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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01224

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Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-B inducing kinase NIK

¹ Department of Vascular Biology and Thrombosis Research, University of Vienna Medical School and Competence Center Bio-Molecular Therapeutics, Schwarzspanierstr. 17, 1090 Vienna, Austria
² Room S620, Section of Immunobiology, Yale University Medical School, 300 Cedar Street, New Haven, CT 06520, USA

View larger version (62K): [in a new window]   Fig. 1. Nuclear and nucleolar localization of NIK. (A) Immunofluorescent staining of endogenous NIK in the breast cancer cell line MB-MDA-231 indicates predominant nuclear localization of NIK in these cells. (B) Top: schematic drawing of NIK showing the predicted nuclear localization sequence (amino acids 143-149, black box) and two potential nuclear export sequences, as predicted by computer search (hatched boxes). Bottom: truncation mutants of YFP-NIK lacking either the first or both of the putative nuclear export sequences accumulate in the nucleus and nucleolar-like structures as assessed by confocal laser scanning microscopy. 293 cells were transiently transfected with the fusion constructs using the calcium phosphate method and moderately expressing cells were assessed by confocal laser scanning microscopy. (C) Verification of the nucleolar localization of YFP-NIK truncation mutants. 293 cells were transfected as described before using YFP-NIK(1-769), YFP-NIK(1-794) or full length YFP-NIK together with CFP-L23, a ribosomal protein accumulating in nucleoli. (D) Immunofluorescent staining of endogenous NIK in the breast cancer cell line MB-MDA-231 and counterstaining with DAPI. Nucleolar structures are indicated by arrows.

View larger version (68K): [in a new window]   Fig. 2. Identification of a nucleolar localization signal. (A) Mutation of the basic amino acids in the region 143-146 (RKKR) to alanine residues prevents localization of YFP-NIK1-794 in nucleoli while still retaining its nuclear accumulation. Moderately expressing cells were analyzed by confocal microscopy. (B) Fusion of the seven basic amino acids 143-149 of NIK to either the C terminus of GFP (GFP-RKKRKKK) or the N terminus of GFP (RKKRKKK-GFP) induces accumulation of the modified GFP in nucleoli. An additional accumulation in the nucleoplasm is observed for the C-terminally tagged GFP-RKKRKKK.

View larger version (13K): [in a new window]   Fig. 3. Fluorescence loss in photobleaching (FLIP) proves that the putative first NES is not active. 293 cells were grown on coverslips and transfected (top) with YFP-NIK(1-794), which contains the first predicted NES, or with (bottom) YFP-NIK(1-769) lacking this domain. The coverslips were mounted onto an incubation chamber filled with medium and live cells were examined by laser scanning microscopy. The cytosol was bleached with high laser power and cytosolic as well as nuclear fluorescence intensities were recorded over time as described in the Materials and Methods section. Fluorescence decreased significantly in the cytosol whereas it remained constant in the nucleus for both constructs indicating that nuclear NIK did not shuttle into the cytosol.

View larger version (45K): [in a new window]   Fig. 4. Characterization and verification of nuclear export and nucleolar localization sequences by point mutations in full-length NIK. (A) To verify the functionality of the NES at position 795-805 that was previously identified with truncation mutants, point mutations of the conserved leucine or isoleucine residues were generated. Mutation of leucines at positions 803 and 805 to alanine induced a significant shift from the cytosol into the nucleus and accumulation in nucleoli. The same pattern was observed for variants in which isoleucine-800 or all hydrophobic amino acids of the predicted NES (NESmut) were replaced by alanine. (B) The NES mutant of full-length NIK accumulates in nucleoli and colocalizes with nucleolar CFP-L23 chimera. (C) Replacement of the basic amino acids RKKR at positions 143-146 with alanines in the NES-mutant form of NIK leads to nuclear localization with exclusion of nucleoli. (D) Mutation of all seven basic amino acids RKKRKKK at positions 143-149 of the NES mutant results in a predominant cytosolic localization.

View larger version (15K): [in a new window]   Fig. 5. Fluorescence loss in nucleoli upon bleaching in the nucleoplasm reveals dynamic distribution of NIK between nucleoli and nucleoplasm. FLIP experiments were performed with (A)YFP-NIK(1-769) and (B) YFP-NIK(1-794) as described in the Materials and Methods section. A bleach region was defined in the nucleoplasmic area, distant from nucleoli, and fluorescence intensities were measured in the nucleoplasm and in nucleoli over time. The decrease in nucleolar fluorescence indicates rapid exchange of nucleolar proteins with the nucleoplasm.

View larger version (24K): [in a new window]   Fig. 6. FRAP analysis to determine diffusion and mobility of NIK variants within a compartment. (A) Mobility of NIK in cytosol (cyt.), nucleoplasm (nuclear) and nucleoli (nucleolar) as determined by FRAP. Variants of YFP-NIK with the respective major localization were analyzed for fluorescence recovery after photobleaching as described in the Materials and Methods section. The values for the calculated maximum recovery (max) indicating the fraction of mobile molecules and the calculated halftime (t1/2) of diffusion are specified. Representative curves are shown. Mean values of halftimes normalized for the bleaching area and standard deviations are specified in the text. (B) Mobility of nucleolar YFP-NIK compared to the GFP-chimera of the ribosomal protein L23 and nucleolar GFP. Halftimes of diffusion and maximum values are given.

View larger version (15K): [in a new window]   Fig. 7. Effect of NIK variants on NF-B activation in reporter gene assays. 293 cells were transfected with a NF-B-dependent luciferase reporter (containing 5 NF-B binding sites) and a constitutively expressed ß-galactosidase construct for normalization together with NIK variants exhibiting distinct intracellular localizations. The fold induction over the vector control is given for nuclear/non-nucleolar NIK (NES mutant, 143-146A), nuclear/nucleolar NIK (NES mutant), for cytoplasmic NIK that shuttles between cytosol and nucleus (cyt./shuttling; YFP-NIK wild type) and for cytoplasmic NIK that has both NES and NLS mutated (NES mutant, 143-149 A; cyt./non-shuttling).

View larger version (6K): [in a new window]   Fig. 8. Schematic illustration of the localization signals of NIK. From the results obtained with various mutants of YFP-NIK, a scheme of the targeting domains is depicted, showing the nucleolar localization sequence (NoLS, amino acids RKKR at position 143-146), the nuclear localization sequence (NLS, amino acids KKK at 146-149), and the functional nuclear export sequence (NES, amino acids 795-805).

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