Nuclear accumulation of interferon y

Examination of the interferon y (IFN-y) amino acid sequence revealed two conserved basic amino acid clusters similar to the prototype nuclear llizatin signal. We followed the fate of cedl surace receptor-bound IFN-y in murine leukemia L1210 cells. A timeand temperaturedependent accumulation of murine IFN-y in the cell nucleus could be demonstrated by autoradiography and Inirect immunofluorescence after the rapid isolation of nuclei. Human IFN-y was lso internalized and transiocated to the nucleus of murine L1210 cell transfeced with and exp g the human IFN-'y receptor, but it appeared to be retained by the nucleus only transiently. IFN-y mole chemically rosslinked to their cell surface receptor remaln capable ofbeing transocated to the nucleus even as part ofa receptor-ligand complex. Thus, the bipartite nuclear lztion signal sequence appears to be functional and suggests that nuclear targetingcould participate in IFN-y signal transduction. Murine and human interferon y (MuIFN-y, HuIFN-y) elicit similar cellular responses after binding to their speciesspecific cell surface receptors and subsequent internalization. Among these effects are antiproliferative and antiviral activity, modulation of surface antigens, and immunoregulation (1-3). Whether internalization of IFN-y is required for biological activity is still unclear. It was previously shown that binding to its receptor is not sufficient for HuIFN-y to elicit a physiologic response in murine cells (4, 5). Nevertheless, biological activity was observed after either microinjection ofHuIFN-y(6) or the direct intracellular production of HuIFN-y in murine cells (7). Earlier work suggested that interferon would bind to nuclear membrane receptors (8, 9). More recently, our understanding of post-cell-membrane receptor signaling pathways has increased and nuclear targeting of hormones and growth factors has been demonstrated (10). Analysis of nuclear protein structure has uncovered a consensus sequence of basic amino acids required for efficient transport from the cytosol to the nucleus. This amino acid sequence, the prototype of which is the basic sequence found in the large tumor antigen of simian virus 40 or nucleoplasmin (reviewed in ref. 10), is termed the nuclear localization signal (NLS). Close examination of murine and human IFN-y sequences revealed two conserved stretches of basic amino acids similar to the NLS (11). Furthermore, crystallographic analysis of HuIFN-y indicated that these stretches of basic amino acids are exposed on the surface of the protein (12). Antibodies directed against the first basic epitope, Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Gln-Lys [residues 84-94 of HuIFN-y) (12), abolish biological activity without inhibiting binding to its receptor (11). Recent analysis of HuIFN-y analogs showed that the second (C-terminal) basic cluster (residues 128-131 of HuIFN-y) is equally essential for a biological response and that partial or complete removal of this cluster results in drastic loss of activity (13). Thus, we asked whether the conserved clusters of basic amino acids function as NLSs in vivo-that is, whether IFN-'y is targeted into the nucleus. EXPERIMENTAL PROCEDURES Cell Culture. L1210 murine leukemia cells and transfected L1210 cells [clone 2/10-14, containing the entire gene for the HuIFN-y receptor (4)] were cultured as previously described (3). Binding Assays. 1251-labeled IFN-y (125I-IFNy) binding assays and acid elutions were performed on 2-5 x 107 cells as previously described (2, 3). Isolation of Nuclei. Suspensions of 2-5 x 107 cells in 1 ml of culture medium were laid over 2 ml of 20%o Ficoll-Paque (Pharmacia) in lysis buffer [PBS/10 mM MgCl2/10 mM CaCl2/0.1% dimethyl sulfoxide/2% (vol/vol) Nonidet P-40/ 1.6% (vol/vol) Triton X-100, pH 7.4] all Sigma, supplemented with 5% fetal bovine serum (FBS) and centrifuged at 200 x g for 8 min at 40C. After aspiration of supernatant, pellets containing nuclei were resuspended in lysis buffer without FBS. At this point preparations were examined for cytoplasmic lactate dehydrogenase (LDH) as an indicator of contaminating cytoplasm and were found to be LDH negative. Autoradiography. Cells or nuclei were incubated at saturating (-3 nM) concentrations of 1251-MuIFN-y or 1251HuIFN-y at 40C or 370C for 2 h. Cells and nuclei were subsequently washed in PBS, pH 7.4, containing 10 mM MgCl2 and 10mM CaCl2, and allowed to adhere to polylysinecoated glass chamber slides (Nunc) for 15 min. After removal of supernatant, cells were fixed with paraformaldehyde (4% in PBS) for 3 min, rinsed copiously with 5% FBS in PBS, and dried. Slides were coated with NTB-2 photographic emulsion (Kodak), developed at 48 h intervals, and counter-stained with Giemsa. Indirect Immunofluorescence. Cells fixed as above were permeabilized (0.1% Nonidet P-40, 10 min at 40C) and incubated with a rabbit anti-recombinant MuIFN-'y polyclonal antiserum raised in our laboratory at a dilution of 1:100. The secondary antibody, a fluorescein isothiocyanate (FITC)goat anti-rabbit antibody (Sigma), was used at a 1:50 dilution. DAPI (4',6-diamidino-2-phenylindole) solutions for staining nuclei were adjusted to 10 ng/ml to give similar light intensity as specific signals. Prior to microscopy (Olympus IMT2), cells were embedded in 50% glycerol in PBS, pH 8, containing p-phenylenediamine at 1 mg/ml to reduce photobleaching (14). Photographs are pseudocolor representations of digitized images acquired after intensification (equal gain for all pictures) and background subtraction as described (15). Crosslinking of IN-y to Its Receptor. Cells were treated with saturating concentrations (-3 nM) of murine or human 125I-IFN-y (2 h, 40C), washed twice with PBS (pH 7.4, Abbreviations: FITC, fluorescein isothiocyanate; Hu, human; IFN-y, interferon y; LDH, lactate dehydrogenase; Mu, murine; NLS, nuclear localization signal. *To whom reprint requests should be addressed at the present address: National Institute of Environmental Health Sciences, Laboratory of Pulmonary Pathobiology, 111 T. W. Alexander Drive, Building 101, D-224, Research Triangle Park, NC 27709. 11831 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11832 Cell Biology: Bader and Wietzerbin supplemented with 10 mM I crosslinked with disuccinimi (2, 3). After 15 min cells wer( ml of medium. Nuclei were irn time point (0.25 h) together w other samples were cultured i nuclei were isolated. After two pellets were frozen at -20' autoradiography (3).