A Carboxyl Leucine-Rich Region of Parathyroid Hormone-Related Protein Is Critical for Nuclear Export
http://www.100md.com
《内分泌学杂志》
Division of Biological Sciences (J.C.P.), Division of Endocrinology, Department of Medicine (D.W.B., L.J.D.)
Department of Anesthesiology (R.H.H.), University of California, San Diego, California 92161
Medicine Service (D.W.B., L.J.D.) and Anesthesiology Service (R.H.H.), Veterans Affairs San Diego Healthcare System, San Diego, California 92161
Abstract
PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein’s nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109–139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1–108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109–139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126–136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126–136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.
Introduction
TRANSPORT OF LARGE macromolecules into and out of the nucleus is a regulated process that requires binding to specific transport proteins to traverse the nuclear pore complex (NPC). The ability of a protein to complex with import and export proteins is endowed by nuclear localization signals (NLS) or nuclear export signals (NES), respectively. NLSs are frequently monopartite or bipartite clusters of basic residues that mediate binding to a member of the karyopherin nuclear transport family, such as importin- (1). On the other hand, an NES is a loosely conserved motif typically consisting of specifically spaced sequences of leucines or other hydrophobic amino acids, typically separated by charged or polar spacer amino acids. A protein containing a leucine-rich NES can form a complex with nuclear export receptor chromosome region maintenance protein 1 (CRM1)/exportin-1 and the small GTPase protein Ran in its GTP bound form (2). This complex is then translocated from the nucleus to the cytoplasm by facilitated diffusion through the NPC. RanGAP, a cytoplasmic protein, activates the intrinsic GTPase activity of Ran; hydrolysis of the terminal phosphate of GTP disrupts the NES-CRM1-Ran complex and releases the shuttled protein into the cytoplasm. CRM1 then cycles back to the nucleus for another round of NES export. Leptomycin B (LMB), a small fungicide derived from a strain of Streptomyces, inhibits CRM1-mediated transport by interfering with the association of CRM1 and the NES (3, 4).
PTHrP is best known for mediating hypercalcemia when secreted by solid tumors (5). However, the protein can act locally at the cells of many normal tissues and cancers not connected with hypercalcemia. The local effects include paracrine or autocrine actions on growth, apoptosis, and smooth muscle relaxation as well as calcium transport (6, 7, 8, 9, 10). Several portions of the molecule act at the cell surface, presumably through receptor binding, but the only known receptor is the PTH/PTHrP receptor that is shared between PTHrP 1–34 and PTH 1–34. The protein can also escape the secretory pathway to remain inside the cell (11, 12). Intracellular retention has functional significance; PTHrP exerts intracrine effects in the cell nucleus, predominantly affecting growth and apoptosis (13, 14).
Nuclear localization of PTHrP represents a balance between nuclear import and export and appears to be regulated in some cell types (15). PTHrP possesses an NLS within residues 67–94 that is capable of binding to importin- directly without an intervening role for importin- (11, 16). Inside the nucleus, PTHrP targets the nucleoli in most tissues, likely through the auspices of additional basic residues close to the NLS (13, 17). The protein resides in the nuclei during the G1 phase of the cell cycle, but phosphorylation of threonine 85 by cdk2 during S phase reduces the rate of nuclear import and shifts PTHrP to the cytoplasm (15, 18). Cell cycle-based redistribution of nuclear PTHrP could not occur if the protein did not shuttle back and forth from cytoplasm to nucleus.
PTHrP fused to green fluorescent protein (GFP) has been demonstrated to undergo nuclear export, and the rate of export is sensitive to LMB, demonstrating that transport depends on CRM1 (12). However, the precise location of the NES of PTHrP has not been identified. Our goal in this study was to determine whether PTHrP is exported from the nucleus directly via a leucine-rich NES. Based on the primary structure, we considered the amino acid 109–139 sequence, which contains many leucine residues, as the most likely region of PTHrP to contain an NES. We performed sequence analyses comparing PTHrP 109–139 with known NESs from other proteins and selected a smaller domain to test as a putative NES. We then evaluated the importance of this region for nuclear export of GFP-containing PTHrP chimeras that contained the full sequence or various deletion mutations.
Materials and Methods
Chemicals
Hoechst 33342, LMB, and cycloheximide were purchased from Calbiochem (San Diego, CA). The restriction enzymes and Quick ligation kit were purchased from New England Biolabs (Beverly, MA). The mutagenic primers were purchased from Proligo (Boulder, CO). All other chemicals, unless specified, were obtained from Sigma Chemicals (St. Louis, MO).
Construction of chimeras containing GFP and PTHrP
The pEGFP-C1 vector was obtained from CLONTECH (Palo Alto, CA) and was used as the template for the GFP-PTHrP chimeras. The PTHrP 1–139 and PTHrP 1–108 fragments were generated from the human PTHrP expression plasmid, pCi-neo-PTHrP1–173 (19), using specific primers flanked by SalI and XbaI restriction sites. Both pEGFP-C1 vector and the PCR products were digested with SalI and XbaI, ligated together using a Quick ligation kit and transformed into XL-1 blue cells (Stratagene, La Jolla, CA). PTHrP immunoassays and DNA sequencing confirmed the integrity of the plasmid preparations.
The entire gene encoding EGFP, except for the ATG start codon, was amplified via PCR from the pEGFP-C1 vector using PCR primers containing 5' BglII and 3' XhoI restriction sites (lowercase letters): forward, 5'-CTCagatctGTGAGCAAGGGCGAGGAGCTG-3', and reverse, 5'-GAGctcgagCTTGTACAGCTCGTCCATGCC-3'. This PCR product was used as the insert to make the GFP2 construct. Next, the EGFP gene was PCR amplified using a second set of PCR primers containing 5' XhoI and 3' SalI restriction sites (lowercase letters; forward, 5'-CTCagatctGTGAGCAAGGGCGAGGAGCTG-3', and reverse, 5'-ACCgtcgacCTTGTACAGCTCGTCCATGCC-3') to make the GFP3 PCR product. After digesting both the pEGFP-C1 vector and the 5' BglII and 3' XhoI PCR product with BglII and XhoI, the fragments of interest were gel purified from a 1% SeaPlaque low-melting-point agarose gel (Cambrex, Baltimore, MD), extracted using a QIAquick gel extraction kit (QIAGEN Inc., Valencia, CA), and ligated together in a 1:1 molar ratio to make the pEGFP2 vector. To convert the pEGFP2 vector to a pEGFP3 vector, the same cloning procedure was followed using the second PCR insert containing the 5' XhoI and 3' SalI sites and the pEGFP2 vector with XhoI and SalI as restriction enzymes.
The GFP3-PTHrP 1–108, 139, and 173 constructs were created by transferring the PTHrP insert from their respective single GFP expression vectors using SalI and XbaI with the same reagents and cloning procedures as described above.
Mutagenesis
To generate the mutations within PTHrP 126–139, we used the Quick change XL kit (Stratagene) as per the manufacturer’s recommendations. Mutations were confirmed by DNA sequencing.
Cell culture
HeLa, COS-1, and 293 cells, derived from human ovarian cancer, African green monkey kidney, and human embryonic kidney, respectively, were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in monolayer using DMEM (Cellgro, Herndon, VA) supplemented with 5–10% fetal bovine serum (Gemini Bio-Products, Woodland, CA) in a humidified incubator at 37 C with 95% air and 5% CO2.
Transfections
For transfection studies, the cells were plated at 60–70% confluency and incubated overnight as described above. The 293 and HeLa cells were transfected using GenePORTER-2 (Gene Therapy Systems, San Diego, CA) and the COS-1 cells were transfected using GenePORTER-1 according to the manufacturer’s instructions. Typical transfection efficiencies were approximately 75, 55, and 90% for 293, HeLa, and COS-1 cells, respectively, as measured by the percentage of cells fluorescing in pEGFP-C1-transfected cell preparations.
Immunoblotting
COS-1 cells were trypsinized after transfection and pelleted by centrifugation at 200 x g for 5 min. The cell pellets were sonicated for 10 sec in cell lysis buffer (PBS with 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate) and total protein concentrations in the cell lysates were measured using a BCA protein assay (Pierce Chemical Co., Rockford, IL). The proteins (20 μg/lane) were separated using a 4–12% Bis-Tris SDS-PAGE gel in 3[N-morpholino]propanesulfonic acid buffer (Invitrogen Corp., Carlsbad, CA) and then transferred to a nitrocellulose membrane. The membrane was blocked for nonspecific binding by incubating in 10% nonfat dry milk (NFM) in Tris-buffered saline [10 mM Tris-HCl (pH 8.0), 150 mM NaCl] for 30 min, washed three times in Tris-buffered saline, and incubated in the presence of 1 μg/ml anti-GFP monoclonal antibody (Molecular Probes, Eugene, OR) in 5% NFM overnight at 4 C on a rotating shaker. Between multiple washes, the membrane was incubated in goat-antimouse IgG conjugated to horseradish peroxidase (Calbiochem) and diluted in 5% NFM overnight at 4 C on a shaker. The immunoreactive proteins were detected using a chemiluminescence reaction kit (Pierce Chemical) and exposed for 1–2 min on a Kodak MIN-R 2000 x-ray film (Eastman-Kodak Co., Rochester, NY). To control for protein loading, the membranes were subsequently immunostained for -tubulin levels (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
PTHrP immunoassays
Conditioned media and cell extracts from transfected cells were assayed for PTHrP expression by region-specific RIAs (20, 21). In brief, human PTHrP 1–34, 38–64, or 109–141 peptides were used as standards, and PTHrP 1–86 and tyr-108–141 peptides were radioiodinated by the chloramine T method. Rabbit PTHrP antibodies directed against PTHrP 1–34, 38–64, or 109–141 were used in a 3-d nonequilibrium immunoassay format, as previously described (20, 21). All samples were assayed in triplicate in multiple dilutions that paralleled the corresponding PTHrP standard curve and the intra- and interassay variations were approximately 7–12%.
Static fluorescence microscopy and image analysis
To assess steady-state cellular distributions of PTHrP, 293 cells were plated in 35-mm glass bottom culture dishes (MatTek, Ashland, MA) and transfected with GFP3-PTHrP chimeras. Forty-eight hours after transfection, the media were replaced with normal growth media with or without 4 ng/ml LMB and incubated for 3 h. Thirty minutes before the end of this incubation, Hoechst 33342 was added to the media to stain the nuclei. The cells were then fixed in 3.7% paraformaldehyde in PBS for 30 min, washed three times with PBS, and mounted with antifade media (Biomeda Corp., Foster City, CA).
The cell fluorescence was analyzed using a DMLB microscope (Leica Microsystems, Buffalo, NY) equipped with a 100-W mercury lamp and standard fluorescein isothiocyanate and 4',6'-diamino-2-phenylindole filter cubes (Chroma Technology Corp., Rockingham, VT). The GFP and Hoechst 33342 fluorescence images were captured with a Spot RT 220–3 digital camera (Diagnostic Instruments, Sterling Heights, MI). The images were analyzed and calibrated using Image-Pro Plus version 4.5.1.29 software (Media Cybernetics, Silver Spring, MD). Average pixel intensity measurements were calculated for a 400 square pixel area (16 μm2) area of interest (AOI) as follows: first, the AOI was positioned over a nuclear, nonnucleolar region and the average intensity was recorded. Next, the AOI was repositioned to a representative region of the cytoplasm of the same cell and the average intensity was recorded. Correct placement of the cytoplasmic AOI relative to the nucleus was confirmed using the Hoechst 33342 image to define the nuclear boundary. The data were exported into Microsoft Excel (Microsoft, Redmond, WA), and the distribution of the fluorescent PTHrP protein between the nucleus and cytoplasm was quantified by the ratio of the fluorescence of the two compartments, Fn/c.
Confocal microscopy
All time course experiments were performed with a Zeiss Axiovert 110M microscope equipped with an LSM 510 confocal laser scanning system using the LSM software version 3.2sp2 (Carl Zeiss MicroImaging, Inc., Thornwood, NY). The in vitro export assay and fluorescence recovery after photo bleaching (FRAP) studies used a Plan Neofluor 40x/1.3 oil objective, whereas fluorescence loss in photobleaching (FLIP) used the Plan Apochromat 63x/1.4 oil objective. Scan settings were the same for all experiments: GFP was excited with 5% of Ar/Kr laser output on the 488-nm wavelength laser line coupled to a BP505–550 emission filter. Propidium iodide (PI) was excited using 40% of the Ar/Kr laser output on the 568-nm laser line coupled to a LP585 emission filter. Two 12-bit 1024 x 1024 images were obtained in line mode at a rate of 3.9 sec per image (7.8 sec total scan time) and averaged to reduce background noise. Generally, six 1-μm Z stacks were obtained for each scan to adjust for focal drift. At each time point, the analysis used the Z stack with the optimal plane of focus.
FLIP
Before microscopy, HeLa cells transfected with single GFP-PTHrP chimera expression plasmids were incubated for 30 min with 50 μg/ml cycloheximide in 25 mM HEPES (pH 7.4). Each FLIP assay focused on an individual transfected cell. The cell was brought into focus on the stage of the confocal microscope preheated to 37 C using an air stream stage incubator (Nevtek, Burnsville, VA). It was scanned with the settings described in the previous section and the pinhole wide open at 1 mm. Between each scan, the cytoplasm AOI was photobleached for 20 iterations using 50% laser output of both 488 and 568 laser lines. Three rounds of scanning and bleaching reduced cytoplasmic fluorescence to background levels. Nuclear export was assayed beginning with the very next scan. Nuclear fluorescence was quantified with ImagePro software and expressed as a percentage of the fluorescence on the initial scan.
The LSM software (Carl Zeiss MicroImaging) has a photobleaching technology that deserves further description. The laser scans in a raster pattern and uses a shutter guided by a user-defined bleaching AOI to control whether an individual point in the field is excited. The AOI is selected with the polygon drawing tool and can take an irregular shape (22). Thus, the bleaching template can include the entire cytoplasm but exclude the nucleus (as shown in Fig. 8A) using a FRAP experiment as an example. We typically use the phase contrast image to aid in drawing the AOI. The area of the template was kept nearly constant from cell to cell and was set slightly larger than each of the cells studied. Thus, the laser dwell time and the interval between images was relatively even among the analyses.
The purpose of the FLIP studies was to determine the importance of sequences within the PTHrP 109–139 region for nuclear export of PTHrP. We compared export rates for GFP-PTHrP 1–108 and GFP-PTHrP 1–139 because the two proteins are close in size, 39 vs. 42 kDa, respectively. The upper size limit at which passage through the NPC is restricted is only slightly larger. In contrast, GFP at 27 kDa is well below the size limit for passive diffusion through the NPC. In FLIP studies, it escapes the nucleus nearly as rapidly as cytoplasmic GFP can be photobleached (23).
In vitro nuclear export assay
This assay was adapted from protocols described by others (3, 19). Basically, cells transfected with the fluorescent-labeled protein of interest are first permeabilized. This step eliminates the protein from the cytoplasmic compartment and depletes the cell of endogenous RanGTP, a necessary cofactor for nuclear transport (3). We use PI, which is excluded from intact cells, to identify permeabilized cells. Export of the protein from the nucleus is held in check and begins when exogenous RanGTP is added to the assay buffer. Replacement of CRM1 is unnecessary because the large protein is not depleted by permeabilization (24).
COS-1 cells were used 48 h after transfection with GFP3-PTHrP chimera expression plasmids. The transfected cells were washed with transport buffer [TB: 20 mM HEPES-KOH (pH 7.3), 110 mM KOAc, 2 mM Mg(OAc)2, and 1 mM EGTA] supplemented with 10% fetal bovine serum and then washed in ice-cold complete TB (cTB: TB supplemented with 2 mM dithiothreitol; 1 mM phenylmethylsulfonyl fluoride; and 1 μg/ml each of pepstatin, leupeptin, and aprotinin). Permeabilization was performed with 40 μg/ml digitonin in ice-cold cTB for 5 min on ice. Next, cells were washed twice with ice-cold cTB, incubated on ice for 15 min with cTB containing 4 μg/ml PI, washed again, and left in 100 μl cTB in the central cavity of the MatTek 35-mm, glass-bottom culture dish. The cells were brought into focus on the preheated stage. Finally, 20 μl 5x assay buffer (5 μM Ran, 5 μM GTP, 1 μM ATP, 2.5 mg/ml BSA) were added to the cell suspension, the lid was replaced, and imaging began immediately. The two fluorophores were imaged on separate tracks to prevent cross talk of the fluorophores. Data were collected only for cells that were PI positive. The pinholes of each channel were wide open (1 mm). Nuclear fluorescence was measured and presented as in the FLIP experiments.
FRAP
Cells were transfected with GFP3-PTHrP chimeras and treated before the experiment with cycloheximide as described for FLIP. The PI track was disabled and the pinhole size for the GFP track was adjusted to 1.5 airy units. One image was acquired before photobleaching to outline an AOI including the entire cytoplasm as described above. The selected region was bleached for 80 iterations with a laser output of 100%. Bleaching the GFP3 chimeras required a higher output than the 50% used for the single GFP constructs that were used for FLIP. Scans were then collected with the standard scan settings on a continuous cycle for the remained of the assay with no further bleaching.
Fluorescence in nuclear and cytoplasmic compartments were measured with ImagePro, exported to Excel, and expressed as Fn/c. The kinetics of export were analyzed by fitting data points exponentially using SigmaPlot software (SPSS, Chicago, IL) to a three-parameter model adapted from models used by others (25): Fn/c = span (1 – e–kt) – top, where top is the relative Fn/c ratio immediately after photobleaching and span represents the extent of exported protein. Half-time (t1/2) of nuclear export was defined as ln(2)/k.
Results
GFP-PTHrP 1–108 exports from the nucleus at a slower rate than GFP-PTHrP 1–139
HeLa cells transiently transfected with GFP-PTHrP 1–139 or GFP-PTHrP 1–108 demonstrated cytoplasmic and nuclear GFP fluorescence, with signal localizing to nucleoli as well (Fig. 1A, left panels). Nucleolar fluorescence for the truncation mutant chimera was greater than for the wild-type construct. Continual photobleaching with the FLIP protocol reduced cytoplasmic fluorescence to background after 100 sec (image not shown). After 600 sec, nuclear fluorescence had been lost, compared with baseline for both constructs. The decrement was noticeably greater for GFP-PTHrP 1–139, compared with GFP-PTHrP 1–108 (Fig. 1A, right panels). Bleaching maintained cytoplasmic fluorescence at background levels. Image analysis showed that nuclear fluorescence decreased linearly with time for both GFP-PTHrP chimeras, but GFP PTHrP 1–108 exported at a significantly (P < 0.01) slower rate than GFP PTHrP 1–139 (Fig. 1B).
Sequence alignments of known NESs with PTHrP 109–139
We compared experimentally verified NESs to the PTHrP 109–139 sequence (26) (Fig. 2). Three leucine-rich sequences within the 109–139 region, PTHrP 112–121 (pNES1), PTHrP116–126 (pNES2), and PTHrP126–136 (pNES3), had at least partial homology with other known export signals (Fig. 2). NES consensus sequences are typically approximately 10 amino acids in length with a semidefined consensus of LX2–3LX2–3LXL (27, 28, 29). The last three residues appear to be the most critical for function; typically they consist of a leucine or isoleucine separated by a single polar amino acid from a final leucine (30). The earlier leucine positions in the consensus are not as critical and may be substituted occasionally by other amino acids. Based on these criteria, PTHrP 126–136 was the region most likely to be an NES. That sequence presented significant homology with known NES in human MAP kinase, human p53, human PKI, and other proteins that undergo nucleocytoplasmic shuttling. In particular, it contained the requisite structure in its ultimate tripeptide, LEL134–136. The two upstream candidate sequences lacked the penultimate leucine residue. Jans et al. (15) suggested in a review article that this region was a likely NES for PTHrP, although they provided no supporting data or references. Because of our own sequence analyses and the supporting literature, we focused on PTHrP 126–136 as the potential NES in subsequent mutagenesis studies.
Constructs to test regulated nuclear export of PTHrP
We engineered new chimeric PTHrP forms to study the role of pNES3 in active export of PTHrP. The single GFP chimeric proteins are small enough in mass, less than 48 kDa, to diffuse through the NPC (31) without the assistance of CRM1. To prevent passive mechanisms in the export assays, we made the chimeras large using a triple GFP expression vector (GFP3) (Fig. 3) analogous to one described by a previous investigator (32). We confirmed the identity of the constructs by DNA sequencing, and we demonstrated their functionality with transfection studies. Transfected cells expressed proteins containing amino-terminal, midmolecule, and carboxyl-terminal PTHrP immunoreactivity as expected from the construct coding sequences. For example, the levels of PTHrP 1–34 in conditioned media from cells transfected with GFP3PTHrP 1–108 and GFP3PTHrP 1–139 were 3.2 ± 0.4 and 5.4 ± 0.6 pg/μg protein, respectively, significantly greater than the 1.8 ± 0.4 pg/μg found for cells transfected with vector (P < 0.05, n = 3 cell preparations per group). Also, PTHrP 38–64 levels were 3- to 5-fold greater in cells transfected with the GFP3PTHrP chimeras (data not shown). Finally, cells transfected with GFP3PTHrP 1–139 registered a 5-fold increase in PTHrP 109–139 levels, but cells transfected with GFP3PTHrP 1–108 did not. By immunoblot sizing analysis, the chimeric PTHrP proteins migrated as GFP-positive bands at their expected sizes (Fig. 4). The GFP3 vector protein was effectively restricted from the nucleus, as expected for its size. In contrast, the GFP3-PTHrP chimeric proteins, which include the PTHrP NLS, were distributed to cytoplasm, nucleus, and also nucleoli (Fig. 5).
The nuclear export studies used three chimeric PTHrP variants, GFP3-wild-type PTHrP 1–139, GFP3-PTHrP 1–108, and GFP3-PTHrP 1–139 127–136 (Fig. 3, A–C, respectively). The wild-type chimera would allow us to measure regulated export with an intact PTHrP NES, whereas the PTHrP 1–108 protein presumably had deleted the region containing the NES and would not undergo export. The purpose of the third construct was to refine the location of the NES. It deleted all of the pNES3 sequence except for L126, which remained to avoid disrupting pNES2.
Nuclear level of GFP3-PTHrP chimeras under steady-state conditions
The distribution of GFP3-PTHrP chimeric proteins between cytoplasm and nucleus differed significantly among 293 cells transiently transfected with GFP3-PTHrP 1–139, GFP3-PTHrP 1–139 127–136, and GFP3-PTHrP 1–108 (Fig. 6). The steady-state ratio of nuclear to cytoplasmic fluorescence (Fn/c) was more than 2-fold greater for the deletion and truncation mutants than for the wild-type 1–139 chimera. The constructs also presented different sensitivities to LMB. Treatment with LMB increased the Fn/c for the wild-type construct but had no effect on the two mutants.
In vitro assays of GFP3-PTHrP nuclear export
Nuclear export was measured with an in vitro assay using permeabilized transfected COS-1 cells. The permeabilized cells were bathed in a buffer containing all the components necessary for nuclear export except RanGTP. Export began after adding RanGTP and resulted in a decrease in nuclear GFP fluorescence for the wild-type GFP3-PTHrP 1–139 chimera with levels approaching background after about 15 min (Fig. 7A). In contrast, nuclear fluorescence for GFP3-PTHrP 1–139 127–136 showed almost no change over the same period. The nuclear signal for GFP3-PTHrP 1–139 decreased at a constant rate (Fig. 7B). However, fluorescence did not decline significantly for cells expressing GFP3-PTHrP 1–108, GFP3-PTHrP 1–139 127–136, GFP3-PTHrP 1–139 in the presence of LMB, or fixed cells expressing GFP3-PTHrP 1–139. This experiment was performed twice with nearly identical results.
We also measured the quantity of exported protein present in the transport buffer at the end of the experiment. After an export period of 50 min, the conditioned buffer from permeabilized COS-1 cells transfected with GFP3-PTHrP 1–139 contained approximately 300 pg/ml PTHrP/10,000 cells. In contrast, PTHrP levels were undetectable in the conditioned buffer from cells transfected with either of the mutant chimeras or cells carrying the wild-type chimera but treated with LMB.
FRAP assays of nuclear export of GFP3-PTHrP constructs in intact cells
Intact COS-1 cells expressing GFP3-PTHrP chimeras were incubated with cycloheximide to prevent synthesis of de novo GFP-containing protein and then underwent a period of photobleaching to diminish fluorescence substantially throughout the cytoplasm (Fig. 8). Micrographs in Fig. 8A illustrate how the template was drawn for the bleaching AOI and the course for a typical FRAP assay. The template surrounds the cytoplasm except for a narrow path taken to outline and exclude the nucleus. Figure 8 (middle panel) demonstrates that the photobleaching reduced cytoplasmic fluorescence to background levels in most of the cell but left nuclear fluorescence intact. Figure 8 (right panel) shows that cytoplasmic fluorescence began to recover at the end of the assay due to protein exported out of the nucleus. Nuclear fluorescence was perceptibly diminished. The three micrographs were exposed with same settings and processed to visualize the low levels of cytoplasmic fluorescence after recovery in the final image. The cellular fluorescence before bleaching was high and the prebleach image is saturated.
Nuclear export was quantified by repeated imaging to measure the shift of fluorescent protein from nucleus to cytoplasm. The initial Fn/c ratios after cytoplasmic bleaching were similar for all three chimeras. Nuclear fluorescence diminished and cytoplasmic fluorescence increased for the GFP3-PTHrP 1–139 chimera within only 50 sec (Fig. 8). The ratio decreased progressively with time for the wild-type chimera, but minimal changes were observed for the mutant chimeras. The t1/2 values for the decrease in Fn/c for GFP3-PTHrP 1–139 127–136 and GFP3-PTHrP 1–108 were similar, 433 and 385 sec, respectively, some 2.5 times greater than the t1/2 values for GFP3-PTHrP 1–139, 165 sec.
Discussion
Our first step in searching for the NES of PTHrP was to test whether the export signal depended on the carboxyl-terminal portion of PTHrP. We used FLIP to compare nuclear export of GFP-PTHrP 1–139 and the truncation mutant GFP-PTHrP 1–108. The latter protein lacked several groups of leucine residues that could be involved in export signaling. Both chimeric proteins were small enough to diffuse through the NPC (31). However, the fact that the larger chimera exited the nucleus faster than its smaller truncation mutant was inconsistent with purely diffusive transport; the smaller protein should diffuse faster. The result indicated that active transport contributed to PTHrP shuttling, even though passive egress of protein was possible. The result also suggested that a determinant within the PTHrP 109–139 region augmented the export rate. Interestingly, the two chimeric proteins differed somewhat in cellular distribution. GFP-PTHrP 1–108 labeled nucleoli much more heavily than GFP-PTHrP 1–139. A definitive explanation is not available, but we propose that the reduced rate of nuclear export leads to high nuclear levels of GFP-PTHrP 1–108 (as seen for GFP3-PTHrP 1–108 in Fig. 6), with consequently heavier collections in the nucleoli.
Next we conducted a sequence analysis to define what domains in PTHrP 109–139 might constitute an NES. Of three potential leucine-rich groupings, the 129–136 stretch in pNES3 appeared to fit the characteristics observed for NESs in other proteins (Fig. 2). This agrees with the location of the NES proposed by Jans et al. (15). Based on our analysis, we developed three chimeric PTHrP variants, GFP3-PTHrP 1–139, GFP3-PTHrP 1–108, and GFP3-PTHrP 1–139 127–136 (Fig. 3, A–C, respectively), to study whether pNES3 was important in nuclear export. By including three GFP proteins in series, the chimeras were too large to enter or exit the nucleus passively and required interaction with specialized transport machinery for nuclear transport. The wild-type chimera allowed us to measure regulated export with an intact export signal, whereas the PTHrP 1–108 protein lacked a large region that presumably spanned the putative NES. The third construct specifically deleted the pNES3 sequence but left L126 to avoid disrupting pNES2.
The steady-state nuclear and cytoplasmic levels of the various chimeric proteins contributed to the eventual conclusion that PTHrP 127–136 was important for nuclear export. Proteins with diminished nuclear export would be expected to accumulate in the nucleus to a greater extent than proteins with intact transport. This was the case for PTHrP 1–108 and PTHrP 1–139 127–136, compared with the wild-type PTHrP 1–139. Additional support for the hypothesis emerged from the differing degrees of interaction with CRM1. The Fn/c ratio for the wild-type chimeric protein increased after LMB treatment, indicating that CRM1 was involved in its transport. On the other hand, LMB did not affect the relative nuclear and cytoplasmic levels of the two mutant proteins, suggesting independence from CRM1. Because nuclear export of PTHrP involves CRM1, these data suggest that the carboxyl-terminal portion of PTHrP, particularly PTHrP 127–136 (pNES3), is important for nuclear export.
Direct assays of nuclear export provided confirmation for the proposition. We demonstrated that the export rates of PTHrP depended on an intact pNES3 in two different transport assays, one in vitro and one with intact cells. In the in vitro export assay, the PTHrP 1–108 and the PTHrP 1–139 127–136 chimeras underwent little if no export during the same period that nuclear fluorescence for the wild-type PTHrP 1–139 chimera fell by 80%. Consistent with its effects on steady-state protein levels, LMB reduced the export rate of GFP-PTHrP 1–139 but not the two mutants. The FRAP data showed that PTHrP export depended on carboxyl terminal sequences in intact cells also. The deletion of pNES3 in the GFP3-PTHrP 1–139 127–136 construct appeared to decrease nuclear export to the same extent as truncating the entire PTHrP 109–139 sequence in the GFP3-PTHrP 1–108 construct. Thus, multiple lines of evidence support a critical role for PTHrP 127–136 in nuclear export.
A definitive statement cannot be made whether the other leucine-rich regions, pNES1 and pNES2, contribute to nuclear export based on our data. Because the GFP3-PTHrP 1–139 127–136 construct contained intact pNES1 and pNES2 sequences, the other putative NESs in the 109–139 region may not be as important as pNES3 in determining nuclear export of PTHrP. However, we did not test individual deletion mutations of pNES1 or pNES2 and cannot exclude the possibility that these regions might augment transport in the presence of pNES3. However, pNES1 and pNES2 groups did not support nuclear export of GFP3-PTHrP 1–139 127–136 in the in vitro assay. Also they were not enough to mediate LMB-sensitive changes in steady-state levels of GFP3-PTHrP 1–139 127–136 (Fig. 6). Clearly pNES3 is necessary for CRM1 interaction and PTHrP export, but whether the sequence is sufficient to mediate export is unknown.
Our results are qualitatively similar in several respects to studies presented by Lam et al. (12, 33). They also showed that LMB increases the nuclear level of a GFP-PTHrP fusion protein and decreases the rate of nuclear export of that protein (12, 33). Their work was conducted in COS-1 cells and UMR 106.01 osteogenic sarcoma cells, whereas we studied LMB effects on nuclear PTHrP flux in HeLa cells and 293 cells. Direct quantitative comparisons of kinetic data are not feasible because the constructs and assay techniques differed between the laboratories. The Lam studies used a single GFP protein instead of GFP3 in their chimeric proteins, In addition, they performed FRAP by bleaching a partial region of the cytoplasm rather than the complete area.
Protein expression was decreased for the GFP3-chimeric proteins, compared with the expression of GFP3 protein. We have not established the reason for the difference. One possible explanation is that the presence of nuclear GFP3-PTHrP chimeric protein might decrease protein synthesis through an effect on ribosomal function. PTHrP is known to localize to bind to ribosomal RNA (13, 34). We also found that nucleolar accumulation of GFP-PTHrP chimeras appeared to exceed that of the triple GFP fusion proteins. We have no explanation at this time.
Additional work will be necessary to determine whether CRM1-mediated transport augments nuclear clearance of PTHrP above levels due to passive movement and whether the presence of absence of the PTHrP NES makes a difference in cell function or growth. Nuclear levels of PTHrP vary with cell cycle in UMR106 cells and the protein exerts effects that are specific for particular phases of the cell cycle (11, 35). One might expect that exiting the nucleus at the right time would be important in regulating cell cycle-specific levels of the protein, but whether NES facilitates nuclear escape at specific phases is unknown.
Summary
PTHrP has been shown to export from the nucleus via the CRM1 pathway (12), but details about the signal sequence had not been established. CRM1 typically recognizes a nuclear export signal, a loosely defined consensus sequence that is rich in leucine moieties. The 126–136 region within PTHrP matched this NES consensus pattern, notably in its last three amino acids consisting of two leucines separated by a polar residue. A mutant was created in which the putative NES, pNES3, was disrupted by deleting nine of the 10 residues. Four independent methods were used to assess the impact of this mutation on nuclear export of chimeric GFP3-PTHrP proteins. The GFP series provided a fluorescent tag and restricted passive nuclear shuttling. Transport of the pNES3 mutant was significantly altered, compared with the wild-type PTHrP 1–139 chimera, suggesting that it was not exported by CRM1. These results indicate that PTHrP exports in the CRM1 nuclear export pathway via a leucine-rich NES and that the 126–136 region is a critical component of the NES. The importance of this sequence for nuclear egress of wild-type PTHrP 1–139, which can move in and out of the nucleus passively, and the physiologic significance of NES-mediated export of PTHrP require further study.
Acknowledgments
Dr. Janna Bednenko and Dr. Larry Gerace (The Scripps Research Institute. La Jolla, CA) provided reagents and helpful discussion for the in vitro nuclear export studies.
Footnotes
This work was supported by The Flight Attendants Medical Research Institute, two Department of Veterans Affairs Merit Review Awards (to L.J.D. and R.H.H.), National Cancer Institute Grant CA23100, National Institutes of Health Grant DK60588, and the University of California Tobacco-Related Disease Research Program.
First Published Online November 17, 2005
Abbreviations: AOI, Area of interest; CRM1, chromosome region maintenance protein 1; cTB, complete TB; FLIP, fluorescence loss in photobleaching; Fn/c, ratio of the fluorescence of the nucleus and cytoplasm; FRAP, fluorescence recovery after photo bleaching; GFP, green fluorescent protein; LMB, leptomycin B; NES, nuclear export signal; NFM, nonfat dry milk; NLS, nuclear localization signal; NPC, nuclear pore complex; t1/2, half-time; TB, transport buffer.
Accepted for publication November 7, 2005.
References
Bednenko J, Cingolani G, Gerace L 2003 Nucleocytoplasmic transport: navigating the channel. Traffic 4:127–135
Fornerod M, Ohno M, Yoshida M, Mattaj IW 1997 CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051–1060
Askjaer P, Jensen TH, Nilsson J, Englmeier L, Kjems J 1998 The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP. J Biol Chem 273:33414–33422
Macara IG 2001 Transport into and out of the nucleus. Microbiol Mol Biol Rev 65:570–594
Moseley JM, Gillespie MT 1995 Parathyroid hormone-related protein. Crit Rev Clin Lab Sci 32:299–343
Goltzman D, Hendy GN, Banville D 1989 Parathyroid hormone-like peptide: molecular characterization and biological properties. Trends Endocrinol Metab 1:39–44
Hastings RH 2004 Parathyroid hormone-related protein and lung biology. Respir Physiol Neurobiol 142:95–113
Hastings RH, Quintana RA, Sandoval R, Duey D, Rascon Y, Burton DW, Deftos LJ 2003 Pro-apoptotic effects of parathyroid hormone-related protein in type II pneumocytes. Am J Respir Cell Mol Biol 29:733–742
Hastings RH, Araiza F, Burton DW, Zhang L, Bedley M, Deftos LJ 2003 Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells. Am J Physiol Cell Physiol 285:C1429–C1436
Hastings RH, Summers-Torres D, Yaszay B, LeSueur J, Burton DW, Deftos LJ 1997 Parathyroid hormone-related protein, an autocrine growth inhibitor of alveolar type II cells. Am J Physiol 272:L394–L399
Lam MH, Briggs LJ, Hu W, Martin TJ, Gillespie MT, Jans DA 1999 Importin recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin . J Biol Chem 274:7391–7398
Lam MH, Henderson B, Gillespie MT, Jans DA 2001 Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein. Traffic 2:812–819
Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15:4064–4075
Massfelder T, Dann P, Wu TL, Vasavada R, Helwig JJ, Stewart AF 1997 Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: a critical role for nuclear targeting. Proc Natl Acad Sci USA 94:13630–13635
Jans DA, Thomas RJ, Gillespie MT 2003 Parathyroid hormone-related protein (PTHrP): a nucleocytoplasmic shuttling protein with distinct paracrine and intracrine roles. Vitam Horm 66:345–384
Cingolani G, Bednenko J, Gillespie MT, Gerace L 2002 Molecular basis for the recognition of a nonclassical nuclear localization signal by importin . Mol Cell 10:345–353
Lam MH, Thomas RJ, Martin TJ, Gillespie MT, Jans DA 2000 Nuclear and nucleolar localization of parathyroid hormone-related protein. Immunol Cell Biol 78:395–402
Lam MH, Hu W, Xiao CY, Gillespie MT, Jans DA 2001 Molecular dissection of the importin 1-recognized nuclear targeting signal of parathyroid hormone-related protein. Biochem Biophys Res Commun 282:629–634
Ditmer LS, Burton DW, Deftos LJ 1996 Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1–173 increases production and secretion of the truncated forms. Endocrinology 137:1608–1617
Hastings RH, Asirvatham A, Quintana R, Sandoval R, Dutta R, Burton DW, Deftos LJ 2002 Parathyroid hormone-related protein-(38–64) regulates lung cell proliferation after silica injury. Am J Physiol Lung Cell Mol Physiol 283:L12–L21
Hastings RH, Quintana RA, Sandoval R, Burton DW, Deftos LJ 2003 Amino-terminal and mid-molecule parathyroid hormone-related protein, phosphatidylcholine, and type II cell proliferation in silica lung injury. Am J Physiol Lung Cell Mol Physiol 285:L1312–L1322
Howell JL, Truant R 2002 Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP. Biotechniques 32:80–87
Nicolas FJ, De Bosscher K, Schmierer B, Hill CS 2004 Analysis of Smad nucleocytoplasmic shuttling in living cells. J Cell Sci 117:4113–4125
Kehlenbach RH, Dickmanns A, Gerace L 1998 Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT in vitro. J Cell Biol 141:863–874
Birbach A, Bailey ST, Ghosh S, Schmid JA 2004 Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-B inducing kinase NIK. J Cell Sci 117:3615–3624
la Cour T, Gupta R, Rapacki K, Skriver K, Poulsen FM, Brunak S 2003 NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res 31:393–396
Bogerd HP, Fridell RA, Benson RE, Hua J, Cullen BR 1996 Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay. Mol Cell Biol 16:4207–4214
Kim FJ, Beeche AA, Hunter JJ, Chin DJ, Hope TJ 1996 Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions. Mol Cell Biol 16:5147–5155
Zhang MJ, Dayton AI 1998 Tolerance of diverse amino acid substitutions at conserved positions in the nuclear export signal (NES) of HIV-1 Rev. Biochem Biophys Res Commun 243:113–116
Fornerod M, Ohno M 2002 Exportin-mediated nuclear export of proteins and ribonucleoproteins. In: Weis K, ed. Nuclear transport. Berlin: Springer-Verlag; 67–91
Gorlich D, Kutay U 1999 Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 15:607–660
Brownawell AM, Kops GJ, Macara IG, Burgering BM 2001 Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Mol Cell Biol 21:3534–3546
Lam MH, Thomas RJ, Loveland KL, Schilders S, Gu M, Martin TJ, Gillespie MT, Jans DA 2002 Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules. Mol Endocrinol 16:390–401
Aarts MM, Levy D, He B, Stregger S, Chen T, Richard S, Henderson JE 1999 Parathyroid hormone-related protein interacts with RNA. J Biol Chem 274:4832–4838
Lam MH, House CM, Tiganis T, Mitchelhill KI, Sarcevic B, Cures A, Ramsay R, Kemp BE, Martin TJ, Gillespie MT 1999 Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormone-related protein negatively regulates its nuclear localization. J Biol Chem 274:18559–18566
Nicholas KB, Nicholas Jr HB, Deerfield DW 1997 GeneDoc: analysis and visualisation of genetic variation. EMBNEW. NEWS 4:14(Jared C. Pache, Douglas W. Burton, Leona)
Department of Anesthesiology (R.H.H.), University of California, San Diego, California 92161
Medicine Service (D.W.B., L.J.D.) and Anesthesiology Service (R.H.H.), Veterans Affairs San Diego Healthcare System, San Diego, California 92161
Abstract
PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein’s nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109–139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1–108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109–139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126–136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126–136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.
Introduction
TRANSPORT OF LARGE macromolecules into and out of the nucleus is a regulated process that requires binding to specific transport proteins to traverse the nuclear pore complex (NPC). The ability of a protein to complex with import and export proteins is endowed by nuclear localization signals (NLS) or nuclear export signals (NES), respectively. NLSs are frequently monopartite or bipartite clusters of basic residues that mediate binding to a member of the karyopherin nuclear transport family, such as importin- (1). On the other hand, an NES is a loosely conserved motif typically consisting of specifically spaced sequences of leucines or other hydrophobic amino acids, typically separated by charged or polar spacer amino acids. A protein containing a leucine-rich NES can form a complex with nuclear export receptor chromosome region maintenance protein 1 (CRM1)/exportin-1 and the small GTPase protein Ran in its GTP bound form (2). This complex is then translocated from the nucleus to the cytoplasm by facilitated diffusion through the NPC. RanGAP, a cytoplasmic protein, activates the intrinsic GTPase activity of Ran; hydrolysis of the terminal phosphate of GTP disrupts the NES-CRM1-Ran complex and releases the shuttled protein into the cytoplasm. CRM1 then cycles back to the nucleus for another round of NES export. Leptomycin B (LMB), a small fungicide derived from a strain of Streptomyces, inhibits CRM1-mediated transport by interfering with the association of CRM1 and the NES (3, 4).
PTHrP is best known for mediating hypercalcemia when secreted by solid tumors (5). However, the protein can act locally at the cells of many normal tissues and cancers not connected with hypercalcemia. The local effects include paracrine or autocrine actions on growth, apoptosis, and smooth muscle relaxation as well as calcium transport (6, 7, 8, 9, 10). Several portions of the molecule act at the cell surface, presumably through receptor binding, but the only known receptor is the PTH/PTHrP receptor that is shared between PTHrP 1–34 and PTH 1–34. The protein can also escape the secretory pathway to remain inside the cell (11, 12). Intracellular retention has functional significance; PTHrP exerts intracrine effects in the cell nucleus, predominantly affecting growth and apoptosis (13, 14).
Nuclear localization of PTHrP represents a balance between nuclear import and export and appears to be regulated in some cell types (15). PTHrP possesses an NLS within residues 67–94 that is capable of binding to importin- directly without an intervening role for importin- (11, 16). Inside the nucleus, PTHrP targets the nucleoli in most tissues, likely through the auspices of additional basic residues close to the NLS (13, 17). The protein resides in the nuclei during the G1 phase of the cell cycle, but phosphorylation of threonine 85 by cdk2 during S phase reduces the rate of nuclear import and shifts PTHrP to the cytoplasm (15, 18). Cell cycle-based redistribution of nuclear PTHrP could not occur if the protein did not shuttle back and forth from cytoplasm to nucleus.
PTHrP fused to green fluorescent protein (GFP) has been demonstrated to undergo nuclear export, and the rate of export is sensitive to LMB, demonstrating that transport depends on CRM1 (12). However, the precise location of the NES of PTHrP has not been identified. Our goal in this study was to determine whether PTHrP is exported from the nucleus directly via a leucine-rich NES. Based on the primary structure, we considered the amino acid 109–139 sequence, which contains many leucine residues, as the most likely region of PTHrP to contain an NES. We performed sequence analyses comparing PTHrP 109–139 with known NESs from other proteins and selected a smaller domain to test as a putative NES. We then evaluated the importance of this region for nuclear export of GFP-containing PTHrP chimeras that contained the full sequence or various deletion mutations.
Materials and Methods
Chemicals
Hoechst 33342, LMB, and cycloheximide were purchased from Calbiochem (San Diego, CA). The restriction enzymes and Quick ligation kit were purchased from New England Biolabs (Beverly, MA). The mutagenic primers were purchased from Proligo (Boulder, CO). All other chemicals, unless specified, were obtained from Sigma Chemicals (St. Louis, MO).
Construction of chimeras containing GFP and PTHrP
The pEGFP-C1 vector was obtained from CLONTECH (Palo Alto, CA) and was used as the template for the GFP-PTHrP chimeras. The PTHrP 1–139 and PTHrP 1–108 fragments were generated from the human PTHrP expression plasmid, pCi-neo-PTHrP1–173 (19), using specific primers flanked by SalI and XbaI restriction sites. Both pEGFP-C1 vector and the PCR products were digested with SalI and XbaI, ligated together using a Quick ligation kit and transformed into XL-1 blue cells (Stratagene, La Jolla, CA). PTHrP immunoassays and DNA sequencing confirmed the integrity of the plasmid preparations.
The entire gene encoding EGFP, except for the ATG start codon, was amplified via PCR from the pEGFP-C1 vector using PCR primers containing 5' BglII and 3' XhoI restriction sites (lowercase letters): forward, 5'-CTCagatctGTGAGCAAGGGCGAGGAGCTG-3', and reverse, 5'-GAGctcgagCTTGTACAGCTCGTCCATGCC-3'. This PCR product was used as the insert to make the GFP2 construct. Next, the EGFP gene was PCR amplified using a second set of PCR primers containing 5' XhoI and 3' SalI restriction sites (lowercase letters; forward, 5'-CTCagatctGTGAGCAAGGGCGAGGAGCTG-3', and reverse, 5'-ACCgtcgacCTTGTACAGCTCGTCCATGCC-3') to make the GFP3 PCR product. After digesting both the pEGFP-C1 vector and the 5' BglII and 3' XhoI PCR product with BglII and XhoI, the fragments of interest were gel purified from a 1% SeaPlaque low-melting-point agarose gel (Cambrex, Baltimore, MD), extracted using a QIAquick gel extraction kit (QIAGEN Inc., Valencia, CA), and ligated together in a 1:1 molar ratio to make the pEGFP2 vector. To convert the pEGFP2 vector to a pEGFP3 vector, the same cloning procedure was followed using the second PCR insert containing the 5' XhoI and 3' SalI sites and the pEGFP2 vector with XhoI and SalI as restriction enzymes.
The GFP3-PTHrP 1–108, 139, and 173 constructs were created by transferring the PTHrP insert from their respective single GFP expression vectors using SalI and XbaI with the same reagents and cloning procedures as described above.
Mutagenesis
To generate the mutations within PTHrP 126–139, we used the Quick change XL kit (Stratagene) as per the manufacturer’s recommendations. Mutations were confirmed by DNA sequencing.
Cell culture
HeLa, COS-1, and 293 cells, derived from human ovarian cancer, African green monkey kidney, and human embryonic kidney, respectively, were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in monolayer using DMEM (Cellgro, Herndon, VA) supplemented with 5–10% fetal bovine serum (Gemini Bio-Products, Woodland, CA) in a humidified incubator at 37 C with 95% air and 5% CO2.
Transfections
For transfection studies, the cells were plated at 60–70% confluency and incubated overnight as described above. The 293 and HeLa cells were transfected using GenePORTER-2 (Gene Therapy Systems, San Diego, CA) and the COS-1 cells were transfected using GenePORTER-1 according to the manufacturer’s instructions. Typical transfection efficiencies were approximately 75, 55, and 90% for 293, HeLa, and COS-1 cells, respectively, as measured by the percentage of cells fluorescing in pEGFP-C1-transfected cell preparations.
Immunoblotting
COS-1 cells were trypsinized after transfection and pelleted by centrifugation at 200 x g for 5 min. The cell pellets were sonicated for 10 sec in cell lysis buffer (PBS with 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate) and total protein concentrations in the cell lysates were measured using a BCA protein assay (Pierce Chemical Co., Rockford, IL). The proteins (20 μg/lane) were separated using a 4–12% Bis-Tris SDS-PAGE gel in 3[N-morpholino]propanesulfonic acid buffer (Invitrogen Corp., Carlsbad, CA) and then transferred to a nitrocellulose membrane. The membrane was blocked for nonspecific binding by incubating in 10% nonfat dry milk (NFM) in Tris-buffered saline [10 mM Tris-HCl (pH 8.0), 150 mM NaCl] for 30 min, washed three times in Tris-buffered saline, and incubated in the presence of 1 μg/ml anti-GFP monoclonal antibody (Molecular Probes, Eugene, OR) in 5% NFM overnight at 4 C on a rotating shaker. Between multiple washes, the membrane was incubated in goat-antimouse IgG conjugated to horseradish peroxidase (Calbiochem) and diluted in 5% NFM overnight at 4 C on a shaker. The immunoreactive proteins were detected using a chemiluminescence reaction kit (Pierce Chemical) and exposed for 1–2 min on a Kodak MIN-R 2000 x-ray film (Eastman-Kodak Co., Rochester, NY). To control for protein loading, the membranes were subsequently immunostained for -tubulin levels (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
PTHrP immunoassays
Conditioned media and cell extracts from transfected cells were assayed for PTHrP expression by region-specific RIAs (20, 21). In brief, human PTHrP 1–34, 38–64, or 109–141 peptides were used as standards, and PTHrP 1–86 and tyr-108–141 peptides were radioiodinated by the chloramine T method. Rabbit PTHrP antibodies directed against PTHrP 1–34, 38–64, or 109–141 were used in a 3-d nonequilibrium immunoassay format, as previously described (20, 21). All samples were assayed in triplicate in multiple dilutions that paralleled the corresponding PTHrP standard curve and the intra- and interassay variations were approximately 7–12%.
Static fluorescence microscopy and image analysis
To assess steady-state cellular distributions of PTHrP, 293 cells were plated in 35-mm glass bottom culture dishes (MatTek, Ashland, MA) and transfected with GFP3-PTHrP chimeras. Forty-eight hours after transfection, the media were replaced with normal growth media with or without 4 ng/ml LMB and incubated for 3 h. Thirty minutes before the end of this incubation, Hoechst 33342 was added to the media to stain the nuclei. The cells were then fixed in 3.7% paraformaldehyde in PBS for 30 min, washed three times with PBS, and mounted with antifade media (Biomeda Corp., Foster City, CA).
The cell fluorescence was analyzed using a DMLB microscope (Leica Microsystems, Buffalo, NY) equipped with a 100-W mercury lamp and standard fluorescein isothiocyanate and 4',6'-diamino-2-phenylindole filter cubes (Chroma Technology Corp., Rockingham, VT). The GFP and Hoechst 33342 fluorescence images were captured with a Spot RT 220–3 digital camera (Diagnostic Instruments, Sterling Heights, MI). The images were analyzed and calibrated using Image-Pro Plus version 4.5.1.29 software (Media Cybernetics, Silver Spring, MD). Average pixel intensity measurements were calculated for a 400 square pixel area (16 μm2) area of interest (AOI) as follows: first, the AOI was positioned over a nuclear, nonnucleolar region and the average intensity was recorded. Next, the AOI was repositioned to a representative region of the cytoplasm of the same cell and the average intensity was recorded. Correct placement of the cytoplasmic AOI relative to the nucleus was confirmed using the Hoechst 33342 image to define the nuclear boundary. The data were exported into Microsoft Excel (Microsoft, Redmond, WA), and the distribution of the fluorescent PTHrP protein between the nucleus and cytoplasm was quantified by the ratio of the fluorescence of the two compartments, Fn/c.
Confocal microscopy
All time course experiments were performed with a Zeiss Axiovert 110M microscope equipped with an LSM 510 confocal laser scanning system using the LSM software version 3.2sp2 (Carl Zeiss MicroImaging, Inc., Thornwood, NY). The in vitro export assay and fluorescence recovery after photo bleaching (FRAP) studies used a Plan Neofluor 40x/1.3 oil objective, whereas fluorescence loss in photobleaching (FLIP) used the Plan Apochromat 63x/1.4 oil objective. Scan settings were the same for all experiments: GFP was excited with 5% of Ar/Kr laser output on the 488-nm wavelength laser line coupled to a BP505–550 emission filter. Propidium iodide (PI) was excited using 40% of the Ar/Kr laser output on the 568-nm laser line coupled to a LP585 emission filter. Two 12-bit 1024 x 1024 images were obtained in line mode at a rate of 3.9 sec per image (7.8 sec total scan time) and averaged to reduce background noise. Generally, six 1-μm Z stacks were obtained for each scan to adjust for focal drift. At each time point, the analysis used the Z stack with the optimal plane of focus.
FLIP
Before microscopy, HeLa cells transfected with single GFP-PTHrP chimera expression plasmids were incubated for 30 min with 50 μg/ml cycloheximide in 25 mM HEPES (pH 7.4). Each FLIP assay focused on an individual transfected cell. The cell was brought into focus on the stage of the confocal microscope preheated to 37 C using an air stream stage incubator (Nevtek, Burnsville, VA). It was scanned with the settings described in the previous section and the pinhole wide open at 1 mm. Between each scan, the cytoplasm AOI was photobleached for 20 iterations using 50% laser output of both 488 and 568 laser lines. Three rounds of scanning and bleaching reduced cytoplasmic fluorescence to background levels. Nuclear export was assayed beginning with the very next scan. Nuclear fluorescence was quantified with ImagePro software and expressed as a percentage of the fluorescence on the initial scan.
The LSM software (Carl Zeiss MicroImaging) has a photobleaching technology that deserves further description. The laser scans in a raster pattern and uses a shutter guided by a user-defined bleaching AOI to control whether an individual point in the field is excited. The AOI is selected with the polygon drawing tool and can take an irregular shape (22). Thus, the bleaching template can include the entire cytoplasm but exclude the nucleus (as shown in Fig. 8A) using a FRAP experiment as an example. We typically use the phase contrast image to aid in drawing the AOI. The area of the template was kept nearly constant from cell to cell and was set slightly larger than each of the cells studied. Thus, the laser dwell time and the interval between images was relatively even among the analyses.
The purpose of the FLIP studies was to determine the importance of sequences within the PTHrP 109–139 region for nuclear export of PTHrP. We compared export rates for GFP-PTHrP 1–108 and GFP-PTHrP 1–139 because the two proteins are close in size, 39 vs. 42 kDa, respectively. The upper size limit at which passage through the NPC is restricted is only slightly larger. In contrast, GFP at 27 kDa is well below the size limit for passive diffusion through the NPC. In FLIP studies, it escapes the nucleus nearly as rapidly as cytoplasmic GFP can be photobleached (23).
In vitro nuclear export assay
This assay was adapted from protocols described by others (3, 19). Basically, cells transfected with the fluorescent-labeled protein of interest are first permeabilized. This step eliminates the protein from the cytoplasmic compartment and depletes the cell of endogenous RanGTP, a necessary cofactor for nuclear transport (3). We use PI, which is excluded from intact cells, to identify permeabilized cells. Export of the protein from the nucleus is held in check and begins when exogenous RanGTP is added to the assay buffer. Replacement of CRM1 is unnecessary because the large protein is not depleted by permeabilization (24).
COS-1 cells were used 48 h after transfection with GFP3-PTHrP chimera expression plasmids. The transfected cells were washed with transport buffer [TB: 20 mM HEPES-KOH (pH 7.3), 110 mM KOAc, 2 mM Mg(OAc)2, and 1 mM EGTA] supplemented with 10% fetal bovine serum and then washed in ice-cold complete TB (cTB: TB supplemented with 2 mM dithiothreitol; 1 mM phenylmethylsulfonyl fluoride; and 1 μg/ml each of pepstatin, leupeptin, and aprotinin). Permeabilization was performed with 40 μg/ml digitonin in ice-cold cTB for 5 min on ice. Next, cells were washed twice with ice-cold cTB, incubated on ice for 15 min with cTB containing 4 μg/ml PI, washed again, and left in 100 μl cTB in the central cavity of the MatTek 35-mm, glass-bottom culture dish. The cells were brought into focus on the preheated stage. Finally, 20 μl 5x assay buffer (5 μM Ran, 5 μM GTP, 1 μM ATP, 2.5 mg/ml BSA) were added to the cell suspension, the lid was replaced, and imaging began immediately. The two fluorophores were imaged on separate tracks to prevent cross talk of the fluorophores. Data were collected only for cells that were PI positive. The pinholes of each channel were wide open (1 mm). Nuclear fluorescence was measured and presented as in the FLIP experiments.
FRAP
Cells were transfected with GFP3-PTHrP chimeras and treated before the experiment with cycloheximide as described for FLIP. The PI track was disabled and the pinhole size for the GFP track was adjusted to 1.5 airy units. One image was acquired before photobleaching to outline an AOI including the entire cytoplasm as described above. The selected region was bleached for 80 iterations with a laser output of 100%. Bleaching the GFP3 chimeras required a higher output than the 50% used for the single GFP constructs that were used for FLIP. Scans were then collected with the standard scan settings on a continuous cycle for the remained of the assay with no further bleaching.
Fluorescence in nuclear and cytoplasmic compartments were measured with ImagePro, exported to Excel, and expressed as Fn/c. The kinetics of export were analyzed by fitting data points exponentially using SigmaPlot software (SPSS, Chicago, IL) to a three-parameter model adapted from models used by others (25): Fn/c = span (1 – e–kt) – top, where top is the relative Fn/c ratio immediately after photobleaching and span represents the extent of exported protein. Half-time (t1/2) of nuclear export was defined as ln(2)/k.
Results
GFP-PTHrP 1–108 exports from the nucleus at a slower rate than GFP-PTHrP 1–139
HeLa cells transiently transfected with GFP-PTHrP 1–139 or GFP-PTHrP 1–108 demonstrated cytoplasmic and nuclear GFP fluorescence, with signal localizing to nucleoli as well (Fig. 1A, left panels). Nucleolar fluorescence for the truncation mutant chimera was greater than for the wild-type construct. Continual photobleaching with the FLIP protocol reduced cytoplasmic fluorescence to background after 100 sec (image not shown). After 600 sec, nuclear fluorescence had been lost, compared with baseline for both constructs. The decrement was noticeably greater for GFP-PTHrP 1–139, compared with GFP-PTHrP 1–108 (Fig. 1A, right panels). Bleaching maintained cytoplasmic fluorescence at background levels. Image analysis showed that nuclear fluorescence decreased linearly with time for both GFP-PTHrP chimeras, but GFP PTHrP 1–108 exported at a significantly (P < 0.01) slower rate than GFP PTHrP 1–139 (Fig. 1B).
Sequence alignments of known NESs with PTHrP 109–139
We compared experimentally verified NESs to the PTHrP 109–139 sequence (26) (Fig. 2). Three leucine-rich sequences within the 109–139 region, PTHrP 112–121 (pNES1), PTHrP116–126 (pNES2), and PTHrP126–136 (pNES3), had at least partial homology with other known export signals (Fig. 2). NES consensus sequences are typically approximately 10 amino acids in length with a semidefined consensus of LX2–3LX2–3LXL (27, 28, 29). The last three residues appear to be the most critical for function; typically they consist of a leucine or isoleucine separated by a single polar amino acid from a final leucine (30). The earlier leucine positions in the consensus are not as critical and may be substituted occasionally by other amino acids. Based on these criteria, PTHrP 126–136 was the region most likely to be an NES. That sequence presented significant homology with known NES in human MAP kinase, human p53, human PKI, and other proteins that undergo nucleocytoplasmic shuttling. In particular, it contained the requisite structure in its ultimate tripeptide, LEL134–136. The two upstream candidate sequences lacked the penultimate leucine residue. Jans et al. (15) suggested in a review article that this region was a likely NES for PTHrP, although they provided no supporting data or references. Because of our own sequence analyses and the supporting literature, we focused on PTHrP 126–136 as the potential NES in subsequent mutagenesis studies.
Constructs to test regulated nuclear export of PTHrP
We engineered new chimeric PTHrP forms to study the role of pNES3 in active export of PTHrP. The single GFP chimeric proteins are small enough in mass, less than 48 kDa, to diffuse through the NPC (31) without the assistance of CRM1. To prevent passive mechanisms in the export assays, we made the chimeras large using a triple GFP expression vector (GFP3) (Fig. 3) analogous to one described by a previous investigator (32). We confirmed the identity of the constructs by DNA sequencing, and we demonstrated their functionality with transfection studies. Transfected cells expressed proteins containing amino-terminal, midmolecule, and carboxyl-terminal PTHrP immunoreactivity as expected from the construct coding sequences. For example, the levels of PTHrP 1–34 in conditioned media from cells transfected with GFP3PTHrP 1–108 and GFP3PTHrP 1–139 were 3.2 ± 0.4 and 5.4 ± 0.6 pg/μg protein, respectively, significantly greater than the 1.8 ± 0.4 pg/μg found for cells transfected with vector (P < 0.05, n = 3 cell preparations per group). Also, PTHrP 38–64 levels were 3- to 5-fold greater in cells transfected with the GFP3PTHrP chimeras (data not shown). Finally, cells transfected with GFP3PTHrP 1–139 registered a 5-fold increase in PTHrP 109–139 levels, but cells transfected with GFP3PTHrP 1–108 did not. By immunoblot sizing analysis, the chimeric PTHrP proteins migrated as GFP-positive bands at their expected sizes (Fig. 4). The GFP3 vector protein was effectively restricted from the nucleus, as expected for its size. In contrast, the GFP3-PTHrP chimeric proteins, which include the PTHrP NLS, were distributed to cytoplasm, nucleus, and also nucleoli (Fig. 5).
The nuclear export studies used three chimeric PTHrP variants, GFP3-wild-type PTHrP 1–139, GFP3-PTHrP 1–108, and GFP3-PTHrP 1–139 127–136 (Fig. 3, A–C, respectively). The wild-type chimera would allow us to measure regulated export with an intact PTHrP NES, whereas the PTHrP 1–108 protein presumably had deleted the region containing the NES and would not undergo export. The purpose of the third construct was to refine the location of the NES. It deleted all of the pNES3 sequence except for L126, which remained to avoid disrupting pNES2.
Nuclear level of GFP3-PTHrP chimeras under steady-state conditions
The distribution of GFP3-PTHrP chimeric proteins between cytoplasm and nucleus differed significantly among 293 cells transiently transfected with GFP3-PTHrP 1–139, GFP3-PTHrP 1–139 127–136, and GFP3-PTHrP 1–108 (Fig. 6). The steady-state ratio of nuclear to cytoplasmic fluorescence (Fn/c) was more than 2-fold greater for the deletion and truncation mutants than for the wild-type 1–139 chimera. The constructs also presented different sensitivities to LMB. Treatment with LMB increased the Fn/c for the wild-type construct but had no effect on the two mutants.
In vitro assays of GFP3-PTHrP nuclear export
Nuclear export was measured with an in vitro assay using permeabilized transfected COS-1 cells. The permeabilized cells were bathed in a buffer containing all the components necessary for nuclear export except RanGTP. Export began after adding RanGTP and resulted in a decrease in nuclear GFP fluorescence for the wild-type GFP3-PTHrP 1–139 chimera with levels approaching background after about 15 min (Fig. 7A). In contrast, nuclear fluorescence for GFP3-PTHrP 1–139 127–136 showed almost no change over the same period. The nuclear signal for GFP3-PTHrP 1–139 decreased at a constant rate (Fig. 7B). However, fluorescence did not decline significantly for cells expressing GFP3-PTHrP 1–108, GFP3-PTHrP 1–139 127–136, GFP3-PTHrP 1–139 in the presence of LMB, or fixed cells expressing GFP3-PTHrP 1–139. This experiment was performed twice with nearly identical results.
We also measured the quantity of exported protein present in the transport buffer at the end of the experiment. After an export period of 50 min, the conditioned buffer from permeabilized COS-1 cells transfected with GFP3-PTHrP 1–139 contained approximately 300 pg/ml PTHrP/10,000 cells. In contrast, PTHrP levels were undetectable in the conditioned buffer from cells transfected with either of the mutant chimeras or cells carrying the wild-type chimera but treated with LMB.
FRAP assays of nuclear export of GFP3-PTHrP constructs in intact cells
Intact COS-1 cells expressing GFP3-PTHrP chimeras were incubated with cycloheximide to prevent synthesis of de novo GFP-containing protein and then underwent a period of photobleaching to diminish fluorescence substantially throughout the cytoplasm (Fig. 8). Micrographs in Fig. 8A illustrate how the template was drawn for the bleaching AOI and the course for a typical FRAP assay. The template surrounds the cytoplasm except for a narrow path taken to outline and exclude the nucleus. Figure 8 (middle panel) demonstrates that the photobleaching reduced cytoplasmic fluorescence to background levels in most of the cell but left nuclear fluorescence intact. Figure 8 (right panel) shows that cytoplasmic fluorescence began to recover at the end of the assay due to protein exported out of the nucleus. Nuclear fluorescence was perceptibly diminished. The three micrographs were exposed with same settings and processed to visualize the low levels of cytoplasmic fluorescence after recovery in the final image. The cellular fluorescence before bleaching was high and the prebleach image is saturated.
Nuclear export was quantified by repeated imaging to measure the shift of fluorescent protein from nucleus to cytoplasm. The initial Fn/c ratios after cytoplasmic bleaching were similar for all three chimeras. Nuclear fluorescence diminished and cytoplasmic fluorescence increased for the GFP3-PTHrP 1–139 chimera within only 50 sec (Fig. 8). The ratio decreased progressively with time for the wild-type chimera, but minimal changes were observed for the mutant chimeras. The t1/2 values for the decrease in Fn/c for GFP3-PTHrP 1–139 127–136 and GFP3-PTHrP 1–108 were similar, 433 and 385 sec, respectively, some 2.5 times greater than the t1/2 values for GFP3-PTHrP 1–139, 165 sec.
Discussion
Our first step in searching for the NES of PTHrP was to test whether the export signal depended on the carboxyl-terminal portion of PTHrP. We used FLIP to compare nuclear export of GFP-PTHrP 1–139 and the truncation mutant GFP-PTHrP 1–108. The latter protein lacked several groups of leucine residues that could be involved in export signaling. Both chimeric proteins were small enough to diffuse through the NPC (31). However, the fact that the larger chimera exited the nucleus faster than its smaller truncation mutant was inconsistent with purely diffusive transport; the smaller protein should diffuse faster. The result indicated that active transport contributed to PTHrP shuttling, even though passive egress of protein was possible. The result also suggested that a determinant within the PTHrP 109–139 region augmented the export rate. Interestingly, the two chimeric proteins differed somewhat in cellular distribution. GFP-PTHrP 1–108 labeled nucleoli much more heavily than GFP-PTHrP 1–139. A definitive explanation is not available, but we propose that the reduced rate of nuclear export leads to high nuclear levels of GFP-PTHrP 1–108 (as seen for GFP3-PTHrP 1–108 in Fig. 6), with consequently heavier collections in the nucleoli.
Next we conducted a sequence analysis to define what domains in PTHrP 109–139 might constitute an NES. Of three potential leucine-rich groupings, the 129–136 stretch in pNES3 appeared to fit the characteristics observed for NESs in other proteins (Fig. 2). This agrees with the location of the NES proposed by Jans et al. (15). Based on our analysis, we developed three chimeric PTHrP variants, GFP3-PTHrP 1–139, GFP3-PTHrP 1–108, and GFP3-PTHrP 1–139 127–136 (Fig. 3, A–C, respectively), to study whether pNES3 was important in nuclear export. By including three GFP proteins in series, the chimeras were too large to enter or exit the nucleus passively and required interaction with specialized transport machinery for nuclear transport. The wild-type chimera allowed us to measure regulated export with an intact export signal, whereas the PTHrP 1–108 protein lacked a large region that presumably spanned the putative NES. The third construct specifically deleted the pNES3 sequence but left L126 to avoid disrupting pNES2.
The steady-state nuclear and cytoplasmic levels of the various chimeric proteins contributed to the eventual conclusion that PTHrP 127–136 was important for nuclear export. Proteins with diminished nuclear export would be expected to accumulate in the nucleus to a greater extent than proteins with intact transport. This was the case for PTHrP 1–108 and PTHrP 1–139 127–136, compared with the wild-type PTHrP 1–139. Additional support for the hypothesis emerged from the differing degrees of interaction with CRM1. The Fn/c ratio for the wild-type chimeric protein increased after LMB treatment, indicating that CRM1 was involved in its transport. On the other hand, LMB did not affect the relative nuclear and cytoplasmic levels of the two mutant proteins, suggesting independence from CRM1. Because nuclear export of PTHrP involves CRM1, these data suggest that the carboxyl-terminal portion of PTHrP, particularly PTHrP 127–136 (pNES3), is important for nuclear export.
Direct assays of nuclear export provided confirmation for the proposition. We demonstrated that the export rates of PTHrP depended on an intact pNES3 in two different transport assays, one in vitro and one with intact cells. In the in vitro export assay, the PTHrP 1–108 and the PTHrP 1–139 127–136 chimeras underwent little if no export during the same period that nuclear fluorescence for the wild-type PTHrP 1–139 chimera fell by 80%. Consistent with its effects on steady-state protein levels, LMB reduced the export rate of GFP-PTHrP 1–139 but not the two mutants. The FRAP data showed that PTHrP export depended on carboxyl terminal sequences in intact cells also. The deletion of pNES3 in the GFP3-PTHrP 1–139 127–136 construct appeared to decrease nuclear export to the same extent as truncating the entire PTHrP 109–139 sequence in the GFP3-PTHrP 1–108 construct. Thus, multiple lines of evidence support a critical role for PTHrP 127–136 in nuclear export.
A definitive statement cannot be made whether the other leucine-rich regions, pNES1 and pNES2, contribute to nuclear export based on our data. Because the GFP3-PTHrP 1–139 127–136 construct contained intact pNES1 and pNES2 sequences, the other putative NESs in the 109–139 region may not be as important as pNES3 in determining nuclear export of PTHrP. However, we did not test individual deletion mutations of pNES1 or pNES2 and cannot exclude the possibility that these regions might augment transport in the presence of pNES3. However, pNES1 and pNES2 groups did not support nuclear export of GFP3-PTHrP 1–139 127–136 in the in vitro assay. Also they were not enough to mediate LMB-sensitive changes in steady-state levels of GFP3-PTHrP 1–139 127–136 (Fig. 6). Clearly pNES3 is necessary for CRM1 interaction and PTHrP export, but whether the sequence is sufficient to mediate export is unknown.
Our results are qualitatively similar in several respects to studies presented by Lam et al. (12, 33). They also showed that LMB increases the nuclear level of a GFP-PTHrP fusion protein and decreases the rate of nuclear export of that protein (12, 33). Their work was conducted in COS-1 cells and UMR 106.01 osteogenic sarcoma cells, whereas we studied LMB effects on nuclear PTHrP flux in HeLa cells and 293 cells. Direct quantitative comparisons of kinetic data are not feasible because the constructs and assay techniques differed between the laboratories. The Lam studies used a single GFP protein instead of GFP3 in their chimeric proteins, In addition, they performed FRAP by bleaching a partial region of the cytoplasm rather than the complete area.
Protein expression was decreased for the GFP3-chimeric proteins, compared with the expression of GFP3 protein. We have not established the reason for the difference. One possible explanation is that the presence of nuclear GFP3-PTHrP chimeric protein might decrease protein synthesis through an effect on ribosomal function. PTHrP is known to localize to bind to ribosomal RNA (13, 34). We also found that nucleolar accumulation of GFP-PTHrP chimeras appeared to exceed that of the triple GFP fusion proteins. We have no explanation at this time.
Additional work will be necessary to determine whether CRM1-mediated transport augments nuclear clearance of PTHrP above levels due to passive movement and whether the presence of absence of the PTHrP NES makes a difference in cell function or growth. Nuclear levels of PTHrP vary with cell cycle in UMR106 cells and the protein exerts effects that are specific for particular phases of the cell cycle (11, 35). One might expect that exiting the nucleus at the right time would be important in regulating cell cycle-specific levels of the protein, but whether NES facilitates nuclear escape at specific phases is unknown.
Summary
PTHrP has been shown to export from the nucleus via the CRM1 pathway (12), but details about the signal sequence had not been established. CRM1 typically recognizes a nuclear export signal, a loosely defined consensus sequence that is rich in leucine moieties. The 126–136 region within PTHrP matched this NES consensus pattern, notably in its last three amino acids consisting of two leucines separated by a polar residue. A mutant was created in which the putative NES, pNES3, was disrupted by deleting nine of the 10 residues. Four independent methods were used to assess the impact of this mutation on nuclear export of chimeric GFP3-PTHrP proteins. The GFP series provided a fluorescent tag and restricted passive nuclear shuttling. Transport of the pNES3 mutant was significantly altered, compared with the wild-type PTHrP 1–139 chimera, suggesting that it was not exported by CRM1. These results indicate that PTHrP exports in the CRM1 nuclear export pathway via a leucine-rich NES and that the 126–136 region is a critical component of the NES. The importance of this sequence for nuclear egress of wild-type PTHrP 1–139, which can move in and out of the nucleus passively, and the physiologic significance of NES-mediated export of PTHrP require further study.
Acknowledgments
Dr. Janna Bednenko and Dr. Larry Gerace (The Scripps Research Institute. La Jolla, CA) provided reagents and helpful discussion for the in vitro nuclear export studies.
Footnotes
This work was supported by The Flight Attendants Medical Research Institute, two Department of Veterans Affairs Merit Review Awards (to L.J.D. and R.H.H.), National Cancer Institute Grant CA23100, National Institutes of Health Grant DK60588, and the University of California Tobacco-Related Disease Research Program.
First Published Online November 17, 2005
Abbreviations: AOI, Area of interest; CRM1, chromosome region maintenance protein 1; cTB, complete TB; FLIP, fluorescence loss in photobleaching; Fn/c, ratio of the fluorescence of the nucleus and cytoplasm; FRAP, fluorescence recovery after photo bleaching; GFP, green fluorescent protein; LMB, leptomycin B; NES, nuclear export signal; NFM, nonfat dry milk; NLS, nuclear localization signal; NPC, nuclear pore complex; t1/2, half-time; TB, transport buffer.
Accepted for publication November 7, 2005.
References
Bednenko J, Cingolani G, Gerace L 2003 Nucleocytoplasmic transport: navigating the channel. Traffic 4:127–135
Fornerod M, Ohno M, Yoshida M, Mattaj IW 1997 CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051–1060
Askjaer P, Jensen TH, Nilsson J, Englmeier L, Kjems J 1998 The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP. J Biol Chem 273:33414–33422
Macara IG 2001 Transport into and out of the nucleus. Microbiol Mol Biol Rev 65:570–594
Moseley JM, Gillespie MT 1995 Parathyroid hormone-related protein. Crit Rev Clin Lab Sci 32:299–343
Goltzman D, Hendy GN, Banville D 1989 Parathyroid hormone-like peptide: molecular characterization and biological properties. Trends Endocrinol Metab 1:39–44
Hastings RH 2004 Parathyroid hormone-related protein and lung biology. Respir Physiol Neurobiol 142:95–113
Hastings RH, Quintana RA, Sandoval R, Duey D, Rascon Y, Burton DW, Deftos LJ 2003 Pro-apoptotic effects of parathyroid hormone-related protein in type II pneumocytes. Am J Respir Cell Mol Biol 29:733–742
Hastings RH, Araiza F, Burton DW, Zhang L, Bedley M, Deftos LJ 2003 Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells. Am J Physiol Cell Physiol 285:C1429–C1436
Hastings RH, Summers-Torres D, Yaszay B, LeSueur J, Burton DW, Deftos LJ 1997 Parathyroid hormone-related protein, an autocrine growth inhibitor of alveolar type II cells. Am J Physiol 272:L394–L399
Lam MH, Briggs LJ, Hu W, Martin TJ, Gillespie MT, Jans DA 1999 Importin recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin . J Biol Chem 274:7391–7398
Lam MH, Henderson B, Gillespie MT, Jans DA 2001 Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein. Traffic 2:812–819
Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15:4064–4075
Massfelder T, Dann P, Wu TL, Vasavada R, Helwig JJ, Stewart AF 1997 Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: a critical role for nuclear targeting. Proc Natl Acad Sci USA 94:13630–13635
Jans DA, Thomas RJ, Gillespie MT 2003 Parathyroid hormone-related protein (PTHrP): a nucleocytoplasmic shuttling protein with distinct paracrine and intracrine roles. Vitam Horm 66:345–384
Cingolani G, Bednenko J, Gillespie MT, Gerace L 2002 Molecular basis for the recognition of a nonclassical nuclear localization signal by importin . Mol Cell 10:345–353
Lam MH, Thomas RJ, Martin TJ, Gillespie MT, Jans DA 2000 Nuclear and nucleolar localization of parathyroid hormone-related protein. Immunol Cell Biol 78:395–402
Lam MH, Hu W, Xiao CY, Gillespie MT, Jans DA 2001 Molecular dissection of the importin 1-recognized nuclear targeting signal of parathyroid hormone-related protein. Biochem Biophys Res Commun 282:629–634
Ditmer LS, Burton DW, Deftos LJ 1996 Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1–173 increases production and secretion of the truncated forms. Endocrinology 137:1608–1617
Hastings RH, Asirvatham A, Quintana R, Sandoval R, Dutta R, Burton DW, Deftos LJ 2002 Parathyroid hormone-related protein-(38–64) regulates lung cell proliferation after silica injury. Am J Physiol Lung Cell Mol Physiol 283:L12–L21
Hastings RH, Quintana RA, Sandoval R, Burton DW, Deftos LJ 2003 Amino-terminal and mid-molecule parathyroid hormone-related protein, phosphatidylcholine, and type II cell proliferation in silica lung injury. Am J Physiol Lung Cell Mol Physiol 285:L1312–L1322
Howell JL, Truant R 2002 Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP. Biotechniques 32:80–87
Nicolas FJ, De Bosscher K, Schmierer B, Hill CS 2004 Analysis of Smad nucleocytoplasmic shuttling in living cells. J Cell Sci 117:4113–4125
Kehlenbach RH, Dickmanns A, Gerace L 1998 Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT in vitro. J Cell Biol 141:863–874
Birbach A, Bailey ST, Ghosh S, Schmid JA 2004 Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-B inducing kinase NIK. J Cell Sci 117:3615–3624
la Cour T, Gupta R, Rapacki K, Skriver K, Poulsen FM, Brunak S 2003 NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res 31:393–396
Bogerd HP, Fridell RA, Benson RE, Hua J, Cullen BR 1996 Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay. Mol Cell Biol 16:4207–4214
Kim FJ, Beeche AA, Hunter JJ, Chin DJ, Hope TJ 1996 Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions. Mol Cell Biol 16:5147–5155
Zhang MJ, Dayton AI 1998 Tolerance of diverse amino acid substitutions at conserved positions in the nuclear export signal (NES) of HIV-1 Rev. Biochem Biophys Res Commun 243:113–116
Fornerod M, Ohno M 2002 Exportin-mediated nuclear export of proteins and ribonucleoproteins. In: Weis K, ed. Nuclear transport. Berlin: Springer-Verlag; 67–91
Gorlich D, Kutay U 1999 Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 15:607–660
Brownawell AM, Kops GJ, Macara IG, Burgering BM 2001 Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Mol Cell Biol 21:3534–3546
Lam MH, Thomas RJ, Loveland KL, Schilders S, Gu M, Martin TJ, Gillespie MT, Jans DA 2002 Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules. Mol Endocrinol 16:390–401
Aarts MM, Levy D, He B, Stregger S, Chen T, Richard S, Henderson JE 1999 Parathyroid hormone-related protein interacts with RNA. J Biol Chem 274:4832–4838
Lam MH, House CM, Tiganis T, Mitchelhill KI, Sarcevic B, Cures A, Ramsay R, Kemp BE, Martin TJ, Gillespie MT 1999 Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormone-related protein negatively regulates its nuclear localization. J Biol Chem 274:18559–18566
Nicholas KB, Nicholas Jr HB, Deerfield DW 1997 GeneDoc: analysis and visualisation of genetic variation. EMBNEW. NEWS 4:14(Jared C. Pache, Douglas W. Burton, Leona)