- Research article
- Open Access
Subcellular distribution of nuclear import-defective isoforms of the promyelocytic leukemia protein
© Jul-Larsen et al; licensee BioMed Central Ltd. 2010
- Received: 27 June 2010
- Accepted: 21 November 2010
- Published: 21 November 2010
The promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein.
Expression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain.
This study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering.
- Nuclear Localization Signal
- Early Endosome
- U2OS Cell
- Nuclear Periphery
- Nuclear Lamina
The PML protein participates in several different cellular functions, including transcription regulation, differentiation, virus defence and tumour suppression [1–4]. In addition, this protein represents one of the two fusion partners in the PML/retinoic acid receptor alpha (RARA) fusion oncoprotein, which supports tumorigenesis in patients with acute promyelocytic leukemia [5, 6].
PML belongs to a group of more than 70 different human proteins commonly referred to as the TRIM family of proteins. These proteins are characterized by the presence of a tripartite motif (TRIM) at their N-terminus, which generally comprises three different structural elements, including a RING domain, one or two B-boxes and a coiled coil. The C-terminal region of these proteins typically contains different types of functional domains and may vary between protein isoforms due to alternative pre-mRNA splicing [7, 8]. Some common functions of TRIM family members have been identified. For example, a number of members appear to function in the innate immune defence against viruses and several have been shown to possess ubiquitin ligase activity [7, 9, 10]. In addition, TRIM family proteins appear to have a general propensity to form macromolecular protein assemblies at various subcellular compartments [8, 11, 12]. It is not clear, however, how the conserved structural organization of TRIM family members contributes to these functions at the molecular level.
A unique feature of the PML protein is its ability to support the structural integrity of nuclear compartments called PML nuclear bodies (PML NBs). These structures can readily be detected by immunofluorescence microscopy as numerous foci within the nucleus, and they recruit a multitude of different proteins with diverse cellular functions . The ability of PML to induce the formation of these structures is facilitated by the TRIM domain, SUMO conjugated residues and a SUMO interacting motif [13–15].
The PML protein is expressed as several alternatively spliced isoforms, and a selected group of these have been designated PML I through PML VII [16, 17]. The PML splice variants identified to date contain identical N-termini, including the TRIM domain, whereas the C-termini vary considerably among different isoforms. It is therefore likely that the N-terminus performs a function that is shared by the different isoforms and that the C -terminal variable domain contributes to isoform-specific functions. In agreement with this, some isoform-specific functions of PML have been identified [18–21]. The variability of the PML C-termini probably contributes significantly to the ability of PML to participate in a large variety of different cellular processes.
Although most PML isoforms target PML NBs, splice variants lacking the central nuclear localization signal (herein referred to as NLS6 because it originates from exon 6 of the PML pre-mRNA) have also been identified  and may therefore have cytoplasmic functions [16, 23, 24]. In addition, the PML I isoform is known to contain a nuclear export domain at its variable C-terminus, suggesting that it may shuttle between the nucleus and the cytoplasm . To identify potential cytoplasmic PML targets, we have analysed the subcellular distribution of different PML isoforms containing a mutated NLS6 in the osteosarcoma cell line U2OS. Our analyses show that PML has the potential to target different subcellular compartments beside PML NBs, including early endosomes, late endosomes/lysosomes and the inner nuclear membrane. Subcellular targeting by PML is determined by the isoform specific C-terminal sequence as well as by the presence or absence of a functional NLS6. In addition, the PML TRIM domain is found to have a general role in protein clustering at each of the different target compartments.
Differential compartment targeting of import-defective PML isoforms
Targeting of early endosomes and late endosomes/lysosomes by cytoplasmic PML
Targeting of the nuclear periphery by PML II
To determine if targeting of PML II to the nuclear periphery merely represented a phenomenon caused by PML II overexpression or if also endogenous PML has the capacity to target these nuclear structures, we examined endogenously expressed PML in U2OS cells. By immunofluorescence analysis of untransfected U2OS cells using antibodies directed against the constant part of the PML protein (thereby detecting all isoforms), we observed a small sub-fraction (between 1.5 and 2.5%) of an asynchronously growing population of U2OS cells that contained detectable PML lining the nuclear periphery. Interestingly, the number of cells containing detectable PML at nuclear membrane proximal sites increased following incubation of the cells with DRAQ5 or Actinomycin D, two reagents that are known to induce genotoxic stress (Figure 3B). This effect was, however, not observed following treatment with other genotoxic stressors/RNA synthesis inhibitors, including DRB, α-amanitin or hydroxyurea (data not shown), suggesting that recruitment of PML to the nuclear periphery is not caused by all types of genotoxic drugs or RNA synthesis inhibitors. Importantly, these results show that endogenous PML has the capacity to target the nuclear periphery and that recruitment of PML to these nuclear sites may be induced by certain types of genotoxic stress.
We also determined the ability of PML II to target the nuclear periphery in three other cell lines, including HeLa, GM847 and HaCaT. Interestingly, HaCaT and HeLa cells did not support re-localization of PML to the nuclear periphery upon PML II overexpression. Instead, these cells showed accumulation of PML II in seemingly normal PML bodies at nuclear sites distal to the nuclear periphery (Additional file 2). GM847 cells, on the other hand showed a peripheral localization of overexpressed PML II that was similar to that seen in U2OS cells (Additional file 2). Further, the two drugs DRAQ5 and Actinomycin D were found to significantly induce re-localization of PML to nuclear periphery only in U2OS and GM847 cells (the same cell lines as those supporting nuclear periphery targeting of PML II) but not in HaCaT or HeLa Cells (data not shown). This result shows that the ability of PML to target nuclear membrane proximal sites is largely cell-type dependent.
PML II contains functional domains at the C-terminus that facilitate transport across the nuclear membrane and targeting of the nuclear periphery
Targeting of early endosomes, late endosomes/lysosomes and the nuclear periphery by PML depends on a functional TRIM domain
Target compartments of wild-type and nuclear import-defective PML isoforms in U2OS cells
Nuclear import-defective mutant
The ability of PML to target early and late endosomes may reflect a role for PML in endosome trafficking. A functional association of PML with early endosomes has previously been reported for a splice variant of PML that lacks exon 5 and 6 and that contains an N-terminal configuration similar to that of PML IV. In this case, cytoplasmic PML was found to function in TGFβ-mediated signaling through interactions with SMAD1, SMAD2 and SARA at early endosomes . Further, since several viruses and bacteria are known to exploit endosomal trafficking routs as a means to invade their host, the ability of PML to target these cytoplasmic organelles may also reflect a role of this protein in the cellular defense against pathogens. In agreement with this, PML represents an interferon responsive gene (which is characteristic for genes involved in the innate immune response) and has been shown to restrict replication of certain viruses [3, 31, 32]. In a recent study, production of splice variants of PML lacking exon 5 and 6 was shown to be increased in interferon treated and HSV1-infected cells . This finding suggests the existence of a regulatory mechanism whereby cells respond to virus infection by altering the splice pattern of PML to obtain increased expression of cytoplasmic versus nuclear PML. The present study indicates that PML proteins produced by mRNA species lacking exon 5 and 6 will be expected to target late endosomes/lysosomes. Further studies are needed to determine if PML exerts its antiviral property by interfering with endosomal or lysosomal functions.
A limitation of the present study is that we were unsuccessful in detecting endogenous PML within early or late endosomes by immunofluorescence labeling using anti-PML antibodies. In fact, PML in most types of cultured mammalian cells are primarily detected within PML NBs where it is most highly concentrated. We cannot fully exclude the possibility that the ectopically expressed NLS6-defective PML detected in endosomes represents protein aggregates that are in the process of being cleared from the cell by lysosome-mediated degradation. However, several observations suggest a functional role of PML at these locations. First, the observed subcellular distribution of PML to late endosomes is dependent on a functional RING motif. This suggests that a functional TRIM is required for endosome targeting. Second, the cytoplasmic staining of PMLVII as well as PML IIInls, PML IVnls and PML Vnls seem to be present mostly at the exterior of these organelles and not at their interior as would be expected if PML was engulfed by lysosomes. Third, the PML isoforms expressed in the cytoplasm enhances the size of late endosomes/lysosomes, suggesting a stimulatory role of cytoplasmic PML on these compartments. Lastly, overexpressed NLS6-proficient PML proteins can also be observed to form aggregates in the cytoplasm that are readily detected by immunofluorescence, but for these proteins we have never observed co-localization with the lysosomal marker proteins Lamp1 or Rab7. Thus, NLS6 may have a direct role in preventing PML isoforms that are destined to target the nucleus from interacting with late endosome/lysosomes. The PML protein was recently identified in a proteomic screen for phagosomes resident proteins . Thus, even though PML generally is undetectable by immunofluorescence in most cytoplasmic compartments, this protein may be present in organelles such as endosomes and phagosomes at levels that are undetectable by antibodies that we have available.
PMLII overexpressed in U2OS cells was found to primarily target the inner nuclear membrane. Distribution of PMLII to these nuclear sites was also noted in a previous study following expression of this isoform in Chinese hamster ovary cells . This particular distribution of PML appears to be highly cell type specific as PMLII expressed in HaCaT or HeLa cells exhibited PML clusters at more central regions of the nucleus consistent with normal PML NB morphology. Interestingly, both cell lines (U2OS and GM847) that were found to support targeting of PML to the nuclear periphery are ALT cells, cells that use alternative lengthening of telomeres (ALT) and not telomerase as their primary mechanism for telomere elongation [35, 36]. It has previously been shown that ALT cells contain special PML NBs termed ALT associated PML bodies (APBs) that sequester DNA repair proteins and telomeric DNA . Thus, the ability to direct PML to the nuclear periphery may represent an additional phenotype that accompanies ALT cells.
So far we have not been able to detect structural components of the nuclear periphery that co-localized with PML at the edge of the nucleus. In fact, both nuclear lamina as well as nuclear pore complexes (as detected with the nucleoporin-specific antibody Mab414; data not shown) were found to be excluded from sites containing overexpressed PMLII. The association of PML with the nuclear periphery may reflect a role of this protein in cellular processes such as transcription regulation, DNA replication or DNA repair since these cellular processes are known to be active at nuclear envelope proximal sites [38–40]. Interestingly, the region of PMLII that we found to be responsible for inducing nuclear periphery targeting overlaps the amino acid sequences previously shown to interact directly with the adenovirus protein E4 Orf3, which is known to be involved in PML NB disruption during adenovirus infection . This may indicate that the activity supported by PMLII at the nuclear periphery represents a barrier that certain viruses need to overcome in order to achieve successful infection.
The presence of an extra NLS at the C-terminal variable domain of PMLII suggests that some splice variants of this protein may enter the nucleus even in the absence of amino acid sequences encoded by exon 6. Analysis of the C-terminal region of PMLII does not reveal peptide sequences that match the consensus sequence of any known NLSs . However, the region between aa 717 to 767, which in the present study was shown to be important for import activity, is rich in arginines and serines. This may suggest the presence of a serine arginine-rich NLS similar to that used by SR-proteins, a group of proteins involved in pre-mRNA splicing [27–29]. The lack of sequence similarity between NLS6 and the NLS present within PMLII C-terminal variable region, suggests that PML II uses two distinct nuclear import routes.
NLS6 seems to represent an unconventional NLS that may play a central regulatory role in several aspects of PML trafficking and subcellular localization. Besides playing an important role in nuclear import, this peptide sequence has also been shown to be required for targeting PML to cytoplasmic PML-containing compartments referred to as cytoplasmic assemblies of PML and nucleoporins (CyPNs) . These structures form during the mitosis-to-G1 transition of the cell cycle and seem to be derived directly from post mitotic PML NBs . NLS6 may also be regulated by SUMOylation since this peptide sequence is known to contain one of the three lysine residues that represent SUMO conjugation sites in PML. However, abrogation of this SUMOylation site by mutagenesis was found not to affect PML nuclear import, suggesting a role of this SUMOylation event that is not directly related to import . Finally, genome sequencing analysis of patients with an aggressive ATRA-resistant form of APL revealed mutations in the non-rearranged PML gene that is predicted to cause premature translation termination of the PML protein upstream of NLS6 . Based on the data presented in the present study, such mutants may contribute to tumorigenesis through interference with late endosomes/lysosomes functions.
In this study we have shown that forced expression of PML in the cytoplasm leads to clustering of this protein at different types of cellular compartments, including early endosomes, late endosomes and the inner nuclear membrane. Since the ability of PML to cluster at each of these different sites depends on a functional TRIM domain, our data support the notion that the TRIM domain plays a general role in protein clustering and that the alternatively spliced C-terminus of the protein has a specific role in compartment selection. Further studies are needed to elucidate potential functions of PML at early endosomes, late endosomes and the nuclear periphery.
Cell-lines and transfection
U2OS cells (human osteosarcoma), GM-847 cells (SV40 transformed human fibroblasts), HaCaT (human keratinocyte) and HeLa (human cervical cancer) were maintained in Iscove's modified Dulbecco's medium (IMDM; Bio Whittaker, Belgium) containing 10% foetal calf serum (PAA, Austria) at 37°C and 5% CO2. Cells were transfected using the FuGENE6 transfection reagent (Roche, Switzerland) according to the manufacturer's protocol. U2OS cells stably expressing Flag-tagged PML I, II or III were generated using lentivirus constructs generously provided by Dr Roger D Everett at MRC virology unit, Glasgow, UK.
His-tagged PML I through V expressed from a pcDNA3 vector were kindly provided by Dr. K-S Chang at the University of Texas, Austin, Texas . His-tagged PML VII and NLS mutants (K486A_K487A) of PML I through V were described in . The PML II truncation mutants were made by PCR cloning using the forward primer (gaagcccagcctatggctgtg) in combination with the reverse primers (tatgaattcatgcctccccggcgccactggc (1-552), tatgaattcaagactggactggcgaggagtg (1-602), tatgaattcagtggacggcagggcgctc (1-652), tatgaattcattgcagctgggcaggatgttc (1-716) or tatgaattcacaccacggaagacatgtcaag (1-767)). The PCR product was then substituted for the PML II specific sequence of pcDNA3 His-PML II or pcDNA3 His-PML IInls using the Van91I (Fermentas, Canada) and Eco RI (NEB, MA) restriction sites. The 1-681 mutant was constructed by digesting the pcDNA3 His-PML II vector with Apa I restriction enzyme (Takara, Japan) and re-ligation. This resulted in the loss of PML II aa 682-829 and formation of a short, 12 aa, nonspecific tail at the end of the protein.
RING finger mutants of PML I, II and VII were constructed by introducing point-mutations (C57S;C60S) to the respective His-tagged PML isoforms using the QuikChange kit (Stratagene, CA). The His-PML I-RN and His-PMLII-RN, RING finger and NLS double mutants, were constructed by introducing the RING finger and NLS mutations in two subsequent reactions using the QuikChange kit. The plasmid expressing GFP-tagged Rab7 was kindly provided by Dr. Harald Stenmark at Rikshospitalet, Oslo, Norway.
Cell fixation and immunofluorescence labeling was performed as described previously . Primary antibodies used were mouse anti-His (HIS-H8), mouse anti-PML (PG-M3), rabbit anti-PML (H238) mouse anti-Lamp1 (H5G11) and mouse anti-Lamin A/C (636) (all from Santa Cruz, CA), rabbit anti-Lamin B1 (Abcam, UK) and mouse anti-Flag (F3165) (Sigma, MO). Secondary antibodies were FITC or Texas-Red labeled goat antibodies against mouse immunoglobulin subtypes or against rabbit antisera (Southern Biotech, AL).
Microscopic images were obtained using a Zeiss LSM510 Meta laser confocal microscope (Zeiss, Germany) with a 63× oil immersion lens (Zeiss).
Chemicals and treatment
DRAQ5 treatment was performed by adding DRAQ5 (Biostatus, UK) to a final concentration of 2 μM, to the medium for two hours before fixation. Actinomycin D treatment was performed by adding Actinomycin D (Sigma), to a final concentration of 5 μg/ml, for four hours before fixation. To quantify cells with PML at the nuclear periphery, cells were counted manually in the microscope. For each sample, four hundred cells were counted and scored for the presence of PML staining at the nuclear periphery. In each case, two independent parallels were counted.
The authors would like to thank Professor Roger Everett at MRC Virology unit in Glasgow, Scotland for generously providing the lentivirus vectors expressing FLAG-tagged PML. ÅJL was funded by the Norwegian Cancer Society, AG was funded by the University of Bergen and SOB was funded by the Norwegian Research Council. The work was further supported by the Cancer Gene Therapy Program funded by the Norwegian Health Department and Helse-Vest.
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