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Mol Cell Biol. Oct 2002; 22(19): 6788–6796.
PMCID: PMC134045

Nuclear Targeting by the Growth Factor Midkine

Abstract

Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.

Extracellular signaling molecules such as growth factors and cytokines bind to plasma membrane receptors that activate their own kinases and/or recruit adapter proteins. These events on the plasma membrane have been regarded as the onset of signaling. Subsequent to receptor binding of ligands, the ligand-receptor complexes are internalized and delivered to specific vesicular compartments (e.g., early and late endosomes and lysosomes), leading to desensitization. While the ligands are often degraded, the receptors themselves are either degraded or recycled back to the cell surface.

Mounting evidence indicates that nuclear targeting by extracellular signaling molecules plays an indispensable role in their biological activities. For example, acidic fibroblast growth factor (aFGF) and Schwannoma-derived growth factor need nuclear localization for their mitogenic activity (28, 33, 76). For basic FGF (bFGF), increases in ribosomal gene transcription and cell proliferation are tightly correlated to the nuclear translocation of bFGF (1, 5). Thus, signals from the cell surface receptor and translocation of the ligand to the nucleus cooperate and play roles in the biological activities of many extracellular signaling molecules. The translocation of ligands across the plasma membrane is dependent on their own plasma membrane receptors. Nuclear localization signals (NLSs) of ligands themselves have been implicated in nuclear translocation for many ligands, such as platelet-derived growth factor A (PDGF A) (11), PDGF B (41), aFGF (28, 76), gamma interferon (82), interleukin 1α (75), interleukin 1β (24), and interleukin 5 (29). However, the precise mechanism of nuclear targeting by extracellular signaling molecules is poorly understood.

Midkine was first identified as the product of a retinoic acid-responsive gene that is up-regulated in the differentiation system of embryonal carcinoma cells (32, 70). Its important roles have been implicated in various aspects of biology, such as neuronal survival and differentiation (48, 73, 79), carcinogenesis (10, 30, 51, 72), and tissue remodeling (31, 53, 80). At the cellular level, midkine promotes cell growth (47, 48, 68), cell survival (54, 58, 73), cell migration (26, 43, 59, 66), and plasminogen activator activity (38). Although midkine does not have an apparent NLS, it is localized in the nucleus in hemangioma cells (67) and in cells in a variety of tumor tissues (data not shown). Recently, we identified low-density lipoprotein (LDL) receptor-related protein (LRP) as a midkine-binding protein (49). Because the LRP antagonist receptor-associated protein (RAP) suppresses midkine-mediated neuronal cell survival, it has been suggested that LRP is a component of the functional midkine receptor (49).

LRP belongs to the LDL receptor family. There are five prototype members of the family: LDL receptor, ApoE receptor 2, very low-density lipoprotein (VLDL) receptor, LRP, and LRP2/Megalin. The major functions of these receptors are to endocytose and deliver their ligands to lysosomes for degradation or catabolism (27, 39, 65). There are over 30 identified ligands of these receptors, including ApoE lipoproteins, α2-macroglobulin, plasminogen activator, and plasminogen activator inhibitor-1 complexes, lipoprotein lipase, and thrombospondin-1 (21). Among them, ApoE lipoproteins are common ligands for all members, whereas α2-macroglobulin is a specific ligand for LRP (22). In addition, it was recently reported that some members of the LDL receptor family function as signaling membrane receptors. ApoE receptor 2 and VLDL receptor are reelin receptors, which play a crucial role in neuronal-cell migration during embryogenesis and which utilize adapter protein Disabled-1 for intracellular signaling (12, 25, 71). Recently identified members of the family LRP5 and -6 function, together with Frizzled, as Wnt receptors, which are important for body axis determination, neuronal differentiation, and carcinogenesis (45, 56, 69, 74). These findings suggest that LRP may also function as a signaling membrane receptor for midkine. However, taking into account that LRP exhibits the strongest endocytosis activity among the LDL receptor family members (42), it is also an intriguing hypothesis that LRP mediates midkine internalization and is involved in nuclear targeting by midkine. In this paper we report the involvement of LRP and nucleolin, a nucleocytoplasmic shuttle protein, in the nuclear targeting of midkine. We found that (i) LRP delivers exogenous midkine not only to the endocytosis/degradation system but also to the nuclear transport system, (ii) midkine endocytosis via LRP is followed by cytoplasmic nucleolin-midkine association, which leads to the nuclear translocation of midkine, (iii) NLS-deficient nucleolin traps midkine in the cytoplasm and inhibits its nuclear localization, and (iv) nuclear targeting is needed for the full antiapoptotic activity of midkine.

MATERIALS AND METHODS

Reagents.

Yeast-produced human midkine protein (26) was a generous gift from S. Sakuma (Meiji Milk Co. Ltd., Odawara, Japan). N-terminally biotinylated human midkine was prepared as described previously (61). Rabbit antibasigin antibodies were raised against the extracellular domain of mouse basigin (15). 125I-Na, ECL, protein A-Sepharose CL-4B, and protein G-Sepharose 4 fast flow were purchased from Amersham Pharmacia Biotech. DNase I and the pGEM-T Easy Vector System I were from Promega. pcDNA3.1+ was from Invitrogen. Lipofectamine Plus was from Life Technologies. Fluorescein isothiocyanate (FITC)-conjugated streptavidin, the anti-FLAG M2 monoclonal antibody, the tetramethyl rhodamine isocyanate-conjugated anti-mouse immunoglobulin G (IgG) antibody, the peroxidase-conjugated monoclonal antibiotin antibody (BN-34), and propidium iodide were from Sigma. The antinucleolin mouse monoclonal antibody (MS-3) was from Santa Cruz Biotechnology. The antihemagglutinin (HA) rat monoclonal antibody (3F10) was from Roche. N-Nitrosocysteine was from Dojin. The Lab-Tek chamber slides were from Nunc. Restriction enzymes and a ligation kit were from Takara. The horseradish peroxidase-conjugated anti-mouse IgG antibody was from Seikagaku. Cisplatin was from Bristol-Myers Squibb.

Cells and culture.

Mouse L cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and were seeded onto dishes, chamber slides, or coverslips a day before each experiment. Mouse embryonic fibroblasts (MEF) (PEA13 [LRP−/−, LRP deficient, homozygous, CRL-2216], PEA10 [LRP+/−, LRP deficient, heterozygous, CRL-2215], and MEF-1 [LRP+/+, simian virus 40-transfected, CRL-2214]) (77) were obtained from the American Type Culture Collection and cultured as described above.

Radioiodination of midkine.

The purified midkine protein was radioiodinated by the chloramine-T method. Ten micrograms of midkine in 90 μl of 50 mM sodium phosphate buffer, pH 7.2, was added to 1 mCi of 125I-Na and 50 μl of a freshly prepared solution of chloramine-T (1 mg/ml in 50 mM sodium phosphate buffer, pH 7.2). After 1 min at 20°C, the reaction was stopped by adding 50 μl of 5 mM sodium bisulfite and 100 μl of 150 mM potassium iodide. Free 125I-Na was separated from 125I-midkine by chromatography on Sephadex G-25 (PD10; Amersham Pharmacia Biotech) equilibrated with 50 mM sodium phosphate buffer, pH 7.2, containing 0.25% bovine serum albumin. Analysis of 125I-midkine by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE) followed by autoradiography gave a single band of 17 kDa. The specific activity of 125I-midkine was 5 × 107 cpm/μg.

Overexpression of nucleolin and nucleolin mutants.

Mouse nucleolin cDNA (nucleotides [nt] 1 to 2124) (6) tagged with the coding sequence for a FLAG epitope sequence was obtained from the RNA of mouse fibroblasts by means of PCR with reverse transcription. Several nucleolin mutants, in which the NLS (encoded by nt 841 to 897), N terminus (encoded by nt 1 to 831), or C terminus (encoded by nt 907 to 2124) was deleted were designed as shown in Fig. Fig.6.6. For mutants with acidic stretches, acidic stretches were tentatively defined as follows: AS1, encoded by nt 1 to 135; AS2, encoded by nt 136 to 510; AS3, encoded by nt 511 to 654; AS4, encoded by nt 655 to 831. Subcloning of PCR products was performed with pGEM-T Easy Vector System I. After DNA sequencing, nucleolin expression vectors were constructed in pcDNA3.1(+). Each vector was transiently transfected into cells with Lipofectamine Plus a day before each experiment. Cells were lysed with a lysis buffer (20 mM Tris-HCl [pH 7.5], 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 0.5 mM EDTA, 0.5% [vol/vol] Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 μg of aprotinin/ml, 10 μg of leupeptin/ml, and 1 mM sodium vanadate) and then centrifuged at 10,000 × g at 4°C for 10 min. A supernatant (cell lysate) and its immunoprecipitate with the anti-FLAG M2 monoclonal antibody were prepared and analyzed by Western blotting with the anti-FLAG M2 monoclonal antibody, the horseradish peroxidase-conjugated anti-mouse IgG antibody, and the ECL system.

FIG. 6.
Mapping of midkine-binding sites in nucleolin. (A) Structures of nucleolin and its derivatives. AS, acidic stretch; GAR, glycine- and arginine-rich domain; MK, midkine; RBD, RNA-binding domain. At the right, the ability to bind to midkine (+ or ...

Kinetic analysis of endocytosis and cell fractionation.

The binding of 125I-midkine was carried out for 2 h at 4°C with 20 ng of 125I-midkine/ml in culture medium. Unbound ligand was removed by washing with ice-cold culture medium, and midkine-bound cells were incubated for various times at 37°C in prewarmed medium. At each time point, the plates were quickly placed on ice and the medium was replaced with ice-cold phosphate-buffered saline (PBS). Cells were harvested and fractionated into detergent-soluble and nuclear fractions by a method previously described (1, 17, 81). Briefly, the cells were harvested by trypsinization (0.125% trypsin, 0.025% EDTA in PBS at 37°C for 10 min) and then lysed in 1 ml of 20 mM Tris-HCl, pH 7.5, containing 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 0.5 mM EDTA, 0.5% (vol/vol) Triton X-100, 1 mM PMSF, 2 μg of aprotinin/ml, 10 μg of leupeptin/ml, and 1 mM sodium vanadate (nuclear preparation buffer). The detergent-soluble fraction was obtained by removal of nuclei with low-speed centrifugation (800 × g for 5 min at 4°C) (57). The pellet was further washed three times with the same buffer to obtain nuclei. Microscopic analysis demonstrated the absence of cytoplasmic membranes and organelles in the nuclear fraction. The nuclei were solubilized in 0.25 ml of nuclear preparation buffer comprising 0.4% (wt/vol) SDS, 1 mM MgCl2, and 10 U of DNase I for 3 min at 4°C, followed by the addition of 0.75 ml of nuclear preparation buffer. The nuclear extract was incubated at 37°C for 5 min and at room temperature for an additional 10 min and then centrifuged at 14,000 × g for 10 min (57). Both fractions were analyzed by SDS-15% PAGE, followed by autoradiography.

For some assays, cells were fractionated into membrane and cytosolic fractions. After harvest by trypsinization, cells were washed with ice-cold culture medium and then with the nuclear preparation buffer without Triton X-100 and homogenized in the nuclear preparation buffer without Triton X-100. After removal of the nuclear pellet by low-speed centrifugation (800 × g for 5 min at 4°C), high-speed centrifugation (100,000 × g for 1 h at 4°C) was performed to separate membranes and vesicles from soluble proteins. The soluble fraction was regarded as the cytosolic fraction. The pellet was resolved in nuclear preparation buffer containing 0.5% Triton X-100 (membrane fraction).

Cell surface biotinylation.

Cell surface biotinylation was performed with EZ-link (Pierce) in accordance with the manufacturer's protocol. After biotinylation, cells were lysed with ice-cold radioimmunoprecipitation assay buffer (10 mM sodium phosphate [pH 7.0], 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 2 mM EDTA, 1 mM PMSF). Proteins were subjected to SDS-PAGE and Western blotted with the mouse monoclonal antibiotin antibody.

Indirect immunofluorescence staining.

To visualize the subcellular localization of midkine, nucleolin, and Cy3-labeled RAP, indirect immunofluorescence staining was performed. For these experiments, cells were grown on glass coverslips or chamber slides and then treated with 200 ng of biotinylated midkine/ml and/or 1 μg of Cy3-labeled RAP/ml. The cells were washed three times with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 10 min at 4°C, permeabilized with 1 or 4% Triton X-100 in PBS at 4°C for 20 min, and then blocked with 1% bovine serum albumin in PBS at 4°C for 30 min. The treated cells were incubated at 20°C for 1 h with FITC-conjugated streptavidin. Finally, they were extensively washed with PBS and then examined with a confocal microscopic system (MRC1024 system). For counterstaining, detection of the nuclei was performed with 0.5 μg of propidium iodide/ml, which detected double-stranded nucleic acids. For the immunodetection of endogenous nucleolin or overexpressed nucleolin and a mutant nucleolin tagged with FLAG, cells were stained first with the antinucleolin mouse monoclonal antibody or the anti-FLAG M2 monoclonal antibody and then with the tetramethyl rhodamine isocyanate-conjugated anti-mouse IgG antibody.

Cell survival assay.

Cells transfected with the vector for NLS-deficient nucleolin (ΔNLS) or the control vector were seeded on chamber slides in DMEM supplemented with 10% fetal bovine serum. After overnight incubation, culture medium was replaced with fresh DMEM without serum. After a further 48 h of incubation, cells were pretreated with or without midkine (100 ng/ml) for 3 h and then treated with 100 μM cisplatin (an anticancer drug) or 200 μM S-nitrosocysteine (a donor of NO) for 24 h. Percentages of apoptotic cells were estimated with the In Situ apoptosis detection kit (Takara) based on the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) method (18). Cells were counterstained with 0.2 μg of propidium iodide/ml. Apoptotic and healthy cells were counted in five fields at 200× magnification (total cell number per field was approximately 500). Statistical analysis was performed with a paired t test. P values <0.05 were regarded as significant.

RESULTS

Midkine is internalized and translocated to the nucleus.

The kinetics of exogenous midkine internalization were studied by exposing cells to 125I-labeled midkine at 20 ng/ml. We first compared the efficiencies of two available methods (low pH and trypsinization) for removing cell surface-bound 125I-midkine. 125I-midkine was allowed to bind to the cell surface at 4°C for 2 h. Unbound midkine was removed by washing the cells with ice-cold medium. The cell surface-bound fraction represented approximately 50% of the applied 125I-midkine. To remove the cell surface-bound fraction, cells were incubated with either a low pH buffer (150 mM NaCl, 50 mM acetic acid) or 0.125% trypsin and 0.025% EDTA in PBS. Most of the cell surface-bound ligands were removed by either method, but approximately 1% of the applied 125I-midkine remained with the cells, which probably reflected the not-removed cell surface 125I-midkine fraction (see the band at 0 min in Fig. Fig.1A).1A). We thus used the trypsin method in the subsequent experiments.

FIG. 1.
Time course of midkine internalization. L cells (a mouse fibroblast cell line) were incubated with 125I-midkine at 4°C for 2 h. Cells were washed with ice-cold medium to remove unbound 125I-midkine and then incubated at 37°C to initiate ...

By a shift of the temperature to 37°C after the cells were washed with ice-cold medium, the cells were induced to internalize cell surface-bound 125I-midkine. The time course of 125I-midkine internalization is shown in Fig. Fig.1.1. The amount of internalized midkine in the detergent-soluble fraction reached a peak at around 10 min (Fig. 1A and C), whereas the nuclear fraction did not peak until 20 min (Fig. 1B and D). Note that the recovered midkine appeared to be intact because it was indistinguishable in size from the intact form (Fig. 1A and B). The amount of internalized midkine decreased after these points, reaching the level at 0 min at around 60 min. Consistent with continuous intracellular midkine degradation, 125I counts released into the medium increased until 60 min (data not shown).

The subcellular localization of midkine was determined by means of indirect immunofluorescence using N-terminally biotinylated midkine and FITC-conjugated streptavidin. Cytoplasmic midkine was detected within 5 min of incubation at 37°C, whereas the intensity of the signal in the nucleus reached a maximum at 20 min (Fig. (Fig.2),2), which is consistent with the results in Fig. Fig.1.1. The internalized midkine was detected not only homogeneously, reflecting cytoplasmic localization, but also in vesicular compartments.

FIG. 2.
Spatial and temporal profiles of midkine internalization. L cells were exposed to midkine, which was labeled with biotin at its N terminus, as in Fig. Fig.1.1. The localization of midkine (green; top and bottom panels) was determined by indirect ...

Midkine internalization is dependent on LRP.

Since midkine binds to LRP (49), we examined if midkine internalization depends on LRP. Wild-type MEF (LRP+/+) internalized midkine, but LRP-deficient MEF (LRP−/−) did not (Fig. (Fig.3).3). LRP+/− cells, which are heterozygous for the LRP locus, gave the same results as LRP+/+ cells (data not shown).

FIG. 3.
Suppression of midkine internalization in LRP-deficient cells. Wild-type (LRP+/+) and LRP-deficient (LRP−/−) embryonic fibroblasts were used to assess the effect of LRP on midkine internalization. The procedure was the ...

Differential intracellular trafficking of midkine and RAP.

The profile of midkine internalization was compared with that of RAP, since both of the molecules are ligands for LRP. After 5 min of incubation at 37°C, midkine and RAP were mostly colocalized inside the cells, probably in early endosomes (Fig. (Fig.4).4). However this colocalization disappeared afterward. While midkine was translocated to the nucleus, RAP remained in the vesicular compartments in the cytoplasm (Fig. (Fig.4).4). Thus, LRP-mediated endocytosis does not always induce nuclear targeting by its ligands. The data also suggest that there is a mechanism to promote the nuclear translocation of midkine.

FIG. 4.
Differential internalization of midkine and RAP. Both midkine and RAP are ligands for LRP. Their internalization profiles were compared. The procedure was as described in Fig. Fig.2.2. Bar, 10 μm.

Cytoplasmic midkine binds to nucleolin.

We previously observed that midkine binds to nucleolin in a ligand blot assay (67). Nucleolin has been implicated in various aspects of ribosome biogenesis (rDNA transcription, rRNA maturation, and ribosome assembly) and nucleocytoplasmic transport (20). Surprisingly, nucleolin was also reported to be located on the cell surface (8, 13, 35, 40). We therefore localized nucleolin in cells used in this study.

As a monoclonal antibody against nucleolin that can be used for immunocytochemistry was not good for immunoprecipitation, we generated a full-length nucleolin construct tagged with FLAG at its C terminus. Figure Figure5A5A shows proteins extracted by cell fractionation. LRP and basigin (a glycoprotein belonging to the Ig superfamily) are cell surface membrane proteins. Calnexin is a membrane protein of the endoplasmic reticulum. These three were mainly detected in the membrane fraction (Fig. (Fig.5A).5A). Nucleolin was detected not only in the nuclear fraction but also in the cytosolic and membrane fractions (Fig. (Fig.5A).5A). When the cell surface was labeled by biotinylation, cell surface membrane protein basigin was biotinylated but endoplasmic reticulum membrane protein calnexin was not (Fig. (Fig.5B).5B). Nucleolin was itself biotinylated (Fig. (Fig.5B);5B); thus a part of the nucleolin was located on the cell surface.

FIG. 5.
Midkine binds to LRP and then to nucleolin. (A) Cell fractionation for detection of protein location. Cyt, cytosol; Memb, membrane; Nuc, nucleus. (B) Western blot analysis after cell surface biotinylation. Cell surface membrane protein basigin was biotinylated, ...

The roles of two key molecules (LRP and nucleolin) in midkine internalization were then determined. Consistent with the data shown in Fig. Fig.3,3, L cells (an LRP-expressing mouse fibroblast cell line) and LRP+/+ cells internalized midkine but LRP−/− cells did not (Fig. (Fig.5C,5C, lysate). When nucleolin-FLAG was overexpressed, 125I-midkine was coprecipitated with nucleolin-FLAG in L and LRP+/+ cells but not in LRP−/− cells (Fig. (Fig.5C).5C). Therefore, midkine endocytosis depends on LRP and midkine binds to nucleolin only when midkine is endocytosed. Figure Figure5D5D supports these data. As midkine binds to the second and fourth ligand-binding domains of LRP (K. Kadomatsu, unpublished data), we used HA-mLRP4T100. This construct is a miniform of LRP containing the fourth ligand-binding domain with a HA tag at the N terminus, which is functional in endocytosis if an appropriate ligand is applied (52). In LRP−/− cells, 125I-midkine was internalized only when HA-mLRP4T100 was overexpressed (Fig. (Fig.5D,5D, lysate). Cotransfection of nucleolin-FLAG and HA-mLRP4T100, but not nucleolin-FLAG alone, enabled nucleolin to bind midkine (Fig. (Fig.5D,5D, IP: α-FLAG).

These data were further supported by Fig. Fig.5E.5E. In this experiment, we examined the temporal profile of midkine internalization by cell fractionation. In the membrane fraction (probably mainly endosomes), the amount of 125I-midkine was relatively constant from 5 to 20 min on incubation at 37°C. From this fraction, midkine was coimmunoprecipitated with LRP but not nucleolin. In contrast, the amount of midkine in the cytosolic fraction reached a maximum at 10 min. Midkine was coimmunoprecipitated with nucleolin in this fraction. Taken together, the data indicate that midkine is endocytosed by LRP and is translocated to the cytosol, where it binds to nucleolin. As nucleolin is known as a nucleocytoplasmic shuttle protein, it is possible that midkine is translocated to the nucleus as a cargo of nucleolin.

A nucleolin mutant affects midkine localization.

We next determined the midkine-binding site in nucleolin. The structure of nucleolin can be divided into four parts (Fig. (Fig.6).6). The N-terminal domain contains four acidic stretches (AS1, -2, -3, and -4), major phosphorylation sites, and sites for nucleolin-protein interaction. The second part is a bipartite NLS, which is essential for the nuclear translocation of nucleolin. The third part contains the four RNA-binding domains (RBD1, -2, -3, and -4), essential for RNA binding. At the C terminus is the GAR domain, which is rich in glycine, arginine, and phenylalanine residues and which is important for both the nucleolin-protein interaction and the function of the RNA-binding domains (Fig. (Fig.6).6). We generated various nucleolin mutant constructs (Fig. (Fig.6).6). The results in Fig. Fig.6B6B indicate that the N-terminal domain of nucleolin, particularly the acidic region, is responsible for midkine binding. One acidic stretch among the four was sufficient for midkine binding.

The localization of midkine and endogenous nucleolin was then examined. Figure Figure77 shows pictures of LRP+/+ cells after 20 min of 37°C incubation. Following serum stimulation of starved cells, 90% of cells showed a predominantly nucleolar localization for nucleolin and nuclear localization for midkine (Fig. (Fig.7,7, left). The remaining 10% of cells showed only a nuclear and cytoplasmic localization for nucleolin and a cytoplasmic localization for midkine (Fig. (Fig.7,7, left). This close relation of midkine and nucleolin in intracellular localization suggests the possible involvement of nucleolin in nuclear targeting by midkine.

FIG. 7.
Effect of an NLS-deficient nucleolin on midkine subcellular localization. (Left) Localization of endogenous nucleolin and exogenously added midkine. LRP+/+ cells were incubated under serum starvation for 2 days and then stimulated with ...

Nucleolin expression vectors were next transfected into LRP+/+ cells. Since LRP+/+ cells have endogenous nucleolin, midkine localization may be under the control of both endogenous nucleolin and overexpressed mutant nucleolin. Overexpression of the full-length nucleolin did not affect midkine localization (Fig. (Fig.7,7, right). However, the NLS-deficient mutant (ΔNLS) showed a striking phenomenon: midkine appeared to be trapped in the cytoplasm and was colocalized with ΔNLS (Fig. (Fig.7,7, right). The results indicate that ΔNLS has a dominant-negative effect on midkine subcellular localization and support the idea that the endogenous nucleolin functions in the nuclear transport of midkine.

Nuclear targeting is necessary for the antiapoptotic activity of midkine.

When LRP+/+ cells were treated with cisplatin (an anticancer drug), apoptosis was induced: 100 μM cisplatin caused the death of approximately 60% of the cells within 24 h. If cells were pretreated with 100 ng of midkine/ml for 3 h and then treated with cisplatin, cell death was significantly suppressed [Fig. [Fig.8A,8A, LRP+/+ (CV) cells; P < 0.01]. This suppression was significantly blocked in ΔNLS-overexpressing cells (P < 0.05) [compare LRP+/+ (CV) and LRP+/+ (ΔNLS) in Fig. Fig.8A].8A]. In LRP−/− cells, midkine could not protect cells from cisplatin-induced apoptosis [Fig. [Fig.8A,8A, LRP−/− (CV)]. However, if a miniform of LRP was overexpressed, midkine significantly suppressed cisplatin-induced apoptosis [Fig. [Fig.8A,8A, LRP−/− (mLRP4T100)]. Similar results were obtained when NO-induced apoptosis was examined (Fig. (Fig.8B).8B). These results suggest that the nuclear targeting is essential for the full activity of midkine in cell survival.

FIG. 8.
Effect of overexpression of ΔNLS and LRP on antiapoptotic activity of midkine. Cells were transfected with the indicated vectors. Control vectors (CV) used were pcDNA3.1+ for the nucleolin ΔNLS expression vector (ΔNLS) ...

DISCUSSION

The present findings indicate that LRP-mediated endocytosis not only leads to ligand degradation but also confers a pathway for the nuclear localization of a ligand. Midkine is internalized in an LRP-dependent manner and is further transported to the nucleus depending on nucleolin. The nuclear targeting of midkine is indispensable for the full activity of midkine in the promotion of cell survival.

It has been reported that nucleolin is also localized on the cell surface (8, 13, 35, 40). For example, cell surface nucleolin has been implicated in virus infection (8, 13). During human immunodeficiency virus (HIV) infection of T lymphocytes, the V3 loop of HIV binds to cell surface nucleolin, facilitating HIV attachment to cells, which can be counteracted by exogenous midkine (9). Indeed, nucleolin was detected at the cell surface in the present study. However, the cell surface nucleolin was not involved in midkine endocytosis, since only cytosolic nucleolin associated with midkine. The laboratories of Olson and Melese originally proposed a model of nucleolin as a scaffold protein, that is, the N-terminal domain of nucleolin recruits proteins important in ribosome biogenesis, while RNA-binding domains interact with rRNA (46, 62). This model was confirmed and extended by recent data. The N-terminal domain is needed for the first step in rRNA processing (19). Topoisomerase I binds to the first, second, and third, but not the fourth, acidic stretches in the N-terminal domain of nucleolin (14). The idea that nucleolin recruits topoisomerase I to sites of rDNA transcription in the nucleolus (4, 14) is supported by the findings that Saccharomyces cerevisiae nucleolin ortholog Nsr1p binds to yTop1p (yeast topoisomerase I ortholog) and is important in the subcellular localization of yTop1p (14). The present study has further extended the model and demonstrated for the first time that nucleolin functions in the nuclear transport of a growth factor. It is possible that other ligands, besides midkine, may bind to and utilize nucleolin for their nuclear targeting. The following two findings support this idea: the N-terminal domain of nucleolin binds NLSs (41, 78), and many ligands known to be localized in the nucleus contain NLSs (11, 24, 28, 29, 41, 75, 76, 82).

We failed to detect a physical association between LRP and nucleolin (data not shown). Thus, there may be some distance between these two molecules. We speculate that LRP-bound midkine is internalized and transported to the early or late endosomes. Since midkine is functional even at low pH, as assessed by using a system involving midkine-induced plasminogen activator activity (37), the function of midkine may remain intact in the endosomes. Pseudomonas exotoxin (PE) provides one model for the translocation of an exogenous protein into the cytosol via LRP-mediated internalization (16, 36, 52). After binding to LRP on the cell surface, PE is translocated as a cargo of endosomes to the endoplasmic reticulum, where furin cleaves it, producing an ADP-ribosylating fragment. This fragment is translocated to the cytosol from the endoplasmic reticulum and inactivates elongation factor 2 through ADP ribosylation, shutting down protein synthesis. Although diphtheria toxin also employs receptor-mediated internalization, proteolytic processing, translocation to the cytosol, and ADP-ribosylation of elongation factor 2, this toxin is translocated to the cytosol from acidic endosomes. As full-length midkine was recovered from the nucleus, it is not likely that dynamic processing, as for PE, for midkine translocation to the cytosol takes place. Midkine may provide another mechanism of cytoplasmic translocation via LRP-mediated internalization.

Nucleolin is known to be phosphorylated in its N-terminal domain by casein kinase II and p34cdc2 (3, 7, 55). In a study involving Xenopus laevis egg extracts, it was shown that p34cdc2 phosphorylation sites improve the nuclear translocation of nucleolin when they are dephosphorylated and enhance cytoplasmic localization when they are phosphorylated (63). Therefore, nuclear localization of nucleolin depends on both its NLS and phosphorylation status. Nucleolin-bound midkine may be transported to the nucleus following this mechanism.

With respect to nuclear targeting by midkine, we recently found that the 37-kDa laminin-binding protein precursor binds midkine and is cotranslocated with midkine into the nucleus (61). It is possible that midkine uses both nucleolin and laminin-binding protein precursor as shuttle proteins but that, in the cells used in the present study, the nucleolin shuttle is the predominant one.

The intracellular domain of LRP can recruit many cytosolic signaling molecules, including DAB1, JIP-1, PSD-95, and SHC (2, 23). Thus, we cannot exclude the possibility that LRP functions as a signaling membrane receptor for midkine. It is rather likely that signaling from both the cell surface and the nucleus cooperates for the full activity of midkine, as reported for other growth factors, such as aFGF, bFGF, and Schwannoma-derived growth factor (28, 33, 76). The results in Fig. Fig.88 support this idea.

We previously reported that pleiotrophin/HB-GAM (heparin-binding growth associated molecule) also binds nucleolin in a ligand blot assay (67). Midkine and pleiotrophin/HB-GAM exhibit about 50% homology and form a family of heparin-binding growth factors that is distinct from the FGF family and other growth factor families (50). These two factors share several biological activities, such as the enhancement of neurite extension and plasminogen activator activity and the promotion of carcinogenesis. It is thus plausible that pleiotrophin/HB-GAM uses the same pathway as midkine does for its nuclear targeting. It is possible that LRP (49; K. Kadomatsu, unpublished data) and receptor-type protein tyrosine phosphatase ζ (43, 44, 59) are shared by midkine and pleiotrophin/HB-GAM. Additionally, it has been proposed that membrane heparan sulfate proteoglycan N-syndecan (34, 60) and receptor tyrosine kinase ALK (64) are receptors for pleiotrophin/HB-GAM. Cell surface complex formation from these molecules and its cooperation with nuclear signaling are the next subjects of investigation.

Acknowledgments

We thank Justin Weinberg for reading and commenting on the manuscript.

This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and grants-in-aid for Center of Excellence Research and by grants from the National Institutes of Health.

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