Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10*

Trinette L. Chuang and Raymond R. SchleefDagger

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single Mr 80,000 EGFP·PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology.

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Serine proteinase inhibitors (serpins)1 are a large superfamily of homologous proteins that resemble alpha 1-proteinase inhibitor in overall structure and form stoichiometric 1:1 inhibitory complexes with target proteases that are typically stable to treatment with denaturants (e.g. SDS) (1-3). Serpins play crucial roles in the neutralization of serine protease activities that are involved in a wide variety of vital processes including blood coagulation, fibrinolysis, complement activation, inflammation, and cell migration (2). Within the serpin superfamily, ovalbumin represents the parent prototype of a currently emerging family of structurally related proteins (ov-serpins) (4). Human members of the ov-serpin family include plasminogen activator inhibitor-2 (PAI-2) (5), an elastase inhibitor isolated from monocyte-like cells (6), squamous cell carcinoma antigen (7), cytoplasmic antiproteinase (i.e. protease inhibitor-6) (8, 9), and a tumor suppressor called maspin (10). Two serpins related to protease inhibitor-6 have been cloned from a placental lambda gtII library (i.e. protease inhibitors -8 and -9) (11), and data of Sun et al. (12) suggest that the latter molecule is an intracellular granzyme B inhibitor that is associated with cytotoxic lymphocytes.

During studies investigating the presence of protease inhibitors in hematopoiesis, our group utilized a polymerase chain reaction (PCR)-based homology cloning strategy to identify a novel ov-serpin, which exhibited a high amino acid homology (48%) with PAI-2, protease inhibitor-6, and human leukocyte elastase inhibitor (13). The isolated cDNA contains a single large open reading frame that encodes a 397-amino acid protein (13). Northern blotting analysis with this cDNA revealed a single 2.3-kilobase transcript that is expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. This molecule was designated bone marrow-associated serpin (bomapin) and assigned the systematic title of protease inhibitor 10 (PI-10) by the Genome Data Base Collaboration (13). Recent data obtained in our laboratory (14) revealed that transcripts for this protease inhibitor are elevated in the bone marrow and peripheral blood of patients with acute myeloid leukemia and in chronic myelomonocytic leukemia, suggesting that PI-10 may be expressed preferentially in hematopoietic progenitor cells of monocytic lineage.

PI-10 exhibits all the structural features that distinguish ov-serpins from the larger family of serpin proteins (13), which is typified by the absence of an N-terminal signal peptide extension that results in an intracellular distribution for many of these molecules (4). Another feature that is observed only in ov-serpins is the presence of an insertion between helices C and D that is believed to form a loop between these two helixes (4). For example, exon 3 in the PAI-2 gene codes for a 33-amino acid residue sequence between helices C/D (5), which contains the substrate sites within PAI-2 for intracellular enzyme transglutaminase (15) and a domain responsible for the binding of PAI-2 to several cytosolic proteins (e.g. annexin I) (16). In comparison, the C/D-interhelical loop of PI-10 contains a series of basic amino acids (KKRK77) (13) that exhibit homology to known nuclear targeting signals (e.g. SV40 antigen, PKKKRKV; underlined amino acids have been shown to be particularly important for signal function; Ref. 17). Hypothesizing that the interhelical loop may direct PI-10 to the nucleus, we examined the role of this region in the cellular distribution of this molecule by generating expression constructs of PI-10 fused to a reporter tag. Transfection experiments using a eukaryotic model cell system (i.e. HeLa cells) revealed that the C/D-interhelical loop contains sufficient and necessary information to target a reporter to the nucleus.

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Plasmid Constructions-- The entire coding region for PI-10 was excised from pBluescript SK(+)/PI-10 (13) using the 5'-SacI site and the 3'-ApaI site and subcloned into a SacI/ApaI-digested pEGFP-C3 vector (CLONTECH, Palo Alto, CA). To mutate four basic amino acids in the interhelical loop of PI-10, the 871-nucleotide region between the two EcoRI sites in PI-10 (i.e. nucleotides 234 and 1105) was first removed from pEGFP·PI-10 by digestion with EcoRI, and the plasmid was gel-isolated and religated, thus generating a cDNA construct with a single EcoRI site immediately downstream of the region encoding the interhelical loop. This construct (i.e. pEGFPC3·PI-10 down-triangle 234-1105) was subjected to PCR amplification using 20 pmol of SacI-containing forward primer 5'-TCAGATCTCGAGCTCCACC-3' and 20 pmol of mutagenic EcoRI-containing reverse primer 5'-CGCCTTCGAATTCCATTGCCGCTGCTGCTTCACTTTCAGGGTCAC-3' (underlined sequence indicates mutated region) in combination with conditions described previously (13). The PCR product was gel purified, digested with SacI and EcoRI, and ligated into an SacI/EcoRI-digested pEGFP·PI-10 down-triangle 234-1105 to generate pEGFP·PI-10 K74A, K75A, R76A, K77A, down-triangle 234-1105. In a separate reaction, the 871 bp region between nucleotides 234-1105 was gel-isolated following EcoRI digestion of pEGFP·PI-10 and religated to an EcoRI-digested pEGFP·PI-10 K74A, K75A, R76A, K77A, down-triangle 234-1105. The resulting construct (i.e. pEGFP·PI-10 K74A, K75A, R76A, K77A) will be subsequently referred to as pEGFP·PI-10 A4.

To subclone the interhelical loop of PI-10 and the PI-10 A4 mutant into pEGFP, primers flanking the cDNA encoding the interhelical loop of PI-10 between Arg-63 to Glu-87 (i.e. forward primer, 5'-TAAGCAGAGCTCAGAGACCAGGGAGTCAAATGTG; reverse primer, 5'-TCCGACGGGCCCTTCCGAGTTGCTCAAGTTGAATTCC) were prepared that contained sites for restriction enzymes to facilitate subcloning (SacI and ApaI, respectively). The interhelical loop of PI-10 and PI-10 mutant A4 were separately amplified, and the PCR products were subcloned in-frame into the pEGFP-C3 vector. Sequencing was performed by the dideoxy termination method as described previously (13).

The cDNA encoding PAI-2 in pUC8 (i.e. pPAI J7) (18) was provided by E. K. O. Kruithof (University Hospital of Geneva, Geneva, Switzerland). The PAI-2 cDNA was excised using EcoRI and initially subcloned into an EcoRI-digested pBluescript SK(+). The PAI-2 cDNA was subsequently excised using SalI and BamHI and subcloned into a SalI/BamHI-digested pEGFP-C3.

Growth and Transfection of HeLa Cells-- HeLa cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transfection studies, HeLa cells were plated into either 8-well cell culture slides (5 × 104/well) or 60-mm diameter culture dishes (5 × 105/dish) in growth media and cultured for 24 h. The cells were washed and transfected with 0.25 µg/ml of a DNA construct by utilizing 2 µg/ml LipofectAMINE according to the manufacturer instructions (Life Technologies, Inc). Transiently transfected cells were washed and fixed, and 200 EGFP-expressing transfected cells were examined for nuclear or cytoplasmic staining using a Leitz Diaplan microscope. Data are represented as the percentage of cells with fluorescent nuclear staining. For generation of stable cell lines, the transfected cells were washed after 48 h and incubated in media supplemented with the antibiotic Geneticin (G-418, Life Technologies) at 0.5 µg/ml, and individual clones were isolated by ring cloning. Stable lines were analyzed for the production of recombinant proteins by subjecting 106 cells to SDS-polyacrylamide gel electrophoresis (PAGE)/immunoblotting using previously described protocols (14, 19) with the modification that a monoclonal antibody to GFP (1:500 dilution, product 8362-1, CLONTECH) was the primary detecting antibody.

In Vitro Transcription/Translation of EGFP·PI-10 and EGFP·PI-10 A4-- The cDNA inserts of EGFP·PI-10 and EGFP·PI-10 A4 in the vector pBluescript were expressed using a coupled in vitro transcription and translation reticulocyte lysate system (Promega, Madison, WI) in the presence of [35S]methionine (Amersham Pharmacia Biotech) and T3 RNA polymerase (Promega) using conditions previously described (13). For analysis, samples (2.5 µl) of the reaction mixtures generated in the presence of pBluescript-EGFP·PI-10 or pBluescript-EGFP·PI-10 A4 were incubated (15 min, 37 °C) in the absence or presence of thrombin (product T6759, Sigma). Samples were heated to 100 °C in the presence of 2% SDS and 100 mM dithiothreitol, subjected to SDS-PAGE, and analyzed by fluorography. Quantitations of bands in the x-ray film were performed by densitometry using an AlphaImager 2000 Documentation and Analysis System.

Isolation of Nuclei and Immunoanalysis-- Nuclei of EGFP·PI-10-transfected cells were prepared according to the procedure of Benz and Strominger (20). Briefly, washed cells (107) were resuspended in 2 ml of 0.25 M sucrose, 5 mM CaCl2, 25 mM Hepes, pH 8 (4 °C), and incubated (4 °C, 5 min) with an equal volume of 0.15 M sucrose, 5 mM CaCl2, 25 mM Hepes containing 0.5% Brij 58 (Sigma). The mixture was diluted 30-fold with 0.25 M sucrose buffer, and the nuclei were washed twice by centrifugation. Alternatively, the transfected cells were disrupted by 40 strokes in a Dounce homogenizer, and the nuclei were pelleted by centrifugation (600 × g, 10 min) as described previously (21). The pellet was resuspended in 0.25 M sucrose buffer, the homogenization repeated, and the preparation examined in a phase-contrast microscope to ensure lysis of the cells. Nuclei were harvested and washed by centrifugation (600 × g, 10 min). Washed nuclei were resuspended in 0.15 M NaCl at 107/ml, lysed by three freezing/thawing cycles, and clarified by centrifugation (10,000 × g, 10 min). The nuclear lysates were subjected to immunoprecipitation using previously described protocols (14, 19). Briefly, rabbit antisera to GFP (product 8363, CLONTECH) or non-immune rabbit serum (1 µg/ml, respectively) were incubated (16 h, 4 °C) with protein A-Sepharose beads. The beads were washed and incubated with 100 µg of nuclear lysates from EGFP·PI-10-transfected cells. The beads were washed by centrifugation, eluted with SDS-sample buffer, and subjected to SDS-PAGE/immunoblotting.

    RESULTS AND DISCUSSION
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To gain information on the cellular distribution of PI-10, we prepared eukaryotic expression vectors containing the cDNA encoding PI-10 fused to a tag (i.e. enhanced green fluorescent protein, EGFP). A cell line (i.e. HeLa cells) was selected that does not constitutively express PI-10 and has been shown to be a useful model system for the transfection/high-level expression of another ov-serpin (i.e. PAI-2) (22). Fig. 1 indicates that incubation of HeLa cells in the presence of LipofectAMINE and pEGFP·PI-10 resulted in intense fluorescence of the nucleus in comparison to the weak staining of the cytoplasm (panel B) and the negative fluorescence of cells incubated with LipofectAMINE in the absence of a plasmid (panel A). Control experiments with plasmids encoding EGFP (vector alone, panel C) resulted in a general staining of the cytoplasm. Quantitative analysis revealed that over 94.62 ± 2.4 (mean ± S.D.) of the pEGFP·PI-10-transfected cells were positive for nuclear staining, whereas only 5.12 ± 1.9 of the nuclei of the pEGFP (vector alone)-transfected cells were positive (Fig. 2A). To demonstrate that localization of the complex to the nucleus was not caused by the formation of a nuclear targeting signal following the fusion of a serpin to EGFP, we subcloned the cDNA encoding the ov-serpin PAI-2 into the vector pEGFP and prepared comparable experiments. Data from a representative transient experiment using plasmid pEGFP-PAI-2 are shown in bar 3 of Fig. 2A and indicate that the expressed PAI-2-containing fusion complex is primarily restricted to the cytoplasm. These transient transfection experiments have been repeated four times with similar results. To examine if the localization to the nucleus is a result of the transient transfection conditions, we also transfected cells with these three EGFP-expressing vectors and isolated single cells in the presence of the antibiotic G418. Eight clones have been isolated from each group, and intense fluorescence was observed in >90% of nuclei in the resulting HeLa clones that were produced by transfection with the EGFP·PI-10 construct, whereas cytoplasmic staining (i.e. <10% nuclear staining) was detected in the stable EGFP vector alone, transfected clones, and in the stable pEGFP·PAI-2-transfected clones.


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Fig. 1.   Immunofluorescent analysis of HeLa cells transfected with expression vectors encoding EGFP-fusion constructs. HeLa cells were incubated with fresh growth media supplemented with LipofectAMINE (2 µg/well) in the absence of a plasmid vector (panel A) or the presence of either pEGFP·PI-10 (panel B, 0.25 µg/well) or pEGFP (panel C, 0.25 µg/well). After 48 h, the cells were washed, fixed, and examined utilizing a Leitz Diaplan microscope equipped for epifluorescence (×1000 magnification).


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Fig. 2.   Evidence for a nuclear targeting signal in helix C/D-interhelical loop of PI-10. Panel A, nuclear targeting of EGFP·PI-10, and mutation of four basic amino acids in the interhelical region of PI-10 alters its cellular distribution. HeLa cells were transfected with LipofectAMINE and either pEGFP·PI-10 (bar 1), pEGFP (bar 2), pEGFP·PAI-2 (bar 3), or pEGFP·PI-10 A4 (bar 4). Twenty-four hours later, the cells were fixed and 200 GFP-expressing transfected cells were examined for nuclear or cytoplasmic staining. Data are represented as the percentage of cells with fluorescent nuclear staining. Inset, molecular species of EGFP·PI-10 in nuclear extracts from transfected cells. Nuclei were isolated from EGFP·PI-10 transfected clone C2E2 (lanes 1 and 2) and EGFP-transfected clone D5 (lanes 3 and 4), lysed by freezing/thawing cycles, and clarified by centrifugation. Nuclear extracts were immunoprecipitated with either normal rabbit IgG (lanes 1 and 3) or rabbit anti-GFP (lanes 2 and 4) coupled to Sepharose A beads and analyzed by SDS-PAGE/immunoblotting. Panel B, formation of complexes between thrombin and either EGFP·PI-10 or EGFP·PI-10 A4 mutant. Samples (2.5 µl) of in vitro transcription/translation reaction mixtures generated in the presence of [35S]methionine and either pBluescript-EGFP·PI-10 (closed symbols) or pBluescript-EGFP·PI-10 A4 (open symbols) were incubated (15 min, 37 °C, n = 4) in the presence of increasing concentrations of thrombin. Samples were subjected to SDS-PAGE and analyzed by fluorography. Relative quantitations of 35S-labeled Mr 120,000 thrombin/EGFP·PI-10 complex were performed by densitometry and expressed as the percent of the initial Mr 80,000 35S-labeled EGFP·PI-10 band. Data (mean ± S.D.) from a representative experiment are shown. Inset, an autoradiogram in which 35S-labeled EGFP·PI-10 (top panel) or 35S-labeled EGFP·PI-10 A4 (bottom panel) was incubated either in the absence (lane 1) or presence (lane 2, 0.05 unit; lane 3, 0.17 unit; lane 4, 0.5 unit; lane 5, 1 unit) of thrombin.

The presence of the fluorescent tag in the nucleus of pEGFP·PI-10-transfected cells suggested that either PI-10 itself contains a nuclear targeting signal or that cytoplasmic PI-10 interacts with another molecule that contains the appropriate signal. Increasing information indicates that proteases are normal constituents of the nucleus and may play a role in various processes. For example, serine proteases have long been recognized to be associated with and can modulate the degradation of chromatin (23, 24). Tokes and Clawson (25) have observed the association of a lamin-specific protease with the nuclear scaffold, and this enzyme appears to participate in the remodeling of the nuclear scaffold after treatment with carcinogens. Granule serine proteases have also been shown to be present within the nuclei of natural killer cells (26). In light of these data, one mechanism to account for the localization of the fluorescent construct to the nucleus could be the interaction between a nuclear targeted protease and EGFP·PI-10 in the cytoplasm and the subsequent routing of this high Mr protease/inhibitor complex to the nucleus. Furthermore, proteasomes have also been detected in the nucleus (for review, see Ref. 27) and could participate in degradation of nuclear targeted PI-10-containing enzyme complexes. However, recent data also indicate that certain proteases are found only within the nucleus. More specifically, Scholtz et al. (28) detailed the characteristics of a cysteine protease that can only be detected in nuclei of mouse embryonic carcinoma cells following their differentiation with retinoic acid, and this group suggested that this protease may play a role in the metabolism of transcription factors. Myer et al. (29) extended this general concept by functionally identifying a serine protease that cleaves one transcription factor (i.e. STAT5beta ) and demonstrating that this enzyme is restricted to the nucleus of early hematopoietic cells. Because the direct targeting of free PI-10 would constitute a mechanism to regulate proteases that are spatially separated from cytoplasmic protease inhibitors, the molecular species of EGFP·PI-10 in the nucleus was characterized to determine whether PI-10 was present in the nucleus in either a free-form or an inactive high Mr protease/inhibitor complex. For this purpose, a protocol (20) for the isolation of intact and functionally active nuclei was selected. This protocol utilizes the lysis of cell membranes and other organelles with the hydrophilic detergent Brij 58, followed by isolation of nuclei by differential centrifugation. Isolated nuclei were extracted by repeated freezing/thawing cycles, and the species of EGFP in the extracts were immunoprecipitated, separated by SDS-PAGE, and detected by immunoblotting using a monoclonal antibody to GFP. The inset to Fig. 2A demonstrates that rabbit antibodies to GFP (lane 2 versus normal rabbit IgG used in lane 1) are able to specifically immunoprecipitate an Mr 80,000 protein in nuclear extracts isolated from EGFP·PI-10-transfected cells but not from EGFP-transfected cells. Furthermore, the size of the molecule immunoprecipitated from nuclei of EGFP·PI-10-transfected cells is in agreement with the combined molecular weights of the Mr 27,000 GFP, a 19-amino acid linker and the Mr 45,000 PI-10 protein. In comparison, immunoprecipitation of nuclear EGFP-transfected cells revealed little free EGFP (Fig. 2A, inset, lane 4). The prominent bands at Mr 55,000-60,000 in lanes 1-4 represent cross-reactivity of the detecting system with rabbit IgG, heavy chain. Similar results were obtained using nuclei that were isolated from cells disrupted with a Dounce homogenizer followed by differential centrifugation (data not shown).

Although a consensus sequence for nuclear localization has not be found, a preponderance of basic amino acids has been shown to be important for this process (for reviews, see Refs. 17 and 30). For example, the sequence PKKKRKV is found in a number of viral proteins (e.g. SV40 T-antigen), the Drosophila Mr 70,000 heat-shock protein, and the tail region of nucleoplasmin (17, 30). We noted that the C/D-interhelical loop of PI-10 contained four basic amino acids that were not only similar to the PKKKRKV sequence in SV40 T-antigen but also identical to the arrangement of the basic amino acids in the proposed nuclear targeting signal within human lamin A (i.e. 423SVTKKRKLE) (30). To investigate the role of charged residues in the C/D-insertion of PI-10 in the targeting of this molecule to the nucleus, we mutated four basic amino acids in this region (i.e. 64QGVKCDPESEKKRK, underlined indicates mutated region) to alanine using a PCR-based strategy. To demonstrate that this mutation did not cause the molecule to fold abnormally, we first characterized one important function of this mutant that is dependent upon the conformation of a serpin. Studies on dysfunctional serpins have revealed that the inhibitory properties of this superfamily are dependent upon both conformation and structural mobility (for review, see Ref. 3). Therefore, we compared the ability of the PI-10 A4 mutant (i.e. pEGFP·PI-10 K74A, K75A, R76A, K77A) and the wild-type PI-10 molecule to form high molecular weight complexes with a protease that are not dissociated by the denaturant, SDS. For this purpose, EGFP·PI-10 and EGFP·PI-10A4 were expressed by in vitro transcription/translation utilizing conditions previously described (13). In the absence of information on a cytosolic or nuclear protease that specifically interacts with PI-10, thrombin was selected for these experiments based upon its reported ability to form high Mr SDS-stable complexes with 35S-labeled PI-10 (13). The inset in Fig. 2B shows that the major species of 35S-labeled EGFP·PI-10 (lane 1 in top panel) and 35S-labeled EGFP·PI-10 A4 (lane 1 in bottom panel) migrate primarily on SDS-PAGE with an Mr 80,000, a molecular size similar to the species of EGFP present in the nucleus of EGFP·PI-10-transfected cells (Fig. 2A). In addition, lanes 2-5 of the inset to Fig. 2B indicate that both 35S-labeled EGFP·PI-10 (top panel) and 35S-labeled EGFP·PI-10 A4 (bottom panel) form Mr 120,000 SDS-stable complexes with thrombin that are dependent on protease concentration. Densitometric scanning of the autoradiograms revealed that similar amounts of Mr 120,000 complexes are formed using either 35S-labeled EGFP·PI-10 (Fig. 2B, closed symbols) or 35S-labeled EGFP·PI-10 A4 (open symbols) at each thrombin concentration. Because these data suggest that the PI-10 A4 mutant is not abnormally folded but rather exhibits a similar conformation as the wild-type molecule with respect to its ability to form a complex with the protease thrombin, we examined the ability of the PI-10 A4 to be targeted to the nucleus. Fig. 2A indicates that transient transfection of HeLa cells with EGFP·PI-10 A4 resulted in the distribution of the fluorescent label to be confined to the cytoplasm (bar 4) in comparison with the nuclear localization of wild type PI-10 coupled to the fluorescent tag (bar 1). The data shown are representative of four transient transfection experiments and have also been extended to include four stable cell lines derived using this construct (i.e. <15% nuclear staining/pEGFP·PI-10 A4-transfected clone). This information indicates that four amino acids in the C/D-interhelical loop of PI-10 are necessary for the targeting of this molecule to the nucleus.

These experiments also raise the issue of whether the C/D-interhelical loop of PI-10 contains sufficient information for targeting this protein to the nucleus or if other parts of the molecule are required for this process. Based upon experiments using peptides corresponding to the C/D-interhelical loop of the related ov-serpin PAI-2, Jensen et al. (16) suggested that the insertions in ov-serpins represent independent protein binding domains that evolved for distinct functions. To determine whether the C/D-interhelical loop of PI-10 represents an independent domain capable of targeting a protein to the nucleus, the cDNA encoding this region (i.e. Arg-63 to Glu-87 of PI-10) was PCR-amplified and subcloned into pEGFP. Transient transfection experiments revealed nuclear localization for both EGFP·PI-10 (Fig. 3, bar 1) and EGFP coupled to the C/D-interhelical loop of PI-10 (bar 2). As a control, the cDNA encoding the interhelical loop of PI-10 A4 (i.e. Arg-63 to Glu-87 with mutations K74A, K75A, R76A, and K77A) was also amplified and subcloned into this vector. Transfection experiments revealed that the construct encoding EGFP coupled to the interhelical loop of PI-10 A4 mutant (bar 3), as well as EGFP alone (bar 4), were restricted to the cytosol.


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Fig. 3.   The interhelical loop of PI-10 contains sufficient information to target a reporter to the nucleus. HeLa cells were incubated with media supplemented with LipofectAMINE (2 µg/well) in the presence of 0.25 µg/well of either pEGFP·PI-10 (bar 1), pEGFP·interhelical loop of PI-10 (bar 2), pEGFP·interhelical loop of PI-10 A4 (bar 3), or pEGFP alone (bar 4). Twenty-four hours later, the cells were fixed and 200 GFP-expressing transfected cells were examined for nuclear or cytoplasmic staining. Data are represented as the percentage of cells with fluorescent nuclear staining.

In summary, our data suggest that the C/D-interhelical loop of PI-10 represents an independent nuclear targeting domain. In addition to the interaction of proteins with the C/D-interhelical and reactive site loops, a number of binding sites for other molecules has been identified within the serpin superfamily. Well characterized examples include (i) the interaction of antithrombin III with heparin-containing molecules in the extracellular matrix, cell surface, or solution-phase; and (ii) the interaction of plasminogen activator inhibitor type 1 (PAI-1) with vitronectin present within the extracellular matrix (2, 3). These additional binding sites serve to both localize and regulate the proteolytic inhibitory activity of serpins. If this concept extends to PI-10, one could speculate that additional binding sites exist on PI-10 that serve to localize this inhibitor and hence protect specific nuclear proteins against proteolytic degradation. Current research in our group is directed at clarifying this issue. Furthermore, because a wide variety of proteases are known to converge on the nucleus and affect several processes including proliferation and differentiation, the information presented in this report raises the possibility that other protease inhibitors could be targeted into the nucleus and function in defining and modulating intracellular processes not just in a generalized manner but at their site of action within the nucleus.

    FOOTNOTES

* This research was supported by grants from the National Institutes of Health (HL49563 and HL45954).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Vascular Biology (VB-1), The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7129; Fax: 619-784-7323; E-mail: rschleef{at}scripps.edu.

    ABBREVIATIONS

The abbreviations used are: serpin, serine protease inhibitor; EGFP, enhanced green fluorescent protein; ov-serpin, ovalbumin family of serine protease inhibitors; PAI-2, plasminogen activator inhibitor type 2; PCR, polymerase chain reaction; PI-10, protease inhibitor-10; PI-10 A4, protease inhibitor A4 mutant K74A, K75A, R76A, K77A; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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