell lines. In U937 cells, nearly all PML bodies associate with eIF4E bodies (Figure 2A–C, PML in red and eIF4E in green; G–I, PML in blue and eIF4E in green). Similar results were observed in K562 cells (Figure 2D–F). In addition, these proteins can be co-immunoprecipitated in these cell lines (data not shown). Identical results were obtained using either a combination of the PML polyclonal antibody (Borden et al., 1995) and a monoclonal antibody (mAb) to eIF4E (Campbell Dwyer et al., 2000; Dostie et al., 2000a,b; Lai and Borden, 2000) (Figure 2A–F), or using the combination of a PML mAb to 5E10 (Lai and Borden, 2000; Stuurman et al., 1992) and eIF4E mAb directly conjugated to fluorescein isothiocyanate (FITC) (Figure 2G–I). Both the PML and eIF4E antibodies have been characterized extensively elsewhere (Stuurman et al., 1992; Campbell Dwyer et al., 2000; Lai and Borden, 2000). There appears to be some variability amongst cell types in terms of the frequency of PML and eIF4E co-localization, since a higher frequency of co-localization is observed in myeloid cells as compared with fibroblasts such as NIH 3T3 (Lai and Borden, 2000), or PML+/+ mouse embryo fibroblasts (MEFs; Figure 3J). It is clear from these studies that the majority of PML nuclear bodies co-localize with eIF4E bodies in myeloid cells and that there are some eIF4E bodies present that do not localize with PML, consistent with the fact that in many cell types there are more eIF4E bodies than PML bodies. However, these results indicate that co-localization of PML and eIF4E in nuclear bodies is likely to be a general characteristic of mammalian cell lines. Figure 2.PML and eIF4E co-localize in several cell types. U937 (A–C) and K562 (D–F) cells were stained for eIF4E (in green) and PML (in red) with the overlay in yellow. PML and eIF4E were detected using polyclonal antiPML and eIF4E mAb, respectively. Identical results were obtained using anti-PML 5E10 mAb and eIF4E mAb conjugated directly to FITC when U937 cells (G–I) were stained for eIF4E (in green) and PML (in blue) with the overlay in aqua. The objective is 100×. Scale bars = 10 μM. Confocal micrographs represent single sections through the plane of the cells. Download figure Download PowerPoint Figure 3.eIF4E forms nuclear bodies in the absence of PML. (A) APL patient NB4 cells were stained with eIF4E mAb conjugated directly to FITC (eIF4E mAb-FITC, in green). After ATRA treatment (B), NB4 cells were stained for both eIF4E (eIF4E mAb-FITC, in green) and PML (5E10 mAb, in dark blue), with the overlay in aqua. PML−/− MEFs were stained separately with the polyclonal anti-PML (C) or eIF4E mAb (not conjugated to FITC) (D). (E) A nuclear matrix preparation of PML−/− MEFs was stained with eIF4E mAb. (F) D.melanogaster S2 cells were stained with eIF4E mAb (in green) and with DAPI (in blue) to define the nucleus. (G–I) PML−/− MEFs were transfected with the PML gene, and the localization of endogenous eIF4E (green) and transfected PML (red) were observed with the overlay in yellow. Arrows indicate co-localization. (J) PML+/+ MEFs stained for PML (red) and eIF4E (green), with the overlay in yellow. The objective is 100×. Scale bars = 5 μM. Confocal micrographs represent single optical sections through the plane of the cells. Download figure Download PowerPoint eIF4E forms nuclear structures in the absence of PML bodies We investigated whether eIF4E required PML to form nuclear bodies. In the NB4 cell line developed from an APL patient, PML bodies are disrupted by the PML– RARα fusion protein. Therefore, we monitored the localization of eIF4E as a function of ATRA treatment, which restores normal PML body morphology in NB4 cells. Importantly, eIF4E has body-like morphology in NB4 cells prior to ATRA treatment (Figure 3A, eIF4E in green). After ATRA treatment, PML bodies re-form and co-localize with eIF4E bodies, where co-localization is shown in aqua (Figure 3B, PML in dark blue). Results are similar to those observed in other cell types (Figure 2). Thus, in contrast to other PML partner proteins such as Sp100 (Zhong et al., 2000), eIF4E forms nuclear bodies independently of the distribution of PML. Intriguingly, these data strongly suggest that eIF4E does not require PML for localization into nuclear bodies. To examine whether eIF4E nuclear bodies form in cells lacking PML altogether, we examined the localization of eIF4E in PML−/− MEFs that contain a targeted homozygous deletion of the PML gene (Wang et al., 1998). As expected, no PML is observed in these cells (Figure 3C). Cells stained for eIF4E contain a punctate nuclear pattern of eIF4E bodies that is indistinguishable from those observed in PML-containing cell types (Figure 3D and E). In addition, nuclear eIF4E bodies are detected by immunoelectron microscopy in PML−/− MEFs and U937 cells (data not shown). Therefore, eIF4E does not require PML for assembly into nuclear bodies. If PML assembles onto existing eIF4E nuclear bodies, expression of exogenous PML should reconstitute the PML–eIF4E interaction in PML−/− cells. When transfected, PML associates with endogenous eIF4E bodies (Figure 3G–I), suggesting that eIF4E bodies are sites of PML nuclear body assembly. As observed previously in other fibroblasts, there is an excess of eIF4E bodies relative to PML bodies in both the transfected PML−/− cells and the endogenous material in the PML+/+ cells (Figure 3J), suggesting that eIF4E bodies have a heterogeneous composition and diverse biochemical roles. For these fibroblasts, the frequency of PML bodies associated with eIF4E bodies is similar to results observed in NIH 3T3 cells (Lai and Borden, 2000). PML nuclear bodies are characterized by their association with the nuclear matrix (Melnick and Licht, 1999). Therefore, we examined whether eIF4E bodies are also matrix associated in PML−/− cells. eIF4E bodies are morphologically unaltered by nuclear matrix preparation in PML−/− cells, indicating that eIF4E bodies are nuclear matrix-associated (Figure 3, compare D and E) and that PML is not required for this property. We investigated whether cells from phylogenetically diverse organisms, such as D.melanogaster, which do not possess the PML gene, contain eIF4E bodies. Figure 3F shows that in D.melanogaster S2 cells, eIF4E localizes to nuclear bodies and is present diffusely throughout the cytoplasm. Similarly, eIF4E localizes to nuclear bodies in budding S.cerevisiae, as observed by immunoelectron microscopy (Lang et al., 1994). The nuclear organization of eIF4E in cells from phylogenetically diverse organisms is remarkably similar to that observed in mammalian cells, suggesting that eIF4E body assembly is independent of PML and that eIF4E nuclear bodies are phylogenetically conserved in eukaryotic cells, and may be ancestral to PML. PML is dispersed by the m7G cap analog in cell culture The above results indicate that the integrity of eIF4E bodies is independent of PML. Therefore, we examined the possibility that PML requires eIF4E for association with nuclear bodies. If this hypothesis is true, then disruption of eIF4E bodies should affect the distribution of PML. Previous studies with HeLa cells showed that eIF4E nuclear bodies are specifically disrupted by treatment with an analog of its mRNA substrate, m7GpppG (Dostie et al., 2000b). Thus, the effect of treatment with this analog on the morphology of PML nuclear bodies was examined (Figure 4). U937 cells were permeabilized and incubated with either m7GpppG cap analog or GpppG, which does not bind eIF4E efficiently (Dostie et al., 2000b). Following incubation, cells were washed to remove proteins released from the nucleus upon treatment and then fixed. Incubation with either GpppG or buffer alone does not affect the localization of PML, eIF4E or their co-localization, as seen by comparing Figure 4A and B with Figure 2 (PML in blue, eIF4E in green, co-localization in aqua). Strikingly, treatment with m7GpppG completely disperses these proteins from the bodies (Figure 4C). The same results were observed in K562 cells (Figure 4D–F). Similarly, m7GpppG treatment disassociates PML and eIF4E from bodies in NIH 3T3 cells and eIF4E bodies in PML−/− cells (data not shown). Experiments where each component was stained individually yielded the same results as the double staining experiments. We examined the effect of m7GpppG treatment on another PML body component, Sp100. Sp100, like several PML partner proteins, requires PML for association with bodies (Zhong et al., 2000). We found that Sp100 bodies were completely dispersed by m7GpppG while remaining intact in cells treated with GpppG or buffer (Figure 4G–I). Thus, treatment with the cap analog disrupts assembly of eIF4E, PML and Sp100 bodies in myeloid and non-myeloid cells. The ability of PML to associate with bodies appears to rely on the integrity of eIF4E nuclear bodies. Figure 4.Treatment with the m7GpppG cap analog disrupts the subnuclear distribution of eIF4E, PML and SP100. Cells were treated with buffer, GpppG or m7GpppG as indicated. U937 cells (A–C) and K562 cells (D–F) were stained for PML (dark blue) and eIF4E (green). The overlay is aqua. (G–I) K562 cells were stained for the PML partner protein Sp100. The objective is 100×. Scale bars = 10 μM. Confocal micrographs represent single sections through the plane of the cells. Download figure Download PowerPoint To examine the specificity of these treatments, we investigated the integrity of other nuclear structures. As can be seen from Figure 5A–C, the distribution of Sm proteins, which localize to nuclear speckles and are associated with mRNA splicing, is not altered by cap treatment, in agreement with Dostie et al. (2000b). Furthermore, the distribution of nucleolar and coiled body antigens, nucleolin (Figure 5D–F) and Nopp140 (data not shown) is unchanged by cap treatment. Furthermore, nucleoli appear intact after treatment when examined by phase contrast microscopy. Thus, the m7G cap treatment specifically disperses eIF4E, PML and Sp100 bodies. Figure 5.Treatment with the m7GpppG cap analog does not alter the subnuclear distribution of Sm speckles or nucleoli. Cells were treated with buffer, GpppG or m7GpppG as indicated. U937 and K562 cells were stained for Sm and nucleolin, respectively. The objective is 100×. Scale bars = 5 μM. Confocal micrographs represent single sections through the plane of the cells. Download figure Download PowerPoint Figure 6.(A) m7GpppG-binding dramatically affects the conformation of the eIF4E protein. Far-UV CD spectra of bacterially purified eIF4E at 0.24 μM (black), in complex with 3 μM GpppG (red) and with 3 μM m7GpppG (green). (B) PML dramatically reduces the affinity of eIF4E for m7GTP-Sepharose. Bacterially expressed and purified PML RBB or mutants in site I (PMLRBBI) or site II (PMLRBBII) of the RING were mixed with purified eIF4E and applied to m7GTP-Sepharose beads. (C) The W56A mutation reduces the affinity of eIF4E for m7GTP-Sepharose. The affinity of wild-type and W56A mutant of eIF4E was monitored by m7GTP-Sepharose affinity chromatography. The input for each protein preparation is shown, as is the material that bound m7GTP-Sepharose (lanes 5 and 6) and the unbound fraction (lanes 3 and 4). (D) PML does not reduce the affinity of the W73A eIF4E mutant for m7GTP-Sepharose. Results were monitored by western analysis using eIF4E mAb where WB indicates western blot. Download figure Download PowerPoint eIF4E undergoes a major conformational change upon m7GpppG binding Large-scale conformational changes of eIF4E could alter its association with bodies and provide a mechanism for the dispersal of nuclear bodies upon m7G cap treatment. Therefore, we investigated whether eIF4E undergoes changes in conformation upon m7G cap binding. To monitor the conformation of the polypeptide backbone of eIF4E, we utilized circular dichroism (CD) difference spectroscopy (Fasman, 1996). In agreement with published structures, eIF4E has ∼60% α-helical character (Figure 6A). eIF4E was mixed with a 12.5-fold molar excess of m7GpppG so that all eIF4E molecules
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