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.

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 internalization of exogenous ligands. At the indicated time points, cells were treated with trypsin to remove cell surface-bound proteins, and detergent-soluble and nuclear fractions were isolated. (A and B) Autoradiography after SDS-15% PAGE of detergent-soluble (A) and nuclear (B) fractions. (C and D) Relative densitometrical densities of A and B, respectively, with the maximum value set at 100%.

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. 1. The localization of midkine (green; top and bottom panels) was determined by indirect immunofluorescence staining. The nucleus was stained with propidium iodide (red; top panels). The number in each panel indicates minutes of 37°C incubation. Bar, 10 μm.

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 same as in Fig. 2. The number in each panel indicates minutes of 37°C incubation. Bar, 10 μm.

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. 2. Bar, 10 μm.

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, but not endoplasmic reticulum membrane protein calnexin. Nucleolin was biotinylated. Antibodies used were anti-FLAG (F), anticalnexin (Cal), antibasigin (Bsg), and antibiotin (Bio) antibodies. IP, immunoprecipitation. (C) Cells were allowed to endocytose ¹²⁵I-midkine as described for Fig. 1. Cells were harvested after 10 min of 37°C incubation. ¹²⁵I-midkine (MK) was barely endocytosed by LRP^−/− cells (Lysate) even when nucleolin-FLAG was overexpressed. The association of ¹²⁵I-midkine and nucleolin-FLAG took place only in L and LRP^+/+ cells, not in LRP^−/− cells (IP: α-FLAG). (D) Effect of LRP overexpression on midkine internalization in LRP^−/− cells. ¹²⁵I-midkine was internalized and associated with nucleolin in LRP^−/− cells only when LRP was overexpressed. (E) Temporal profiles of midkine internalization by cell fractionation. LRP^+/+ cells transfected with an LRP expression vector (mLRP4T100) were used. ¹²⁵I-midkine was associated with LRP in the membrane (Mem) fraction. In contrast, ¹²⁵I-midkine was associated with nucleolin in the cytosolic (Cyt), but not membrane, fraction.

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 −) is shown. (B) Various FLAG-tagged nucleolin expression vectors were transfected into LRP^+/+ cells. After incubation with ¹²⁵I-midkine at 4°C, cells were allowed to internalize ¹²⁵I-midkine by incubation at 37°C for 20 min. The binding between ¹²⁵I-midkine and overexpressed FLAG-tagged nucleolin was assessed by precipitation with anti-FLAG antibody as described in Materials and Methods.

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 10% fetal bovine serum for 12 h. The subsequent procedure was the same as for Fig. 2. Pictures of cells at 20 min after 37°C exposure are shown. Arrow, nucleolar localization of nucleolin and nuclear localization of midkine; arrowhead, nuclear and cytoplasmic localization for nucleolin and cytoplasmic localization for midkine. (Right) LRP^+/+ cells were transfected with nucleolin expression vectors. Bar, 10 μm.

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) and pcDNA3 for an LRP expression vector (mLRP4T100). After 48 h of serum starvation, cells were incubated with or without midkine (100 ng/ml; MK) for 3 h. Cells were then incubated with 100 μM cisplatin (an anticancer drug) (A) or 200 μM S-nitrosocysteine (SNOC; a donor of NO) (B). Apoptotic cells were counted as described in Materials and Methods. The y axis represents the relative ratio of apoptotic cell number as revealed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method, normalizing to the value of cisplatin alone. A representative result is shown here (three independent experiments showed similar results). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; n.s., not significant.