We have previously reported the cloning of the Hematopoietic Pbx Interacting Protein (HPIP), a novel protein discovered through its interaction with Pbx1. HPIP is expressed in early hematopoietic precursors, can bind all members of the Pbx family and can inhibit the transcriptional activation of the oncogene E2A-Pbx. To further understand the function of HPIP, we have analysed its cellular localization and characterized its functional localization domains. Using fluorescence microscopy to follow the distribution of different HPIP sequences fused to GFP, we found that HPIP localizes predominantly to cytoskeletal fibers but has the potential ability to shuttle between the nucleus and the cytosol. The cytoskeletal localization of HPIP is mediated by an N-terminal leucine rich region (between aa 190–218) and can be disrupted by the microtubule destabilizing drug vincristine. The HPIP C-terminal domain (aa 443–731) bears a nuclear export activity that is blocked by the CRM1 inhibitor Leptomycin B. In addition, we found two basic amino acid regions located between aa 485–505 and aa 695–720 that contain nuclear import activities attenuated by nuclear export. These observations support a model in which the constitutive attachment of HPIP to the cytoskeleton could be modified by changes in functional domains implicated in nuclear export, import and cytoskeleton binding sequences, allowing the molecule to shuttle between the nucleus and the cytosol.
Pbx1 is a transcription factor originally discovered as the chromosome 1 participant in the translocation t(1;19) that produces the fusion protein E2A-Pbx, seen in 25% of pediatric acute lymphoblastic leukemia (Kamps et al., 1990; Nourse et al., 1990). Pbx1 possesses a divergent homeodomain DNA binding motif and appears to function in collaboration with other homeodomain containing proteins as part of large nucleoprotein complexes (Berthelsen et al., 1998; Goudet et al., 1999; Jacobs et al., 1999; Shen et al., 1999). Normal regulation of the proteins involved in these complexes is essential for both normal embryonic development (Selleri et al., 2001; Veraksa et al., 2000; Capecchi, 1997) and the establishment and maintenance of definitive hematopoiesis (Sauvageau et al., 1995, 1997; Lawrence et al., 1996; Fuller et al., 1999; Buske et al., 2001; Thorsteinsdottir et al., 2001; Dimartino et al., 2001).
Given the important role of Pbx1 in hematopoietic development and in E2A-Pbx associated pre B-acute lymphoid leukemias, we sought to understand further its mechanism of action. As we have previously reported, a search for novel Pbx1 interacting proteins in the hematopoietic system resulted in the identification of the Hematopoietic Pbx Interacting Protein or HPIP (Abramovich et al., 2000). HPIP can bind to the different members of the mammalian Pbx family, inhibit the binding of Pbx1/Hox complex to DNA and block the transcriptional activity of E2A-Pbx (Abramovich et al., 2000). HPIP expression overlaps with that of Pbx1 in multiple tissues and early embryos. Interestingly, both HPIP and Pbx1 have a restricted pattern of expression in hematopoietic cells, with highest expression in early progenitor cells, e.g. CD34+ (Abramovich et al., 2000), which suggests a role for HPIP-Pbx interaction during primitive stages of hematopoiesis. High-density oligonucleotide array analysis has further confirmed the expression of HPIP in CD34+ cells and revealed highest level of expression in CD34+ G-CSF mobilized peripheral blood cells compared with their bone marrow counterparts (Graf et al., 2001). The likely importance of HPIP in hematopoiesis is further supported by our recent findings showing increased numbers of primitive hematopoietic cells active in vitro and in vivo upon retrovirally engineered over-expression of HPIP in CD34+ human cord blood cells (Buske et al., manuscript in preparation).
The molecular mechanism of HPIP function is poorly understood. Our previous data suggest that HPIP activity involves, at least in part, the control of Pbx mediated gene expression (Abramovich et al., 2000). Pbx1 has been shown to constitutively localize to the cytosol by a mechanism that involves its export from the nucleus by the CRM1 receptor (Berthelsen et al., 1999; Abu-Shaar et al., 1999). The presence of Pbx1 in the nucleus is mediated by its association with the homeobox-containing protein Meis1, and depends on the Pbx1 nuclear localization signal (NLS) (Berthelsen et al., 1999; Abu-Shaar et al., 1999). We have previously reported that HPIP localizes mainly to the cytosol (Abramovich et al., 2000). As the subcellular localization usually indicates the potential function of a protein, we set out to further study HPIP localization and to map its localization signals. We show here that HPIP has a complex subcellular distribution; it is largely bound to the cytoskeleton but has the potential ability to shuttle between the nucleus and the cytoplasm by mechanisms that involve nuclear import and export signals.
Results and Discussion
HPIP has two functional nuclear localization signals
Our previous results using conventional fluorescence microscopy and subcellular fractionation of COS and NIH3T3 cells expressing GFP–HPIP and FLAG–HPIP, indicated that while predominantly cytoplasmic, HPIP is found in small amounts in the nucleus (Abramovich et al., 2000). In this study we used confocal microscopy to follow the localization of the fused proteins. The expression of full-length HPIP in the nucleus of COS and NIH3T3 cells was below the level of detection (data not shown), and therefore, we concluded that HPIP is not constitutively present in the nuclear compartment at significant levels.
Nuclear import is usually conferred by a nuclear localization signal, a basic sequence sufficient and necessary for nuclear import of the protein. There are two major types of NLSs: (i) a single stretch of five to six basic amino acids, exemplified by the SV40 large T-antigen NLS; and (ii) a bipartite NLS composed of two basic amino acids, a spacer region of 10–12 amino acids, and a basic cluster in which three of five amino acids must be basic, typified by nucleoplasmin (Silver, 1991; Adam and Adam, 1994). Our initial analysis of the HPIP amino acid sequence revealed the presence of two putative nuclear localization signals (RRRR at 160 and KHKK at 489) (PSORT II). To determine whether their lack of function could explain the exclusion of HPIP from the nucleus, we evaluated if they could promote nuclear localization of the heterologous protein GFP in COS cells. The small size of GFP, 27 kDa, and the lack of endogenous localization signals allow the protein to distribute between the nuclear and cytoplasmic compartments (Figure 1a). As shown in Figure 1b, the localization of GFP fused to HPIP spanning aa 485–505 (containing the sequence KHKK, see Table 1) was nuclear with strong nucleolar staining. In contrast, the localization of GFP fused to HPIP aa 145–167 (containing the sequence RRRR, see Table 1) was similar to that of GFP and was distributed throughout the cell (Figure 1c). These results demonstrate that a functional NLS is resident within HPIP residues 485–505. Surprisingly, a HPIP deletion mutant lacking this NLS, GFP–HPIP505-731 still localized to the nucleus (Figure 1d), suggesting the presence of an additional active NLS. Indeed, further analysis of the HPIP sequence shows the presence of a basic aa-rich region that resembles an NLS at position 695–720. As shown in Figure 1e, GFP–HPIP695-720 (see Table 1) gives strong nuclear and nucleolar staining, demonstrating that HPIP contains at least two functional NLSs. These NLSs are active in the context of a truncated HPIP protein, as HPIP443-731 showed constitutive nuclear and nucleolar localization (Figure 1f). In view of the reported observation of nucleoli localization of the Pbx cofactors Hox B7, C6 and D4 (Corsetti et al., 1995), the presence of signals that allow HPIP to localize to the nucleoli may indicate a specific function for the protein in this compartment. Taken together, our results indicate that the nuclear exclusion of HPIP is not due to the lack of functional NLSs, and that there may be other mechanisms acting on the full‐length protein that facilitate its cytoplasmic localization, including active export of the protein from the nucleus, masking of its NLSs and/or its active retention in the cytoplasm.
HPIP has a functional nuclear export signal
To determine the region(s) of HPIP required for its cytosolic localization, we added HPIP sequences to the N′-terminus of HPIP443-731, and followed the localization of the GFP-fusion proteins. Interestingly, the addition of only 41 aa (HPIP402-731) shifted the majority of the nuclear localization seen for HPIP443-731 to the cytosol (Figure 2a), indicating that sequences located between aa 402–443 are capable of localizing HPIP to the cytosolic compartment.
Active export of proteins from the nucleus requires the presence of nuclear export signal (NES). The most common NES motif in proteins is a stretch of characteristically spaced hydrophobic amino acids, particularly leucine residues, which are requisite for function (Bogerd et al., 1996; Kim et al., 1996). In the nucleus, this motif is recognized by the nuclear export receptor CRM1, which forms a competent nuclear export complex together with RanGTP (Fukuda et al., 1997; Fornerod et al., 1997).
To test whether HPIP402-731 is cytosolic due to the presence of functional NES recognized by the CRM1 receptor, we transfected the GFP-fusion protein into COS cells and followed its localization before and after treatment of the cells with leptomycin B (LMB), a drug that binds CRM1 covalently and inhibits its function (Kudo et al., 1998, 1999). While the vast majority of cells expressing GFP-HPIP402-731 showed cytoplasmic staining, there was a striking shift to nuclear localization after treatment with 10 mM of LMB for 2 h (Figure 2a). This response indicates that HPIP sequences between aa 402–731 contain a functionally active NES and that CRM1 is therefore a likely candidate as the nuclear export receptor for HPIP. Our results also imply that HPIP402-731 can shuttle between the nucleus and the cytoplasm, and that its mainly cytosolic localization can be due to the nuclear export being more efficient than the nuclear import. A similar observation has been reported in a fusion protein carrying the large T NLS of SV40 and the leucine-rich NES of PKI (Stade et al., 1997) as well as in the transcription factor NF-AT (Zhu and Mckeon, 1999) and the STE-20 like kinase MST (Lee and Yonehara, 2002). Interestingly, the HPIP sequence encompassing aa 402–443 contains a leucine-rich region that resembles an NES recognized by the CRM1 receptor. To determine whether this region is responsible for the CRM1 mediated export activity, we tested if it can cause the cytoplasmic accumulation of GFP (GFP–HPIP402-443, see Table 1). Surprisingly, GFP–HPIP402-443 was found expressed throughout the cell and did not have predominant cytoplasmic localization that redistributed throughout the cell after LMB treatment (Figure 2b), a property expected if it had NES activity mediated by the CRM1 receptor. This result implies either that aa 402–443 contain an NES that needs additional aa located C-terminus to aa 443 to be functional, or that addition of aa 402–442 to HPIP443-731 does not add a NES but rather unmasks an otherwise inactive NES contained within aa 443–731. We favor the latter explanation as a longer fusion protein, containing HPIP aa 402–450 (GFP–HPIP402-450) failed to show predominant cytoplasmic localization. In an effort to map the NES, we fused GFP to the leucine-rich region contained within aa 443–731 (GFP–HPIP598-640). This region also failed to drive GFP to the cytosol (Figure 2c), indicating that it is not responsible for the NES activity. Together, these results suggest that the export of HPIP402-731 is not mediated by a typical CRM1 mediated NES. The fact that full-length HPIP did not accumulate in the nucleus after LMB treatment (Figure 2d), implies another mechanism facilitating its cytoplasmic retention; HPIP may not be efficiently transported into the nucleus by the nuclear import machinery or it may interact with proteins that retain it in the cytosol.
HPIP associates with cytoskeletal fibers
GFP–HPIP distribution resembles that of cytoskeletal fibers throughout the cytoplasm with brighter staining surrounding the nucleus (Figure 3a). To test whether HPIP interacts with the cytoskeleton, COS cells were detergent-extracted prior to fixation, a procedure that leaves the cytoskeleton and its associated proteins intact yet removes cytoplasmic and membrane-associated proteins. The same pattern of staining was obtained after detergent extraction, suggesting co-localization of HPIP with cytoskeleton (Figure 3b). As GFP–HPIP seems to decorate fibers that resemble microtubules, we checked its distribution after disruption of the microtubule cytoskeleton by vincristine (Figure 3c right image). This treatment completely abolished the fiber-like staining pattern (Figure 3c left image), suggesting that HPIP is associated with microtubules or that its interaction with other cytoskeletal structures depends on the integrity of the microtubule network. Whether HPIP binds directly to the cytoskeleton or merely to associated proteins cannot be deduced from this study. The association of HPIP with the cytoskeleton may provide a regulatory mechanism controlling the availability of functional protein, as it was suggested for Smad 2, 3 and 4, where microtubules seem to serve as a cytoplasmic sequestering network (Dong et al., 2000). Dissociation from the cytoskeleton following a yet undiscovered stimulus may free HPIP and facilitate its translocation to the nucleus.
To determine which sequences of HPIP are responsible for its co-localization with the cytoskeleton, we looked for cytoskeleton association of GFP–HPIP proteins containing HPIP aa 1–159, aa 160–731, aa 263–731, aa 401–731 and aa 505–731 respectively. Only the GFP fusion containing HPIP sequences between aa 160–731 showed cytoskeletal localization (data not shown), indicating that the cytoskeletal binding region is located between aa 160 and 263. Surprisingly, the leucine rich region spanning aa 190–218 (GFP–HPIP190-218, see Table 1) was enough to localize GFP to the cytoskeleton (Figure 4a). To confirm that this sequence is responsible for HPIP association with the cytoskeleton, we examined the localization of a deletion mutant lacking this region (GFP–HPIPΔ190-218). As shown in Figure 4b, the deletion of aa190–218 abolished the binding of HPIP to the cytoskeleton, confirming that this sequence is responsible for the association. As HPIP190-218 resembles an NES recognized by CRM1, we checked whether LMB could redistribute the cytosolic staining throughout the cell. LMB did not have any effect on the localization of GFP–HPIP190-218 (Figure 5c), indicating that this leucine-rich region either does not contain a functional NES or that it is masked by the binding to the cytoskeleton. Interestingly, LMB did not affect the localization of GFP–HPIPΔ190-218 (Figure 5d) or of full‐length GFP–HPIP (data not shown) treated with vincristine, indicating that the ability of HPIP to shuttle is masked not only by its binding to the cytoskeleton but by some other region(s) within aa 1–189 and 219–401, the latter including the coiled-coil domain (aa 241–389).
In summary, we have defined the sequences of HPIP responsible for its subcellular localization, which include two NLSs, an NES and a cytoskeleton binding region (see Figure 5 for schematic representation of known HPIP functional domains). The evidence presented indicate that these domains confer the potential of HPIP to be a nucleocytoplasmic shuttle protein whose constitutive cytoplasmic localization requires anchoring to cytoskeletal fibers and perhaps nuclear export signal function. The identification and mapping of these localization signals provide a framework for future investigation of the significance of HPIP subcellular localization in its biological functions (e.g. regulation of Pbx), as well as of the signals that facilitate its shuttling between the nucleus and cytosol.
Materials and methods
Cell culture, transient transfections and immunofluorescence studies
COS 7 and NIH3T3 cells were cultured in DMEM supplemented with 10% FCS. For all experiments the cells were plated on coverslips in 6-well plates and 24 h later transfected using Lipofectamine Plus™ Reagent, as specified by the manufacturer (Gibco BRL, Burlington, ON, Canada). Cells were fixed 48 h later with 3% paraformaldehyde in PBS and either stained for tubulin and/or stained with DAPI, mounted and viewed using a Zeiss Axioplan 2 fluorescence microscope or Delta Vision deconvolution microscope system (Applied Precision, Issaquah, WA, USA). Images were acquired using a 12 bit cooled imaging Sensicam camera and MetaSystem software or the Delta Vision system respectively. For tubulin staining, cells were permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with CY3 conjugated monoclonal anti-β-tubulin (Sigma Chemical, St. Louis, MO, USA) at 1 : 50 dilution for 1 h, followed by four washes with PBS.
For removal of soluble proteins, prior to fixation the coverslips were washed twice with cytoskeleton-stabilizing buffer (4 M glycerol, 25 mM PIPES pH 6.9, 1 mM EGTA and 1 mM MgCl2) and incubated for 5 min with cytoskeleton-stabilizing buffer containing 0.2% Triton X-100.
Construction of GFP-HPIP fusion plasmids
Constructs were generated by gene fusion of various parts of HPIP coding sequence to the 3′ end of the gene for enhanced Green Fluorescence Protein (GFP). The recombinant DNA molecules were created using PCR with specific primers containing restriction sites, cut with the respective enzymes and inserted into the pEGF-C1 vector (Clonetech, Palo Alto, CA, USA). The deletion mutant GFP–HPIPΔ190-218 was obtained by simultaneously inserting fragments encoding aa 1–190 and aa 218–731 into the XhoI–BamHI site of pEGC-C1. A HindIII site was added to join the two HPIP fragments.
All the constructs were verified by sequencing and they are available upon request.
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This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Terry Fox Foundation; and the National Institute of Health (Grant Number HL65430). C Abramovich was the recipient of fellowships from the Leukemia Research Society of Canada and the Canadian Institutes for Health Research. We thank Ian Waissbluth for his technical assistance and Drs Minoru Yoshida (Tokyo University, Japan) and Tom Hope (Salk Institute for Biological Studies, La Jolla, CA, USA) for kindly providing LMB.
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Cite this article
Abramovich, C., Chavez, E., Lansdorp, P. et al. Functional characterization of multiple domains involved in the subcellular localization of the hematopoietic Pbx interacting protein (HPIP). Oncogene 21, 6766–6771 (2002). https://doi.org/10.1038/sj.onc.1205784
- hematopoietic Pbx interacting protein
- cellular localization
- nuclear import
- nuclear export
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