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Previous Article | Next Article 
Molecular and Cellular Biology, May 2004, p. 4465-4475, Vol. 24, No. 10
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.10.4465-4475.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Section of Infectious Disease, Department of Medicine,1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030,3 Shanghai Institute of Hematology, Shanghai Rui-Jin Hospital, Shanghai 200025, People's Republic of China2
Received 30 October 2003/ Returned for modification 13 January 2004/ Accepted 12 February 2004
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ABSTRACT |
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-receptor (RAR) genes (X-RAR) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RAR target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RAR proteins was distinct from that of RAR and established which portion(s) of each X-RAR protein—X, RAR, or both—contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RAR was reduced compared to that of RAR. In addition, the mobility of each X-RAR was reduced further by ligand addition, in contrast to RAR, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RAR and the ligand-induced slowing of X-RAR could be attributed to the protein interaction domain contained within X. RXR aberrantly colocalized within each X-RAR; colocalization of RXR with promyelocytic leukemia (PML)-RAR resulted in reduced mobility of RXR. Thus, X-RAR may interfere with RAR through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXR and perhaps other nuclear receptor coregulators critical for myeloid differentiation. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-receptor (RAR) gene at 17q21, resulting in the PML-RAR gene and protein product (7, 35). Four variant translocations or deletions occur in the remaining cases of APL, each involving the RAR locus on chromosome 17 including t(11;17)(q23;q21), t(11;17)(q13; q21), t(5;17)(q35;q21), and del(17) (21, 35). These chromosomal abnormalities produce fusions of RAR with PLZF, NuMA, NPM, and STAT5b respectively (1, 4, 9, 10, 21). It is important to identify APL patients with these alternative chromosomal abnormalities since they may not respond as well as PML-RAR-positive APL patients to treatment regiments with all-trans-retinoic acid (ATRA) and other conventional chemotherapies for APL (17). Nuclear hormone receptors (NRs) comprise a large family of ligand-dependent transcription factors that bind to hormone response elements of target genes and regulate their transcription (3). Type I nuclear hormone receptors such as estrogen receptor (ER), androgen receptor (AR), and glucocorticoid receptor (GR) bind to their response elements as homodimers, whereas type II nuclear hormone receptors, including RAR, thyroid hormone receptor (TR) and vitamin D receptor (VDR), bind to their response elements as heterodimers with retinoid X receptors (RXRs). Both type I and II receptors can recruit coactivators in the presence of ligand. In addition, type II receptors, notably RAR and TR, can recruit corepressors such as SMRT and NCoR in the absence of ligand and repress the transcription of target genes (6).
RARs belong to the superfamily of NRs, which affect many physiological processes including differentiation and growth arrest of various cell types including hematopoietic cells. RARs can dimerize with RXRs and bind to retinoic acid response elements (RAREs) located within promoter regions of specific target genes. In the absence of ligand, RAR-RXR heterodimers associate with nuclear receptor transcriptional corepressors (CoR), SMRT-NCoR, resulting in repression of basal transcription. Ligands, such as retinoic acid (RA), release the CoR complex this is followed by recruitment of transcriptional coactivators to the transcriptional complex, resulting in the activation of gene expression. We and others demonstrated that X-RAR (where X represents PML, PLZF, NPM, NuMA, or STAT5b) could bind to RAREs as homodimers or heterodimers with RXR. These findings and others (reviewed in reference 17) support the concept that X-RAR proteins interfere with normal myeloid differentiation by inhibiting wild-type RAR transcriptional activity. However, our understanding of this process at the molecular level is incomplete.
Recent investigations examining fluorescence-labeled ER in live cells have demonstrated interdependence between their intranuclear dynamics and transcription function (27). Some ligand-dependent variation of AR subnuclear targeting has been shown by similar approaches (24, 32), but recent dynamic assessment of AR expressed at physiological levels indicate clear ligand-dependent differences in AR solubility and mobility (D. L. Stenoien, S. Simeoni, K. Patel, M. G. Mancini, I. Agoulnik, N. L. Weigel, and M. A. Mancini, submitted for publication). Moreover, we have demonstrated that an inactive AR point mutant occurring in androgen-unresponsive prostate cancer cells (19) responded in a ligand-dependent fashion by localizing abnormally within nuclear aggregates and sequestering the bulk of steroid receptor coactivator 1 (SRC-1).
To gain spatiotemporal insight into how X-RAR interferes with normal RAR function, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) of fluorescent protein-tagged RAR, RAR, X-RAR, and each of the X components in fixed and live cells following transient transfection or cotransfection. Our findings demonstrated that intracellular localization of each of the X-RAR proteins was distinct from that of RAR and that this altered localization could be attributed, in each instance, to the truncated X component (
X) or truncated RAR component (RAR) or the fusion of the two. The intranuclear mobility of each X-RAR was reduced compared to that of RAR and was reduced further by addition of ligand. In contrast, RAR showed no change in mobility after addition of ligand. The altered localization and reduced mobility of X-RAR could be attributed to the protein interaction domain of X and could result in the mislocalization and slowing of RXR.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids.
The cyan fluorescent protein (CFP)-tagged RAR, NuMA, NuMA-RAR, and NuMA-RAR(CC) expression constructs (in pECFP-C1 [Clontech]) have been described previously (9). The cDNA sequences for truncated RAR (amino acids [aa] 60 to 462), PML, truncated PML (aa 1 to 552), PML-RAR, PLZF, truncated PLZF (aa 1 to 455), PLZF-RAR, NPM, truncated NPM (aa 1 to 160), NPM-RAR, STAT5b, STAT5b-RAR, and RXR were subcloned from their respective expression constructs (5, 9-11, 25) into pECFP-C1 vector or pEYFP-C1 vector (Clontech) to make CFP-tagged RAR, PML, PML, PML-RAR, PLZF, PLZF, PLZF-RAR, NPM, NPM, NPM-RAR, STAT5b, STAT5b-RAR, and yellow fluorescent protein (YFP)-tagged RXR expression constructs, respectively. The cDNAs for CFP-RAR, CFP-PML-RAR, CFP-PLZF-RAR, CFP-NPM-RAR, CFP-NuMA-RAR, and CFP-STAT5b-RAR contained within the pECFP-C1 construct were subcloned into the pcDNA3.1 expression construct (Invitrogen) to facilitate the in vitro generation of proteins. The (RARE)3-tk-luciferase reporter construct has been reported previously (9). All plasmid constructs were confirmed by DNA sequencing.
Cell transfections and immunoblotting.
For transient transfections, COS-7 or HeLa cells were grown in six-well plates to 50 to 70% confluence. At 12 h later, the cells were transiently transfected with the indicated expression constructs and/or reporter genes using GeneJuice reagent as reported previously (10). After 24 to 48 h of transfection, the cells were lysed in lysis buffer as previously reported (10). Equivalent amounts of protein were electrophoresed on sodium dodecyl sulfate-7.5 or 10% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Millipore). Antibodies used in this study were RAR rabbit polyclonal antibody (C-20; Santa Cruz BioTechnology, Santa Cruz, Calif.) and PLZF mouse monoclonal antibody (Oncogene Research Products, San Diego, Calif.).
In vitro translation and gel shift DNA-binding assays.
CFP-RAR, CFP-PML-RAR, CFP-PLZF-RAR, CFP-NPM-RAR, CFP-NuMA-RAR, and CFP-STAT5b-RAR proteins were generated in vitro using the TNT-coupled rabbit reticulocyte lysate system (Promega) as specified by the manufacturer. Gel shift assays were performed using the in vitro-translated proteins and DR5G RARE as described previously (11).
FRAP.
HeLa cells were cultured in Opti-MEM I medium (Invitrogen) with 4% FBS (30). At 24 h before transfection, the cells were plated onto poly-D-lysine-coated coverslips in 35-mm wells at 105 cells per well for fixation studies or 40-mm coverslips in a 60-mm dish at 2 x 105 cells per dish for FRAP analysis. Transient expression of plasmids (CFP tagged or YFP tagged) was accomplished using GeneJuice transfection reagent (Novegen) as described previously (8, 10). The cells were fed with fresh medium the day after transfection and allowed to recover for approximately 24 h prior to addition of vehicle (ethanol) or ATRA at a final concentration of 10–6 M for 2 h as indicated. The cells were fixed as described previously (26) and imaged with on LSM 510 confocal microscope (Carl Zeiss, Inc.). FRAP analysis has been described previously (28-30). The fluorescent molecules in FRAP experiments are essentially irreversibly photobleached in a small area of the cell by brief (1- to 2-s) exposure to a focused high-power laser beam. Subsequent diffusion of surrounding nonbleached fluorescent molecules into the bleached area leads to a recovery of fluorescence, which is recorded using time-lapse photography at low laser power and quantified using software available with the microscope; this was carried out with an LSM 510 confocal microscope. A single z-section was imaged before and at intervals after the 2-s bleach. The bleach was carried out at a wavelength of 458 nm (CFP) or 514 nm (YFP) and at maximum power for 100 iterations of a box representing 
20% of the nuclear volume. For dual FRAP experiments, both were bleached with the same laser setting and simultaneous images corresponding to CFP and YFP fluorescence were obtained using the multitracking function of the microscope. Fluorescence intensities of regions of interest were obtained using LSM software, and data were analyzed using Microsoft Excel. Representative images from single focal planes were imported as TIFF files (29, 30). To allow pooling of data from each cell analyzed, the initial fluorescence at the end of bleaching was assigned a value of 0 and the final fluorescence recovery was assigned a value of 1.
Statistical analysis. Unless indicated otherwise, data presented are the mean ± standard error of the mean; differences between means were assessed for significance by using Student's t test.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and X partners.
To study the intracellular localization and mobility of RAR, X-RAR, and X partners in living cells, we created constructs expressing CFP chimeras fused to the N-terminal ends of RAR and X-RAR, as well as its normal X partners (Fig. 1). Immunoblot analysis of whole-cell extracts from transfected cells and in vitro translation protein analysis showed that each CFP-tagged construct encoded proteins of the correct size (Figure 1a) (reference 9 and data not shown). To be certain that the addition of CFP to RAR or X-RAR did not significantly alter their functional characteristics compared to untagged RAR or X-RAR, we evaluated the DNA-binding activity of CFP-RAR or CFP-X-RAR by a gel shift assay. Similar to their untagged counterparts, each CFP-X-RAR bound to RARE as a homodimer (Fig. 1b) and each CFP-X-RAR, as well as CFP-RAR, bound to RARE as a heterodimer with RXR (Fig. 1b). To further characterize the functional properties of CFP-RAR or CFP-X-RAR, we examined their transcriptional activity by using the luciferase reporter system as described previously (9-11). CFP-RAR or CFP-X-RAR expression plasmids were cotransfected into HeLa cells with the RARE-containing luciferase reporter construct, and each demonstrated ligand-dependent transactivation to levels equal to or greater than those of their non-CFP-tagged counterparts (Fig. 1c) (reference 9 and data not shown).
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, CFP-X-RAR, and each of their normal CFP-X components (Fig. 2; Table 1). Similar to previous immunohistological observations for wild-type RAR, X-RAR, and the normal X partners (Fig. 2) (12, 14, 15, 22, 33, 34), CFP-RAR and YFP-RXR demonstrated a diffuse and predominantly intranuclear pattern, CFP-PML displayed a typical PML oncogenic domain (POD) structure, CFP-PLZF demonstrated an intranuclear microspeckled pattern, CFP-NPM localized to nucleoli, CFP-NuMA was localized almost exclusively to the nucleoplasm in a reticular pattern, and CFP-STAT5b was localized predominantly within the cytoplasm and demonstrated a diffuse pattern within the nucleus.
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proteins distributed within the nucleus in a pattern distinct from that of RAR, and four of the five CFP-X-RAR proteins (where X is PML, PLZF, NPM, and STAT5b) distributed within the nucleus in a pattern distinct from those of their corresponding CFP-X counterparts (Fig. 2; Table 1), confirming previous immunohistological reports (12, 14, 15, 22, 33, 34). CFP-PML-RAR localized predominantly to the nucleus in a microspeckled pattern distinct from that of PODs. CFP-PLZF-RAR demonstrated a microspeckled pattern similar to that of CFP-PLZF. CFP-NPM-RAR distributed predominantly in a diffuse pattern with occasional microspeckles within the nucleoplasm, distinct from that of CFP-NPM, which localized exclusively to nucleoli. In addition, a small portion of CFP-NPM-RAR colocalized with YFP-NPM within nucleoli (see Fig. 5f), distinct from RAR, as detailed below. CFP-STAT5b-RAR distributed predominantly within the nucleus in a microspeckled pattern along with the more diffuse pattern seen with CFP-STAT5b. As recently reported by us (9), while CFP-NuMA-RAR localized predominantly to the cytoplasm in the absence of ATRA and shifted to predominantly nuclear localization in the presence of ATRA, when present in the nucleus it distributed in a reticular pattern similar to that of CFP-NuMA.
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compared to CFP-RAR or its corresponding CFP-X counterpart, we examined the intracellular distribution of the individual truncated protein components within each fusion protein. When CFP-RAR (aa 60 to 462) and CFP-PML (aa 1 to 552), which are the portions of RAR and PML, respectively, retained in the PML-RAR chimera, were expressed in HeLa cells, each displayed the same cellular localization as its full-length counterpart (Fig. 2; Table 1) i.e., CFP-RAR had a diffuse intranuclear pattern, and CFP-PML had a typical POD structure. When CFP-PML and YFP-PML were cotransfected into HeLa cells, they colocalized within the POD structure (see Fig. 5a), indicating that the RBCC region of PML is sufficient for localization within PODs and that loss of the C-terminal region did not affect POD formation. However, fusion of RAR (aa 60 to 462) to PML (aa 1 to 552) does not result in the POD structure but, rather, leads to the formation of a distinct microspeckled pattern (Fig. 2), indicating that it is the addition of the RAR portion that interferes with POD formation. CFP-PLZF (aa 1 to 455), which is the portion of PLZF retained in PLZF-RAR, localized within the cytoplasm, presumably due to loss of its nuclear localization signal located in the C-terminal end of PLZF and deleted in PLZF. With the fusion of RAR, which contains a nuclear localization signal, to PLZF to generate PLZF-RAR, the ability to localize within the nucleus was regained (Fig. 2). CFP-NPM (aa 1 to 160), which is the portion of NPM retained in NPM-RAR, was localized within the cytoplasm due to deletion of its nuclear localization signal, similar to the findings with PLZF. Also, similar to PLZF-RAR, fusion of RAR to NPM to form NPM-RAR restored its ability to localize to the nucleus. However, unlike NPM, the localization of NPM-RAR within the nucleus is predominantly diffuse and not limited to the nucleolus (Fig. 2). To determine whether NPM-RAR does localize to the nucleolus at all, we cotransfected HeLa cells with YFP-NPM and CFP-NPM-RAR. We found that the CFP-NPM-RAR protein also localized in the nucleolus with wild-type NPM (see Fig. 5f), presumably through heterodimerization mediated by the protein interaction domain within NPM. We previously determined that the coiled-coil domain within the NuMA component of NuMA-RAR was responsible for its colocalization with NuMA in the nucleus (9). C-terminal truncation mutants of STAT5b resembling the STAT5b component of STAT5b-RAR have been identified (2, 13, 16, 23), and while these truncations may function as dominant negatives of full-length STAT5, their intracellular distribution is similar to that of full-length STAT5b, indicating that the altered localization of STAT5b-RAR to microspeckles is a unique feature of the fusion of STAT5b and RAR that cannot be attributed to either component alone.
Reduced mobility of X-RAR in comparison with RAR.
To study the intranuclear mobility of RAR, X-RAR, and X, we performed FRAP of live cells transfected with CFP- and YFP-tagged constructs (Fig. 3 and 4, Table 2). In all FRAP experiments, only cells expressing low levels of protein were examined, to avoid artifacts of overexpression as previously described (30). Specifically, we adhered strictly to the procedure of viewing cells at the lower end of detection and excluded overexpressers (>20-fold) from analysis. Furthermore, we demonstrated, using antibodies to endogenous receptors indirectly detected by Alexa 594, that expression in the CFP-positive cells was within a factor of 2 to 3 of the endogenous expression. Following photobleaching of HeLa cells transfected with CFP-RAR, fluorescence recovery occurred rapidly, with a half-maximal fluorescence recovery time (t1/2) of 0.67 ± 0.15 s. In comparison with wild-type RAR, each of the CFP-X-RAR proteins had reduced mobility in the absence of ligand, ranging from t1/2 of 0.84 ± 0.11 s for CFP-NPM-RAR to t1/2 of >5 min for CFP-PML-RAR. The mobility of CFP-RAR was similar to that of CFP-RAR, indicating that loss of the A domain in RAR did not contribute to the slowing of X-RAR. Rather, in each instance except for STAT5b, the reduction in the mobility of X-RAR compared to RAR could be attributed to reduced mobility of X (Table 2).
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but not RAR.
We demonstrated previously that the addition of estrogen reduced the mobility of ER, a type I nuclear receptor (30). To determine if receptor slowing with ligand is a feature of NR of the type II class, we analyzed the behavior of CFP-RAR after addition of its ligand, ATRA (10–6 M), for 2 h. However, unlike ER, ligand addition did not slow CFP-RAR (Table 2). In contrast to CFP-RAR, however, ATRA did result in the slowing of CFP-PLZF-RAR, CFP-NPM-RAR, CFP-NuMA-RAR, and CFP-STAT5b-RAR, ranging from an 18% reduction of the t1/2 for CFP-NPM-RAR to 30% for CFP-NuMA-RAR (Table 2). Slowing of CFP-PML-RAR with ligand addition could not be assessed because of its markedly reduced baseline mobility (t1/2 >5 min).
The coiled-coil domain of NuMA-RAR is responsible for the reduced mobility of NuMA-RAR compared to RAR and its reduced mobility with ligand.
Protein interaction domains within the X component of X-RAR fusion proteins have been demonstrated by us and others to be responsible for many of the oncogenic properties of X-RAR proteins (9, 10, 17). We previously demonstrated that the coiled-coil domain of NuMA was responsible for NuMA-RAR homodimerization, inhibition of ligand-dependent RAR-mediated gene transcription, and enhancement of Stat3-mediated transcriptional activation (9). To begin to examine the contribution of the protein interaction domains to altered X-RAR mobility, we compared the mobility of CFP-NuMA-RAR with that of CFP-NuMA-RAR(CC), in which the coiled-coil domain of CFP-NuMA-RAR is deleted (Fig. 3; Table 2). While the fluorescence recovery t1/2 of CFP-NuMA-RAR was 1.95 ± 0.41 s, the t1/2 of CFP-NuMA-RAR(CC) was reduced by 59% to 0.80 ± 0.15 s (P < 0.001), which was indistinguishable from the t1/2 of CFP-RAR. In addition to increasing its mobility, deletion of the coiled-coil domain eliminated ligand-induced slowing of CFP-NuMA-RAR (Table 2). Thus, in addition to being essential for its other oncogenic functions, the coiled-coil domain of NuMA is responsible for the reduced mobility of CFP-NuMA-RAR compared to CFP-RAR and its slowing in response to ligand.
Effect of X-RAR on nuclear localization and mobility of RXR.
To determine whether the altered localization and reduced mobility of X-RAR altered the localization and mobility of NR coregulators, we performed fluorescence microscopy and FRAP analysis of HeLa cells transfected with YFP-RXR, without or with CFP-RAR or CFP-X-RAR (Fig. 2, 5, and 6). YFP-RXR alone distributed predominantly within the nucleus in a diffuse fashion similar to that of RAR (Fig. 2). Cotransfection of cells with YFP-RXR and CFP-RAR did not change the nuclear distribution of RXR (Fig. 6). However, when coexpressed with CPF-X-RAR, YFP-RXR changed its nuclear distribution and colocalized with each X-RAR (Fig. 5), presumably due to its ability to heterodimerize with the RAR portion of X-RAR. While YFP-RXR colocalized with CFP-NPM-RAR within the nucleoplasm, we could not detect YFP-RXR within the nucleoli of cells when coexpressed with both CFP-NPM-RAR and CFP-NPM (Fig. 5g).
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, YFP-RXR was rapidly mobile when expressed alone, with a fluorescence recovery t1/2 = 0.97 ± 0.29 s (Fig. 6a). Cotransfection of cells with YFP-RXR and CFP-RAR did not change the nuclear mobility of either YFP-RXR (t1/2 = 0.98 ± 0.23 s) or CFP-RAR (t1/2 = 0.63 ± 0.19 s) (Fig. 6b). In contrast, cotransfection of cells with YFP-RXR and CFP-PML-RAR strikingly reduced the mobility of YFP-RXR by eightfold (t1/2 = 7.81 ± 1.20 s, P < 0.001 [Fig. 6c and d]), indicating that colocalization of RXR with PML-RAR, which is relatively immobile, resulted in its reduced mobility. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferes with normal RAR function, we employed fluorescence microscopy and FRAP of fluorescent protein-tagged RAR, RXR, X-RAR, and each of the X components in fixed and live cells following transient transfection or cotransfection. Our findings demonstrate that (i) the intracellular localization of each X-RAR protein was distinct from RAR and that this altered localization could be attributed, in each instance, to X, RAR, or the fusion of the two; (ii) the intranuclear mobility of each X-RAR was reduced compared to that of RAR; and (iii) in contrast to RAR, which showed no change in mobility with addition of ligand, the mobility of each X-RAR was reduced with ligand. Similar to other oncogenic features of NuMA-RAR, the altered localization and reduced mobility of NuMA-RAR mapped to the coiled-coil domain of NuMA. The altered localization of X-RAR resulted in the mislocalization of RXR, presumably due to the interaction of RXR with the RAR portion of X-RAR. In the case of PML-RAR, which has a fluorescence recovery t1/2 of >5 min, the mislocalization of RXR was accompanied by a dramatic reduction in its mobility.
PML-RAR had previously been demonstrated to sequester RXR within the cytoplasm when overexpressed transiently in COS cells (20) and within microspeckles tightly bound to chromatin within the nucleus of APL cells (33). In transfected COS cells, PML-RAR overexpression prevented the binding of the VDR to a target sequence in vitro and inhibited the vitamin D3-dependent activation of a VDR-responsive reporter gene; in APL cells, ATRA treatment reversed RXR sequestration. These findings raised the possibility that RXR sequestration may be an important mechanism of PML-RAR oncogenesis. Our findings provide additional support for the sequestration hypothesis by demonstrating that RXR and X-RAR, in cells expressing X-RAR at low levels, colocalize within the nucleus within microspeckles and further demonstrate that when PML-RAR and RXR are coexpressed, the mobility of RXR is reduced eightfold compared to its mobility when expressed alone or when coexpressed with RAR. Thus, in addition to sequestering RXR, PML-RAR expression may interfere with normal type II NR functions, including RAR, by reducing the intranuclear mobility of RXR and other critical NR coregulators.
Each of the X-RAR proteins had a nuclear predominance. The RAR component was responsible for this feature in PLZF-RAR, NPM-RAR, and STAT5b-RAR, while the X component mediated nuclear localization of PML-RAR and NuMA-RAR. Each of the X-RAR proteins has a pattern of intranuclear distribution distinct from that of RAR. In all instances except NuMA-RAR, this pattern is microspeckled and distinct from that of X. It is clear from our previously published results (9) that in the case of NuMA-RAR, the NuMA component is responsible for the reticular intranuclear pattern of NuMA-RAR. The microspeckled pattern observed in our studies for each of the other four X-RAR proteins could not be attributed to either X or RAR; rather, it appears to be a unique feature of the chimeric protein.
Studies such as those described in this report, using derivatives of GFP to obtain spatiotemporal information about X-RAR proteins and their individual components singly and in combination, cannot be performed on fixed cells by immunohistological techniques. However, the results are valid only if the fluorescent tag does not interfere with the biological function (if assessable) and the localization of the untagged protein. From previous studies by us and others (reviewed in reference 31), it is clear that attachment of GFP and its derivatives rarely affects the function and localization of the fusion protein. In this work, two lines of evidence provide strong support to the contention that our tagged proteins were not appreciably altered: (i) each tagged X-RAR exhibited biochemical and transcriptional properties similar to those of their untagged counterparts, and (ii) each localized within cells as reported previously in experiments using immunohistological techniques.
Previous studies by us demonstrated that ligand addition can differentially reduce the mobility of type I NR, ER and AR (29, 30; Stenoien et al., submitted), in contrast to the findings of others (24, 32). In our present study, while ligand addition had a modest effect on the mobility of X-RAR, it did not change the mobility of wild-type RAR. These findings raise two possibilities: (i) that the regulation of transcription and/or degradation by type I and type II NRs is distinct and (ii) that by being able to bind to RARE as homodimers, X-RAR may share some additional features with type I NRs involving their regulation and/or degradation. Alternatively, since at least ER and AR undergo posttranslational modification after ligand binding, e.g., ubiquitination, it is possible that the type I NRs are more influenced by ligand than are type II NRs, which are less clearly understood in terms of these modifications.
The reduced mobility of STAT5b-RAR compared to RAR, unlike the other X-RAR fusion proteins, could not be attributed to the reduced mobility of STAT5b, which had a high mobility, similar to that of RAR. However, STAT5b-RAR, like each of the other X-RAR proteins, formed microspeckles. Microspeckles formed by PML-RAR were demonstrated previously to correspond to PML-RAR protein bound tightly to chromatin (33), suggesting that rather than X within X-RAR, it is the ability of each X-RAR to form microspeckles that results in the reduced mobility of X-RAR including STAT5b-RAR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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