Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4290-4294, May 1992 Cell Biology
Nuclear translocation of viral Jun but not of cellular Jun is cell cycle dependent KAZUHIRO CHIDA* AND PETER K. VOGT Department of Microbiology and Norris Cancer Center, University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033-1054
Contributed by Peter K. Vogt, January 27, 1992
The Jun protein is a transcription factor of ABSTRACT the AP-1 complex, and it is concentrated in the cell nucleus. While the cellular Jun protein is transported into the nucleus in a cell-cycle-independent fashion, the oncogenic viral version of the protein translocates into the nucleus most rapidly during the G2 phase of the cell cycle and only slowly during G, and S phases. This cell cycle dependence of nuclear transport has been mapped to the cysteine to serine mutation in the carboxylterminal portion of viral Jun. We have identified a complex nuclear translocation signal located in the bask region of viral Jun. This signal has the sequence ASKSRKRKL. A peptide of this sequence synthesized in vitro and conjugated to IgG can mediate cell-cycle-dependent translocation ofthe microinjected conjugate from the cytoplasm into the nucleus. The nuclear translocation signal has two functional domains. The pentapeptide RKRKL is sufficient as a cell-cycle-independent nucear address. The entire signal is needed for cell-cycledependent nuclear translocation. The amino-terminal tetrapeptide contains the cysteine to serine substitution responsible for cell cycle dependence. Deletion analysis of the Jun protein suggests that the nuclear translocation signal identified In the badc region is required for nuclear translocation of Jun and may be the only such signal in the Jun molecule. The Jun protein is a member of the AP-1 transcription factor family (1, 2). It dimerizes with itself, with other AP-1 proteins, or with transcriptional regulators of the ATF and steroid receptor families (reviewed in ref. 3). These dimers bind to specific enhancer DNA sequences and regulate transcription positively or negatively (3). Immunofluorescent staining shows Jun concentrated in the cell nucleus (4). We describe here the identification of a nuclear localization signal (NLS) in Jun and show that in the oncogenic viral version of Jun (v-Jun) the function of this nuclear addressing peptide is modulated by an amino acid substitution that makes nuclear translocation of v-Jun cell cycle dependent, while cellular Jun (c-Jun) enters the nucleus at approximately constant rates throughout the cell cycle. We also show that the NLS of Jun, conjugated to an unrelated protein, can effect the translocation of that protein into the nucleus. In the case of the v-Jun NLS, this translocation of the heterologous protein becomes cell cycle dependent. The NLS of Jun described here is located in the highly basic DNA contact domain of the molecule, and it appears to be the only NLS of Jun.
MATERIALS AND METHODS Synchronization. Infected or uninfected control chicken embryo fibroblasts (CEF) were grown in medium F10 containing 5x concentrations of vitamins and folic acid and supplemented with 10% fetal calf serum and 4% chicken
serum (CLM). Cells were arrested in Go by reducing the fetal calf serum and chicken serum concentrations to 1% and 0.4%, respectively, for 3 days. Change to CLM was followed by DNA synthesis in more than 90% of the cells 8 hr later. In a second method of synchronization, GO-arrested cells were exposed to aphidicolin at 10 ,ug/ml in CLM, which resulted in arrest at the G1/S boundary. Cells were released from the aphidicolin block by washing with CLM and entered S, which lasted 6 hr. Cytokinesis started 3-4 hr after S.
Immno o
. CEF grown on glass coverslips were
washed with cold phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. The fixed cells were washed once with PBS and then permeabilized with acetone at -20TC for 3 min. All subsequent steps were carried out at room temperature. After another washing with PBS, the cells were incubated with monoclonal antibody to the Jun peptide PEP2 (1:20 dilution) for 1 hr, washed again in PBS, and incubated with anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (1:200, Sigma) for 1 hr. The cells were then washed three times with PBS and once with distilled water and mounted on glass slides with PBS/10%o glycerol. Immune staining was detected by using a Zeiss microscope equipped for epifluorescence. The intensity of fluorescent staining was graded from 1+ (lowest) to 4+ (highest). In Situ Nuclear Fractionation. For use as whole cells, CEF were grown on tissue culture plates (21-mm-diameter multiwells, Falcon). They were washed with cold PBS and then fixed in situ with 3.7% formaldehyde in PBS. The in situ nuclear fraction was prepared by a modification of the method described by Staufenbiel and Deppert (5). Briefly, cells attached to plates were washed twice with cold PBS and then treated with 0.1% Nonidet P-40 in Kern-matrix buffer, which consists of 10 mM N-morpholinoethanesulfonic acid (pH 6.2), 10 mM NaCl, 1.5 mM MgCl2, 10%O (vol/vol) glycerol, and 0.1 mM phenylmethylsulfonyl fluoride, for 10 min at 40C' The extracted cells were washed once with the buffer and once with PBS and then fixed with 3.7% formaldehyde. The fraction remaining in the wells contained nuclei and cytoskeletal proteins but no cytoplasmic soluble materials. ELISA. Whole cells or in situ nuclear fractions prepared as described above were fixed with 3.7% formaldehyde and washed once with PBS, then permeabilized with methanol for 10 min at room temperature. The cells were washed twice with PBS and incubated with 3% bovine serum albumin (BSA) in PBS for 30 min and then with anti-PEP2 monoclonal antibody (1:20 dilution) in 1% BSA in PBS for 1 hr. After three washes with PBS the cells were incubated for 1 hr with anti-mouse IgG antibody conjugated to alkaline phosphatase (1:3000 dilution, Sigma). The cells were then washed twice Abbreviations: NLS, nuclear localization signal; ASV-17, avian sarcoma virus 17; CEF, chicken embryo fibroblasts. *Present address: Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, MinatoKu, Tokyo 108, Japan.
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.
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with PBS and twice with 10 mM diethylamine buffer (pH 9.5) containing 0.5 mM MgCl2 and incubated in 0.5 ml of p-nitrophenyl phosphate at 1 mg/ml in the diethylamine buffer for 4 hr at room temperature. The reaction was stopped by addition of 0.1 ml of 1 M EDTA. Optical absorbance of the solution was measured at 405 nm. Background values were determined by incubation of the cells with the primary antibody in presence of a large excess (200 ,ug/ml) of its antigen, PEP2. Specific absorbance was defined as the difference between values obtained in absence and presence of the competing peptide. Conjugation of Peptides to IgG. Three peptides representing partial Jun sequences were synthesized: PEP5, positions 35-53 of the chicken c-Jun protein (TLNLSDAASSLKPHLRNKN), PEP1, positions 223-239 (PIDMESQERIKAERKRM), and PEP4, positions 245-259 (ASKSRKRKLERIARL). These peptides were conjugated to rabbit IgG with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) by a slight modification of a method previously described (6). IgG was dissolved at a concentration of 6.7 mg/ml in 50 mM sodium phosphate buffer (pH 7.0). MBS dissolved in dimethylformamide (10 mg/ml) was added to the IgG solution to a final concentration of 0.25 mg/ml. The solution was kept for 30 min at room temperature. The MBS-treated IgG was separated from unreacted MBS by gel filtration through a PD10 column (Pharmacia) and then incubated with synthetic peptide at 10 mg/ml for 5 hr. Uncoupled peptide was removed by dialysis against PBS overnight. Microinjection. Microinjections were performed using an Eppendorf microinjector 5242 and micromanipulator 5170 attached to a Zeiss inverted phase-contrast microscope. Peptide-IgG conjugates at 5 mg/ml in PBS were injected into the cytoplasm of 300-500 cells grown on coverslips. During microinjections, cells were kept in F10 medium supplemented with 5% calf serum (GM) at room temperature. The microinjected cells on coverslips were transferred to fresh GM at 37°C and incubated in a CO2 incubator at 37°C. Cells were washed with cold PBS and then fixed with formaldehyde. Microinjected rabbit IgG was detected by direct immunofluorescence using goat anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate. Immunoblotting. Cells were harvested by scraping with a rubber policeman and lysed at 107 cells per ml in an extraction buffer consisting of 10 mM Tris HCl (pH 7.4), 10 mM NaCI, 3 mM MgCl2 and 0.1% Nonidet P-40 at 0°C. Crude nuclei were prepared by using a glass homogenizer. Cytoplasmic and nuclear fractions were obtained by centrifugation at 10,000 X g for 30 min. Immunoblotting was carried out by the procedure of Towbin et al. (7). Proteins were separated by SDS/PAGE (8) and transferred to a nitrocellulose membrane filter (BA85, Schleicher & Schuell). Nonspecific binding sites were blocked with 3% gelatin in Tris-buffered saline (TBS), which consists of 20 mM Tris-HCI (pH 7.5) and 0.5 M NaCI, for 30 min at room temperature. The membrane was incubated with rabbit antiJun polyclonal antibody in 1% gelatin in TBS for 1 hr. The membrane was washed three times with TTBS (TBS with 0.05% Tween 20) and incubated with goat anti-rabbit IgG antibody coupled to alkaline phosphatase (1:3000, Sigma) for 1 hr. The membrane was then washed twice with TTBS and once with TBS and incubated in color development solution consisting of 100 mM Tris-HCl (pH 9.5), 0.4 mM nitroblue tetrazolium, 0.4 mM bromochroloindolyl phosphate, and S mM MgCl2 for 2 min. The reaction was stopped by washing with distilled water. Mutants of jun. Avian sarcoma virus 17 (ASV-17) is the original retrovirus carrying the jun oncogene. It was used in initial experiments. For comparisons, wild-type and mutant Jun proteins were expressed from the RCAS vector, a nondefective retroviral construct based on the genome of
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Rous sarcoma virus (12). DNA constructs of deletion mutants were made by PCR (9) using the v-jun plasmid pAD5-5-2 (10) as a template and following primers: VO-118 VO-119 VO-123 VO-125 VO-126 VO-127
5'-CCCCAAGCTTACAACCTGGCAATCCTITCCAA-3' 5'-TGGCACCTGTGTCCTCCAT-3' 5'-CGCCAAGCTTACGCCGCAATrCTGITTCTCAT-3' 5'-CGATAAGCTTGGGCTGCA-3' 5'-TGGTCGGCCGCAATTCTGTTTCTCAT-3' 5'-GCCACGGCCGAAGAAAAAGTGAAAAC-3'.
Short DNAs of 380 base pairs (bp) for construct A9 (cf. Fig. 9) and 340 bp for construct B2 were produced by using VO-118 and VO-123, respectively, as plus-strand primers and VO-119 as minus-strand primer. The DNAs were digested with Nco I and HindIII and then substituted for the Nco I-HindIII region coding for the carboxyl terminus of v-Jun in pA05-5-2. For making the internal deletion mutant C3, two short DNAs of 330 and 360 bp were produced by using combinations of primers VO-119 and VO-126 and VO-127 and VO-128. The two DNAs were digested with Eag I, mixed, and ligated. The resulting DNA of 650 bp was digested with Nco I and HindIII and substituted for the Nco I-HindIII region of pA05-5-2. GVJ100 was the chimeric construct of VJO (10) and VJ100 [obtained from L. S. Havarstein and I. M. Morgan (11)] using the Nco I site of the jun coding region. Cloning procedures for the expression vector RCAS and transfection of plasmid DNAs to cells were performed by methods previously described (12, 13).
RESULTS Nuclear Translocation of v-Jun but Not of c-Jun Is Cell Cycle Dependent. Immunofluorescent staining of ASV-17-infected CEF or CEF expressing v-Jun from the RCAS vector with an antiserum directed against Jun revealed a conspicuous variability from cell to cell in the intensity ofnuclear fluorescence (Fig. 1A). This heterogeneity persisted in clonal cultures derived from single ASV-17-infected CEF and could therefore not be caused by differences in viral gene expression between proviral integration sites. Immunofluorescent staining did not reveal cytoplasmic Jun protein, possibly because its broader distribution kept it below detectable levels or because it was inaccessible to the monoclonal antibody used. However, immunoblots of CEF lysates, separated into nuclear and cytoplasmic fractions, clearly showed that Jun protein occurred also in the cytoplasm (Fig. 2). Synchronization of ASV-17 infected cultures by serum starvation or aphidicolin then showed that the intensity of
A
B
FIG. 1. Immunofluorescent staining of Jun protein. (A) CEF infected with the RCAS retroviral vector expressing v-Jun. (B) Same vector expressing c-Jun in CEF.
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Cell Biology: Chida and Vogt 2
1
3
Nuclear Accumulation ImmunoflugL ELISA Heterogeneity Cell cycle -ASKIRKRKLI- inacolony dependence
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CJ3-114 |6|
FIG. 2. Distribution of Jun in nuclear and cytoplasmic fractions of CEF infected with ASV-17. Cells (lane 1) were fractionated into nuclear (lane 2) and cytoplasmic (lane 3) fractions and Jun was visualized by immunoblotting (7). Molecular masses (kDa) of marker proteins are shown on the left.
Jun-specific nuclear fluorescence was cell cycle dependent: it was lowest in G1 (90% of nuclei 1-2+, 0o 4+) and highest in G2 (>80o of nuclei 4+). The same observation was made on CEF infected with the v-Jun-expressing retrovirus vector RCAS VJ-0 (10). In contrast to v-Jun, c-Jun did not show this strong cell-cycle-dependent variation. CEF infected with the chicken c-Jun-expressing retrovirus vector RCAS CJ-3 showed c-Jun-specific nuclear immunofluorescent staining of an intensity that was approximately the same in all nuclei in nonsynchronized cultures (Fig. 1B). Amounts of endogenous c-Jun as determined by immunoblotting were too small to contribute to the immunofluorescent staining under these conditions (data not shown). Quantitative measurements of the cell-cycle-dependent nuclear-cytoplasmic distribution of Jun were obtained with the ELISA technique using CEF fractionated in situ (5). Fig. 3A shows the results for CEF infected with RCAS VJ-0 and synchronized by serum starvation. At various times after addition of serum, nuclei were prepared and their v-Jun contents were compared to those of whole cells. While v-Jun in whole cells increased during Go to G1, nuclear translocation took place mainly during S to G2. CEF overexpressing chicken c-Jun from the retrovirus vector RCAS CJ-3 did not show this delay in nuclear translocation of Jun (Fig. 3B). In c-Jun-overexpressing cells the amounts of c-Jun in nuclei paralleled those of c-Jun in whole cells. Synchronization with aphidicolin gave the same results for v-Jun and c-Jun as did serum starvation. Fig. 3 also shows that the time course of total c-Jun synthesis after serum stimulation is the same for VJ-0- and CJ-3-infected CEF, as would be expected because both constructs are driven by the same long terminal repeat 1.0
B
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FIG. 4. Mapping of the cell cycle dependence of nuclear accumulation. The mutational differences that distinguish viral and cellular Jun were tested singly and in combination for their effect on nuclear translocation. (A) Viral Jun + Gag with either S or C at position 248. (B) Cellular Jun with either S or F at position 222 and C or S at 248. Cell cycle dependence is correlated with the C to S mutation at 248.
of the RCAS vector. We conclude that nuclear translocation of v-Jun but not of c-Jun is cell cycle dependent. The v-Jun protein encoded by VJ-0 differs from the c-Jun protein of CJ-3 as follows: (i) Its amino terminus is fused to 220 amino acids coded for by the viral gag gene. (ii) It has suffered a deletion of 27 amino acids in the amino-terminal portion of the molecule ("8 deletion"). (iii) It carries two amino acid substitutions (serine to phenylalanine at position 222 and cysteine to serine at position 248 of v-Jun) (14). To determine which of these differences was responsible for the cell-cycle-dependent nuclear translocation of Jun, we constructed a series of RCAS-Jun expression vectors that carried these changes singly or in combination. CEF were transfected with these constructs, and cell cycle dependency of nuclear translocation was determined by immunofluorescence of clonal cell populations and by ELISA measurements of nuclei and whole cells after serum synchronization (Fig. 4). Cell cycle regulation of nuclear translocation was not correlated with the presence of gag sequences (compare VJ-0 and GVC-100) or with the 8 deletion (compare VJ-1 and VC-100) or the serine to phenylalanine substitution at position 222 (compare CJ3-100 and CV). These observations rule out a role of the size difference between Gag-v-Jun and c-Jun and of two post-translational regulatory regions, the domain and the phosphorylation site at position 222. The cell cycle
Z 0.8 0 =
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FIG. 3. Nuclear translocation of Jun during the cell cycle. Jun concentrations were determined by ELISA tests in CEF synchronized by serum starvation and fractionated in situ. (A) v-Jun. (B) c-Jun. o, Whole cells; *, nuclear fraction.
FIG. 5. Localization of rabbit IgG conjugated to Jun-derived peptides. (A) PEP4. (B) PEP1. (C) PEP5.
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NLS
Peptide ASKS RKRKL IERIARL dependence de c rde eYes PEP4 17////4/ f A ----
--__
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Incubation time (min) FIG. 6. Nuclear translocation of the Pep4-IgG conjugate in CEF synchronized by serum starvation. o, Go; *, G1; o, S; m, G2.
dependence of nuclear translocation was, however, correlated with the presence the cysteine to serine substitution at position 248, located within the highly basic DNA-binding region of the molecule. Therefore we analyzed this domain of Jun for a cell-cycle-dependent nuclear transport signal. A Jun Peptide (PEP4) Coupled to Rabbit IgG Functions as Cell-Cycle-Dependent NLS. By comparing the amino acid sequence of Jun with that of well-known NLSs we selected two peptides as likely candidates for the Jun NLS: a 15residue peptide encompassing positions 245-259 of the chicken c-Jun (PEP4) and a 17-residue peptide from positions 223-239 of c-Jun (PEPi). As control we included a 19-residue peptide, positions 34-53, from within the 6 domain (PEP5). These peptides were synthesized and conjugated by covalent linkage to rabbit IgG. Solutions ofthese conjugates were then microinjected at 1-3 pg in 0.5 pl into the cytoplasm of CEF. One hour after injection the cells were immunofluorescently stained with an IgG-specific serum. The PEP4-conjugated IgG was effectively translocated into the nucleus. The PEP1 and PEP5 conjugates remained cytoplasmic, as did nonconjugated IgG (Fig. 5). Translocation of the PEP4 conjugate could be abolished by coinjection of an excess of free PEP4. To test for possible cell cycle dependence of the IgG nuclear translocation, the PEP4 conjugate was microinjected into CEF synchronized by serum or aphidicolin, and nuclear translocation was assessed during various stages of the cell cycle by IgG-specific immunofluorescence after incubation at 37TC for 15 min (Fig. 6). The rate of nuclear translocation for PEP4-IgG was negligible during Go, slow during G1 and S, and rapid during G2. A conjugate of IgG and the NLS of the simian virus 40 T (tumor) antigen (PKKKRKV) also was translocated into the nucleus but not in a cell-cycledependent manner (data not shown). We conclude that PEP4 Peptide
ASKSUEtRIARL
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D2-6 D3-6 D4-6
////
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A V/////
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FIG. 7. Mapping of the NLS in PEP4. The PEP4 peptides with terminal deletions shown in the figure were conjugated to rabbit IgG and microinjected into serum-synchronized CEF, and the conjugates were localized by immunofluorescence. Each peptide also contained the sequence CGG at the amino terminus and GG at the carboxyl terminus, except for PEP4 and PEP4-Glu C, which contained a single cysteine residue at the amino terminus. The GG sequence at the termini served as a spacer, and the sulfhydryl group of the cysteine was needed for coupling with the crosslinker, maleimidobenzoic acid N-hydroxysuccinimide ester.
No
Yes
FIG. 8. Mapping of the cell cycle dependence of nuclear translocation. The deletion peptides shown in the figure were conjugated to rabbit IgG and microinjected into serum-synchronized CEF, and the conjugates were localized 15 min after injection to reveal cell cycle dependence. Peptide D4 also contained the sequence CGG at the amino terminus. Peptide DO-6 contained the sequence GG at the carboxyl terminus and a single cysteine residue at the amino terminus. See also legend to Fig. 7.
of Jun contains a cell-cycle-regulated NLS that can function when conjugated to an unrelated protein. Cell Cycle Dependence of Nuclear Translocation and Nuclear Addressing Are Determined by Separate Domains of the Jun NLS. To characterize the v-Jun NLS further, we introduced terminal deletions into PEP4 to generate a series of peptides that were conjugated to rabbit IgG and were tested for their ability to direct the conjugate to the nucleus in a cell-cycle-dependent manner. These peptide-IgG conjugates were microinjected at 1-3 pg per cell into the cytoplasm of synchronized CEF, and their nuclear or cytoplasmic localization was determined by IgG-specific fluorescence after incubation at 37rC for 15 min. Under these conditions 70% or more of the nuclei at G2 were IgG positive, but only 20%o or less of the nuclei during Go, G1, or S. The results of these experiments are presented in Figs. 7 and 8 and can be summarized as follows: The minimal NLS contained in PEP4 is the pentapeptide RKRKL. This peptide alone conjugated to IgG could effect nuclear translocation, but the process was not cell cycle dependent. Cell cycle dependence could be induced with a PEP4-derived peptide that encompassed the minimal Jun NLS and the four amino terminally adjacent amino acids ASKS. These data suggest that the nuclear addressing signal of Jun and the determinant for cell cycle dependence of nuclear translocation rest in adjacent but separate domains of the protein. The fourresidue region conferring cell cycle dependence to the nuclear translocation contains the point mutation substituting serine for cysteine at position 248 of v-Jun. This mutation had also been identified in the comparison of Jun constructs carrying various mutations as the one associated with cell cycle dependence of nuclear translocation (compare Fig. 2). The NLS Contained in PEP4 Is Required for Nuclear Translocation of Jun. Amino- and carboxyl-terminal deletion derivatives of v-Jun were generated, inserted into the RCAS expression vector, and transfected into CEF. The transfected cultures were then inspected by Jun-specific immunofluorescence for the cellular localization of the v-Jun protein by SRKKLERIAR
v-jun
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I
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-jun
FIG. 9. Effect of deletion of the Jun NLS. The Jun protein remains cytoplasmic if the sequence SKSRKRKLERIARL containing the NLS is deleted.
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using an antibody directed against Jun PEP2 (15). The results of these tests (Fig. 9) show that the NLS of PEP4 is essential for nuclear localization of Jun and may be the only NLS of the Jun protein. Deletions outside this region did not affect nuclear translocation, while the deletion of the PEP4 region resulted in cytoplasmic accumulation of Jun.
DISCUSSION Nuclear entry ofproteins results from a specific translocation process (reviewed in refs. 16-18). The protein to be transported contains a short stretch of amino acids acting as specific NLS. This signal sequence is recognized by NLSbinding proteins (NBPs) that appear to function as adaptor molecules of the nuclear transport machinery. In a first, ATP-independent, step of nuclear translocation, they effect the movement of the protein to the cytoplasmic side of the nuclear pore complex (NPC). Transit through the NPC and entry into the nuclei are energy dependent. The NLS identified in Jun is similar to the prototypical one of the simian virus 40 large T (tumor) antigen in its high content of basic residues. It mediates nuclear transport that is ATP dependent and can be inhibited by the thiophosphate analogue ATP[y-S] but not by ADPIZI-S], AMP[a-S], or GTP[y-S] (unpublished data). Furthermore, the nuclear transport is inhibited by wheat germ agglutinin, as is the nuclear localization of other proteins, probably reflecting the modification of nucleoporin proteins by 0-linked N-acetylglucosamine. Unlike nuclear translocation of many proteins including c-Jun, that of v-Jun is cell cycle regulated. Amino acids adjacent to the amino terminus of the plain NLS are able to modulate its function. We speculate that this modulation either affects the initial interaction between the Jun NLS with NBP or acts at a later stage, determining passage through the NPC. The mutation in v-Jun that induces cell cycle dependence generates a possible phosphorylation site within a consensus sequence for protein kinase C. This site may be a target of cell-cycle-specific phosphorylation, but other modifications of the v-Jun NLS cannot be ruled out. Cell-cycle-dependent nuclear translocation has recently been reported for the yeast transcription factor SWI 5 (19, 20). It is controlled by the CDC28 kinase. For the simian virus 40 T antigen, the rate of nuclear translocation depends on phosphorylation of two serines near the NLS by casein kinase II (21). Another mechanism by which nuclear entry can be controlled is the masking of the NLS by a cytoplasmic protein. The NLS of the glucocorticoid receptor appears to be silenced in the absence of the hormone by an interaction of the receptor and the heat shock protein hsp90 (22). Factor NF-KB is kept in the cytoplasm by binding to IKB (23). It is striking that by immunofluorescent staining in the absence of detergents Jun cannot be convincingly detected in the cytoplasm, possibly because the epitope reacting with the monoclonal antibody is masked by the association with other proteins that could affect nuclear transport. The cysteine to serine substitution in v-Jun at position 248 is responsible for making the entry of Jun into the nucleus cell cycle dependent. The same substitution has also been shown to affect a redox control mechanism that regulates DNA binding of Jun (24). c-Jun binds to DNA effectively only if the cysteine at position 248 is reduced. The cysteine to serine mutation at this position relieves DNA
Proc. Natl. Acad. Sci. USA 89 (1992)
binding from redox control. Recent studies in our laboratory have also shown that this same mutation alone is sufficient for converting the nontumorigenic c-Jun protein into one that induces tumors in the animal, although the mutation has only a slight effect on transformation in CEF cell culture (ref. 15; I. M. Morgan, L. S. Havarstein, and P.K.V., unpublished data). The altered regulation in nuclear entry of v-Jun may therefore be related to its tumor-inducing capacity. lain Morgan and Sigve Havarstein kindly provided mutant jun constructs, David Gillespie the polyclonal antibody to Jun, and Felipe Monteclaro the monoclonal antibody to PEP2. We thank Mary Anne Galang and Sunee Himathongkham for excellent technical support and Sarah Olivo, Esther Olivo, and Arianne Helenkamp for their help in producing the manuscript. This work was supported by U.S. Public Health Service Research Grant CA 42564 and Grant 1951 from the Council for Tobacco Research. 1. Bohmann, D., Bos, T. J., Admon, A., Nishimura, T., Vogt, P. K. & Tjian, R. (1987) Science 238, 1386-1392. 2. Angel, P., Hattori, K., Smeal, T. & Karin, M. (1988) Cell 55, 875-885. 3. Curran, T. & Vogt, P. K. (1991) in Transcriptional Regulation, eds. McKnight, S. L. & Yamamoto, K. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), in press. 4. Ball, A. R., Jr., Bos, T. J., Loliger, C., Nagata, L. D., Nishimura, T., Su, H., Tsuchie, H. & Vogt, P. K. (1989) Cold Spring Harbor Symp. Quant. Biol. 53, 687-693. 5. Staufenbiel, M. & Deppert, W. (1984) J. Cell Biol. 98, 18861894. 6. Lanford, R. E., Kanda, P. & Kennedy, R. C. (1986) Cell 46, 575-582. 7. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 8. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 9. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239, 487-491. 10. Bos, T. J., Rauscher, F. J., III, Curran, T. & Vogt, P. K. (1989) Oncogene 4, 123-126. 11. Havarstein, L. S., Morgan, I. M., Wong, W.-Y. & Vogt, P. K. (1992) Proc. Natl. Acad. Sci. USA 89, 618-622. 12. Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J. & Sutrave, P. (1977) J. Virol. 61, 3004-3012. 13. Kawai, S. & Nishizawa, M. (1984) Mol. Cell. Biol. 4, 11721174. 14. Nishimura, T. & Vogt, P. K. (1988) Oncogene 3, 659-663. 15. Bos, T. J., Monteclaro, F. S., Mitsunobu, F., Ball, A. R., Jr., Chang, C. H. W., Nishimura, T. & Vogt, P. K. (1990) Genes Dev. 4, 1677-1687. 16. Garcia-Bustos, J., Heitman, J. & Hall, M. N. (1991) Biochim. Biophys. Acta 1071, 83-101. 17. Silver, P. A. (1991) Cell 64, 489-497. 18. Nigg, E. A., Baeuerle, P. A. & Luhrmann, R. (1991) Cell 66, 15-22. 19. Nasmyth, K., Adolf, G., Lydall, D. & Seddon, A. (1990) Cell 62, 631-647. 20. Moll, T., Tebb, G., Surana, U., Robitsch, H. & Nasmyth, K.
(1991) Cell 66, 743-758.
21. Rihs, H.-P., Jans, D. A., Fan, H. & Peters, R. (1991) EMBO J. 10, 633-639. 22. Sanchez, E. R., Toft, D. O., Schlesinger, M. J. & Pratt, W. B.
(1985) J. Biol. Chem. 260, 12398-12401.
23. Ghosh, S. & Baltimore, D. (1990) Nature (London) 344, 678682. 24. Abate, C., Patel, L., Rauscher, F. J., III, & Curran, T. (1990) Science 249, 1157-1161.
Nuclear translocation of viral Jun but not of cellular Jun is cell cycle dependent KAZUHIRO CHIDA* AND PETER K. VOGT Department of Microbiology and Norris Cancer Center, University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033-1054
Contributed by Peter K. Vogt, January 27, 1992
The Jun protein is a transcription factor of ABSTRACT the AP-1 complex, and it is concentrated in the cell nucleus. While the cellular Jun protein is transported into the nucleus in a cell-cycle-independent fashion, the oncogenic viral version of the protein translocates into the nucleus most rapidly during the G2 phase of the cell cycle and only slowly during G, and S phases. This cell cycle dependence of nuclear transport has been mapped to the cysteine to serine mutation in the carboxylterminal portion of viral Jun. We have identified a complex nuclear translocation signal located in the bask region of viral Jun. This signal has the sequence ASKSRKRKL. A peptide of this sequence synthesized in vitro and conjugated to IgG can mediate cell-cycle-dependent translocation ofthe microinjected conjugate from the cytoplasm into the nucleus. The nuclear translocation signal has two functional domains. The pentapeptide RKRKL is sufficient as a cell-cycle-independent nucear address. The entire signal is needed for cell-cycledependent nuclear translocation. The amino-terminal tetrapeptide contains the cysteine to serine substitution responsible for cell cycle dependence. Deletion analysis of the Jun protein suggests that the nuclear translocation signal identified In the badc region is required for nuclear translocation of Jun and may be the only such signal in the Jun molecule. The Jun protein is a member of the AP-1 transcription factor family (1, 2). It dimerizes with itself, with other AP-1 proteins, or with transcriptional regulators of the ATF and steroid receptor families (reviewed in ref. 3). These dimers bind to specific enhancer DNA sequences and regulate transcription positively or negatively (3). Immunofluorescent staining shows Jun concentrated in the cell nucleus (4). We describe here the identification of a nuclear localization signal (NLS) in Jun and show that in the oncogenic viral version of Jun (v-Jun) the function of this nuclear addressing peptide is modulated by an amino acid substitution that makes nuclear translocation of v-Jun cell cycle dependent, while cellular Jun (c-Jun) enters the nucleus at approximately constant rates throughout the cell cycle. We also show that the NLS of Jun, conjugated to an unrelated protein, can effect the translocation of that protein into the nucleus. In the case of the v-Jun NLS, this translocation of the heterologous protein becomes cell cycle dependent. The NLS of Jun described here is located in the highly basic DNA contact domain of the molecule, and it appears to be the only NLS of Jun.
MATERIALS AND METHODS Synchronization. Infected or uninfected control chicken embryo fibroblasts (CEF) were grown in medium F10 containing 5x concentrations of vitamins and folic acid and supplemented with 10% fetal calf serum and 4% chicken
serum (CLM). Cells were arrested in Go by reducing the fetal calf serum and chicken serum concentrations to 1% and 0.4%, respectively, for 3 days. Change to CLM was followed by DNA synthesis in more than 90% of the cells 8 hr later. In a second method of synchronization, GO-arrested cells were exposed to aphidicolin at 10 ,ug/ml in CLM, which resulted in arrest at the G1/S boundary. Cells were released from the aphidicolin block by washing with CLM and entered S, which lasted 6 hr. Cytokinesis started 3-4 hr after S.
Immno o
. CEF grown on glass coverslips were
washed with cold phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 30 min at room temperature. The fixed cells were washed once with PBS and then permeabilized with acetone at -20TC for 3 min. All subsequent steps were carried out at room temperature. After another washing with PBS, the cells were incubated with monoclonal antibody to the Jun peptide PEP2 (1:20 dilution) for 1 hr, washed again in PBS, and incubated with anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (1:200, Sigma) for 1 hr. The cells were then washed three times with PBS and once with distilled water and mounted on glass slides with PBS/10%o glycerol. Immune staining was detected by using a Zeiss microscope equipped for epifluorescence. The intensity of fluorescent staining was graded from 1+ (lowest) to 4+ (highest). In Situ Nuclear Fractionation. For use as whole cells, CEF were grown on tissue culture plates (21-mm-diameter multiwells, Falcon). They were washed with cold PBS and then fixed in situ with 3.7% formaldehyde in PBS. The in situ nuclear fraction was prepared by a modification of the method described by Staufenbiel and Deppert (5). Briefly, cells attached to plates were washed twice with cold PBS and then treated with 0.1% Nonidet P-40 in Kern-matrix buffer, which consists of 10 mM N-morpholinoethanesulfonic acid (pH 6.2), 10 mM NaCl, 1.5 mM MgCl2, 10%O (vol/vol) glycerol, and 0.1 mM phenylmethylsulfonyl fluoride, for 10 min at 40C' The extracted cells were washed once with the buffer and once with PBS and then fixed with 3.7% formaldehyde. The fraction remaining in the wells contained nuclei and cytoskeletal proteins but no cytoplasmic soluble materials. ELISA. Whole cells or in situ nuclear fractions prepared as described above were fixed with 3.7% formaldehyde and washed once with PBS, then permeabilized with methanol for 10 min at room temperature. The cells were washed twice with PBS and incubated with 3% bovine serum albumin (BSA) in PBS for 30 min and then with anti-PEP2 monoclonal antibody (1:20 dilution) in 1% BSA in PBS for 1 hr. After three washes with PBS the cells were incubated for 1 hr with anti-mouse IgG antibody conjugated to alkaline phosphatase (1:3000 dilution, Sigma). The cells were then washed twice Abbreviations: NLS, nuclear localization signal; ASV-17, avian sarcoma virus 17; CEF, chicken embryo fibroblasts. *Present address: Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, MinatoKu, Tokyo 108, Japan.
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.
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with PBS and twice with 10 mM diethylamine buffer (pH 9.5) containing 0.5 mM MgCl2 and incubated in 0.5 ml of p-nitrophenyl phosphate at 1 mg/ml in the diethylamine buffer for 4 hr at room temperature. The reaction was stopped by addition of 0.1 ml of 1 M EDTA. Optical absorbance of the solution was measured at 405 nm. Background values were determined by incubation of the cells with the primary antibody in presence of a large excess (200 ,ug/ml) of its antigen, PEP2. Specific absorbance was defined as the difference between values obtained in absence and presence of the competing peptide. Conjugation of Peptides to IgG. Three peptides representing partial Jun sequences were synthesized: PEP5, positions 35-53 of the chicken c-Jun protein (TLNLSDAASSLKPHLRNKN), PEP1, positions 223-239 (PIDMESQERIKAERKRM), and PEP4, positions 245-259 (ASKSRKRKLERIARL). These peptides were conjugated to rabbit IgG with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) by a slight modification of a method previously described (6). IgG was dissolved at a concentration of 6.7 mg/ml in 50 mM sodium phosphate buffer (pH 7.0). MBS dissolved in dimethylformamide (10 mg/ml) was added to the IgG solution to a final concentration of 0.25 mg/ml. The solution was kept for 30 min at room temperature. The MBS-treated IgG was separated from unreacted MBS by gel filtration through a PD10 column (Pharmacia) and then incubated with synthetic peptide at 10 mg/ml for 5 hr. Uncoupled peptide was removed by dialysis against PBS overnight. Microinjection. Microinjections were performed using an Eppendorf microinjector 5242 and micromanipulator 5170 attached to a Zeiss inverted phase-contrast microscope. Peptide-IgG conjugates at 5 mg/ml in PBS were injected into the cytoplasm of 300-500 cells grown on coverslips. During microinjections, cells were kept in F10 medium supplemented with 5% calf serum (GM) at room temperature. The microinjected cells on coverslips were transferred to fresh GM at 37°C and incubated in a CO2 incubator at 37°C. Cells were washed with cold PBS and then fixed with formaldehyde. Microinjected rabbit IgG was detected by direct immunofluorescence using goat anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate. Immunoblotting. Cells were harvested by scraping with a rubber policeman and lysed at 107 cells per ml in an extraction buffer consisting of 10 mM Tris HCl (pH 7.4), 10 mM NaCI, 3 mM MgCl2 and 0.1% Nonidet P-40 at 0°C. Crude nuclei were prepared by using a glass homogenizer. Cytoplasmic and nuclear fractions were obtained by centrifugation at 10,000 X g for 30 min. Immunoblotting was carried out by the procedure of Towbin et al. (7). Proteins were separated by SDS/PAGE (8) and transferred to a nitrocellulose membrane filter (BA85, Schleicher & Schuell). Nonspecific binding sites were blocked with 3% gelatin in Tris-buffered saline (TBS), which consists of 20 mM Tris-HCI (pH 7.5) and 0.5 M NaCI, for 30 min at room temperature. The membrane was incubated with rabbit antiJun polyclonal antibody in 1% gelatin in TBS for 1 hr. The membrane was washed three times with TTBS (TBS with 0.05% Tween 20) and incubated with goat anti-rabbit IgG antibody coupled to alkaline phosphatase (1:3000, Sigma) for 1 hr. The membrane was then washed twice with TTBS and once with TBS and incubated in color development solution consisting of 100 mM Tris-HCl (pH 9.5), 0.4 mM nitroblue tetrazolium, 0.4 mM bromochroloindolyl phosphate, and S mM MgCl2 for 2 min. The reaction was stopped by washing with distilled water. Mutants of jun. Avian sarcoma virus 17 (ASV-17) is the original retrovirus carrying the jun oncogene. It was used in initial experiments. For comparisons, wild-type and mutant Jun proteins were expressed from the RCAS vector, a nondefective retroviral construct based on the genome of
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Rous sarcoma virus (12). DNA constructs of deletion mutants were made by PCR (9) using the v-jun plasmid pAD5-5-2 (10) as a template and following primers: VO-118 VO-119 VO-123 VO-125 VO-126 VO-127
5'-CCCCAAGCTTACAACCTGGCAATCCTITCCAA-3' 5'-TGGCACCTGTGTCCTCCAT-3' 5'-CGCCAAGCTTACGCCGCAATrCTGITTCTCAT-3' 5'-CGATAAGCTTGGGCTGCA-3' 5'-TGGTCGGCCGCAATTCTGTTTCTCAT-3' 5'-GCCACGGCCGAAGAAAAAGTGAAAAC-3'.
Short DNAs of 380 base pairs (bp) for construct A9 (cf. Fig. 9) and 340 bp for construct B2 were produced by using VO-118 and VO-123, respectively, as plus-strand primers and VO-119 as minus-strand primer. The DNAs were digested with Nco I and HindIII and then substituted for the Nco I-HindIII region coding for the carboxyl terminus of v-Jun in pA05-5-2. For making the internal deletion mutant C3, two short DNAs of 330 and 360 bp were produced by using combinations of primers VO-119 and VO-126 and VO-127 and VO-128. The two DNAs were digested with Eag I, mixed, and ligated. The resulting DNA of 650 bp was digested with Nco I and HindIII and substituted for the Nco I-HindIII region of pA05-5-2. GVJ100 was the chimeric construct of VJO (10) and VJ100 [obtained from L. S. Havarstein and I. M. Morgan (11)] using the Nco I site of the jun coding region. Cloning procedures for the expression vector RCAS and transfection of plasmid DNAs to cells were performed by methods previously described (12, 13).
RESULTS Nuclear Translocation of v-Jun but Not of c-Jun Is Cell Cycle Dependent. Immunofluorescent staining of ASV-17-infected CEF or CEF expressing v-Jun from the RCAS vector with an antiserum directed against Jun revealed a conspicuous variability from cell to cell in the intensity ofnuclear fluorescence (Fig. 1A). This heterogeneity persisted in clonal cultures derived from single ASV-17-infected CEF and could therefore not be caused by differences in viral gene expression between proviral integration sites. Immunofluorescent staining did not reveal cytoplasmic Jun protein, possibly because its broader distribution kept it below detectable levels or because it was inaccessible to the monoclonal antibody used. However, immunoblots of CEF lysates, separated into nuclear and cytoplasmic fractions, clearly showed that Jun protein occurred also in the cytoplasm (Fig. 2). Synchronization of ASV-17 infected cultures by serum starvation or aphidicolin then showed that the intensity of
A
B
FIG. 1. Immunofluorescent staining of Jun protein. (A) CEF infected with the RCAS retroviral vector expressing v-Jun. (B) Same vector expressing c-Jun in CEF.
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Nuclear Accumulation ImmunoflugL ELISA Heterogeneity Cell cycle -ASKIRKRKLI- inacolony dependence
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FIG. 2. Distribution of Jun in nuclear and cytoplasmic fractions of CEF infected with ASV-17. Cells (lane 1) were fractionated into nuclear (lane 2) and cytoplasmic (lane 3) fractions and Jun was visualized by immunoblotting (7). Molecular masses (kDa) of marker proteins are shown on the left.
Jun-specific nuclear fluorescence was cell cycle dependent: it was lowest in G1 (90% of nuclei 1-2+, 0o 4+) and highest in G2 (>80o of nuclei 4+). The same observation was made on CEF infected with the v-Jun-expressing retrovirus vector RCAS VJ-0 (10). In contrast to v-Jun, c-Jun did not show this strong cell-cycle-dependent variation. CEF infected with the chicken c-Jun-expressing retrovirus vector RCAS CJ-3 showed c-Jun-specific nuclear immunofluorescent staining of an intensity that was approximately the same in all nuclei in nonsynchronized cultures (Fig. 1B). Amounts of endogenous c-Jun as determined by immunoblotting were too small to contribute to the immunofluorescent staining under these conditions (data not shown). Quantitative measurements of the cell-cycle-dependent nuclear-cytoplasmic distribution of Jun were obtained with the ELISA technique using CEF fractionated in situ (5). Fig. 3A shows the results for CEF infected with RCAS VJ-0 and synchronized by serum starvation. At various times after addition of serum, nuclei were prepared and their v-Jun contents were compared to those of whole cells. While v-Jun in whole cells increased during Go to G1, nuclear translocation took place mainly during S to G2. CEF overexpressing chicken c-Jun from the retrovirus vector RCAS CJ-3 did not show this delay in nuclear translocation of Jun (Fig. 3B). In c-Jun-overexpressing cells the amounts of c-Jun in nuclei paralleled those of c-Jun in whole cells. Synchronization with aphidicolin gave the same results for v-Jun and c-Jun as did serum starvation. Fig. 3 also shows that the time course of total c-Jun synthesis after serum stimulation is the same for VJ-0- and CJ-3-infected CEF, as would be expected because both constructs are driven by the same long terminal repeat 1.0
B
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FIG. 4. Mapping of the cell cycle dependence of nuclear accumulation. The mutational differences that distinguish viral and cellular Jun were tested singly and in combination for their effect on nuclear translocation. (A) Viral Jun + Gag with either S or C at position 248. (B) Cellular Jun with either S or F at position 222 and C or S at 248. Cell cycle dependence is correlated with the C to S mutation at 248.
of the RCAS vector. We conclude that nuclear translocation of v-Jun but not of c-Jun is cell cycle dependent. The v-Jun protein encoded by VJ-0 differs from the c-Jun protein of CJ-3 as follows: (i) Its amino terminus is fused to 220 amino acids coded for by the viral gag gene. (ii) It has suffered a deletion of 27 amino acids in the amino-terminal portion of the molecule ("8 deletion"). (iii) It carries two amino acid substitutions (serine to phenylalanine at position 222 and cysteine to serine at position 248 of v-Jun) (14). To determine which of these differences was responsible for the cell-cycle-dependent nuclear translocation of Jun, we constructed a series of RCAS-Jun expression vectors that carried these changes singly or in combination. CEF were transfected with these constructs, and cell cycle dependency of nuclear translocation was determined by immunofluorescence of clonal cell populations and by ELISA measurements of nuclei and whole cells after serum synchronization (Fig. 4). Cell cycle regulation of nuclear translocation was not correlated with the presence of gag sequences (compare VJ-0 and GVC-100) or with the 8 deletion (compare VJ-1 and VC-100) or the serine to phenylalanine substitution at position 222 (compare CJ3-100 and CV). These observations rule out a role of the size difference between Gag-v-Jun and c-Jun and of two post-translational regulatory regions, the domain and the phosphorylation site at position 222. The cell cycle
Z 0.8 0 =
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FIG. 3. Nuclear translocation of Jun during the cell cycle. Jun concentrations were determined by ELISA tests in CEF synchronized by serum starvation and fractionated in situ. (A) v-Jun. (B) c-Jun. o, Whole cells; *, nuclear fraction.
FIG. 5. Localization of rabbit IgG conjugated to Jun-derived peptides. (A) PEP4. (B) PEP1. (C) PEP5.
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NLS
Peptide ASKS RKRKL IERIARL dependence de c rde eYes PEP4 17////4/ f A ----
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Incubation time (min) FIG. 6. Nuclear translocation of the Pep4-IgG conjugate in CEF synchronized by serum starvation. o, Go; *, G1; o, S; m, G2.
dependence of nuclear translocation was, however, correlated with the presence the cysteine to serine substitution at position 248, located within the highly basic DNA-binding region of the molecule. Therefore we analyzed this domain of Jun for a cell-cycle-dependent nuclear transport signal. A Jun Peptide (PEP4) Coupled to Rabbit IgG Functions as Cell-Cycle-Dependent NLS. By comparing the amino acid sequence of Jun with that of well-known NLSs we selected two peptides as likely candidates for the Jun NLS: a 15residue peptide encompassing positions 245-259 of the chicken c-Jun (PEP4) and a 17-residue peptide from positions 223-239 of c-Jun (PEPi). As control we included a 19-residue peptide, positions 34-53, from within the 6 domain (PEP5). These peptides were synthesized and conjugated by covalent linkage to rabbit IgG. Solutions ofthese conjugates were then microinjected at 1-3 pg in 0.5 pl into the cytoplasm of CEF. One hour after injection the cells were immunofluorescently stained with an IgG-specific serum. The PEP4-conjugated IgG was effectively translocated into the nucleus. The PEP1 and PEP5 conjugates remained cytoplasmic, as did nonconjugated IgG (Fig. 5). Translocation of the PEP4 conjugate could be abolished by coinjection of an excess of free PEP4. To test for possible cell cycle dependence of the IgG nuclear translocation, the PEP4 conjugate was microinjected into CEF synchronized by serum or aphidicolin, and nuclear translocation was assessed during various stages of the cell cycle by IgG-specific immunofluorescence after incubation at 37TC for 15 min (Fig. 6). The rate of nuclear translocation for PEP4-IgG was negligible during Go, slow during G1 and S, and rapid during G2. A conjugate of IgG and the NLS of the simian virus 40 T (tumor) antigen (PKKKRKV) also was translocated into the nucleus but not in a cell-cycledependent manner (data not shown). We conclude that PEP4 Peptide
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////
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FIG. 7. Mapping of the NLS in PEP4. The PEP4 peptides with terminal deletions shown in the figure were conjugated to rabbit IgG and microinjected into serum-synchronized CEF, and the conjugates were localized by immunofluorescence. Each peptide also contained the sequence CGG at the amino terminus and GG at the carboxyl terminus, except for PEP4 and PEP4-Glu C, which contained a single cysteine residue at the amino terminus. The GG sequence at the termini served as a spacer, and the sulfhydryl group of the cysteine was needed for coupling with the crosslinker, maleimidobenzoic acid N-hydroxysuccinimide ester.
No
Yes
FIG. 8. Mapping of the cell cycle dependence of nuclear translocation. The deletion peptides shown in the figure were conjugated to rabbit IgG and microinjected into serum-synchronized CEF, and the conjugates were localized 15 min after injection to reveal cell cycle dependence. Peptide D4 also contained the sequence CGG at the amino terminus. Peptide DO-6 contained the sequence GG at the carboxyl terminus and a single cysteine residue at the amino terminus. See also legend to Fig. 7.
of Jun contains a cell-cycle-regulated NLS that can function when conjugated to an unrelated protein. Cell Cycle Dependence of Nuclear Translocation and Nuclear Addressing Are Determined by Separate Domains of the Jun NLS. To characterize the v-Jun NLS further, we introduced terminal deletions into PEP4 to generate a series of peptides that were conjugated to rabbit IgG and were tested for their ability to direct the conjugate to the nucleus in a cell-cycle-dependent manner. These peptide-IgG conjugates were microinjected at 1-3 pg per cell into the cytoplasm of synchronized CEF, and their nuclear or cytoplasmic localization was determined by IgG-specific fluorescence after incubation at 37rC for 15 min. Under these conditions 70% or more of the nuclei at G2 were IgG positive, but only 20%o or less of the nuclei during Go, G1, or S. The results of these experiments are presented in Figs. 7 and 8 and can be summarized as follows: The minimal NLS contained in PEP4 is the pentapeptide RKRKL. This peptide alone conjugated to IgG could effect nuclear translocation, but the process was not cell cycle dependent. Cell cycle dependence could be induced with a PEP4-derived peptide that encompassed the minimal Jun NLS and the four amino terminally adjacent amino acids ASKS. These data suggest that the nuclear addressing signal of Jun and the determinant for cell cycle dependence of nuclear translocation rest in adjacent but separate domains of the protein. The fourresidue region conferring cell cycle dependence to the nuclear translocation contains the point mutation substituting serine for cysteine at position 248 of v-Jun. This mutation had also been identified in the comparison of Jun constructs carrying various mutations as the one associated with cell cycle dependence of nuclear translocation (compare Fig. 2). The NLS Contained in PEP4 Is Required for Nuclear Translocation of Jun. Amino- and carboxyl-terminal deletion derivatives of v-Jun were generated, inserted into the RCAS expression vector, and transfected into CEF. The transfected cultures were then inspected by Jun-specific immunofluorescence for the cellular localization of the v-Jun protein by SRKKLERIAR
v-jun
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I
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-jun
FIG. 9. Effect of deletion of the Jun NLS. The Jun protein remains cytoplasmic if the sequence SKSRKRKLERIARL containing the NLS is deleted.
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using an antibody directed against Jun PEP2 (15). The results of these tests (Fig. 9) show that the NLS of PEP4 is essential for nuclear localization of Jun and may be the only NLS of the Jun protein. Deletions outside this region did not affect nuclear translocation, while the deletion of the PEP4 region resulted in cytoplasmic accumulation of Jun.
DISCUSSION Nuclear entry ofproteins results from a specific translocation process (reviewed in refs. 16-18). The protein to be transported contains a short stretch of amino acids acting as specific NLS. This signal sequence is recognized by NLSbinding proteins (NBPs) that appear to function as adaptor molecules of the nuclear transport machinery. In a first, ATP-independent, step of nuclear translocation, they effect the movement of the protein to the cytoplasmic side of the nuclear pore complex (NPC). Transit through the NPC and entry into the nuclei are energy dependent. The NLS identified in Jun is similar to the prototypical one of the simian virus 40 large T (tumor) antigen in its high content of basic residues. It mediates nuclear transport that is ATP dependent and can be inhibited by the thiophosphate analogue ATP[y-S] but not by ADPIZI-S], AMP[a-S], or GTP[y-S] (unpublished data). Furthermore, the nuclear transport is inhibited by wheat germ agglutinin, as is the nuclear localization of other proteins, probably reflecting the modification of nucleoporin proteins by 0-linked N-acetylglucosamine. Unlike nuclear translocation of many proteins including c-Jun, that of v-Jun is cell cycle regulated. Amino acids adjacent to the amino terminus of the plain NLS are able to modulate its function. We speculate that this modulation either affects the initial interaction between the Jun NLS with NBP or acts at a later stage, determining passage through the NPC. The mutation in v-Jun that induces cell cycle dependence generates a possible phosphorylation site within a consensus sequence for protein kinase C. This site may be a target of cell-cycle-specific phosphorylation, but other modifications of the v-Jun NLS cannot be ruled out. Cell-cycle-dependent nuclear translocation has recently been reported for the yeast transcription factor SWI 5 (19, 20). It is controlled by the CDC28 kinase. For the simian virus 40 T antigen, the rate of nuclear translocation depends on phosphorylation of two serines near the NLS by casein kinase II (21). Another mechanism by which nuclear entry can be controlled is the masking of the NLS by a cytoplasmic protein. The NLS of the glucocorticoid receptor appears to be silenced in the absence of the hormone by an interaction of the receptor and the heat shock protein hsp90 (22). Factor NF-KB is kept in the cytoplasm by binding to IKB (23). It is striking that by immunofluorescent staining in the absence of detergents Jun cannot be convincingly detected in the cytoplasm, possibly because the epitope reacting with the monoclonal antibody is masked by the association with other proteins that could affect nuclear transport. The cysteine to serine substitution in v-Jun at position 248 is responsible for making the entry of Jun into the nucleus cell cycle dependent. The same substitution has also been shown to affect a redox control mechanism that regulates DNA binding of Jun (24). c-Jun binds to DNA effectively only if the cysteine at position 248 is reduced. The cysteine to serine mutation at this position relieves DNA
Proc. Natl. Acad. Sci. USA 89 (1992)
binding from redox control. Recent studies in our laboratory have also shown that this same mutation alone is sufficient for converting the nontumorigenic c-Jun protein into one that induces tumors in the animal, although the mutation has only a slight effect on transformation in CEF cell culture (ref. 15; I. M. Morgan, L. S. Havarstein, and P.K.V., unpublished data). The altered regulation in nuclear entry of v-Jun may therefore be related to its tumor-inducing capacity. lain Morgan and Sigve Havarstein kindly provided mutant jun constructs, David Gillespie the polyclonal antibody to Jun, and Felipe Monteclaro the monoclonal antibody to PEP2. We thank Mary Anne Galang and Sunee Himathongkham for excellent technical support and Sarah Olivo, Esther Olivo, and Arianne Helenkamp for their help in producing the manuscript. This work was supported by U.S. Public Health Service Research Grant CA 42564 and Grant 1951 from the Council for Tobacco Research. 1. Bohmann, D., Bos, T. J., Admon, A., Nishimura, T., Vogt, P. K. & Tjian, R. (1987) Science 238, 1386-1392. 2. Angel, P., Hattori, K., Smeal, T. & Karin, M. (1988) Cell 55, 875-885. 3. Curran, T. & Vogt, P. K. (1991) in Transcriptional Regulation, eds. McKnight, S. L. & Yamamoto, K. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), in press. 4. Ball, A. R., Jr., Bos, T. J., Loliger, C., Nagata, L. D., Nishimura, T., Su, H., Tsuchie, H. & Vogt, P. K. (1989) Cold Spring Harbor Symp. Quant. Biol. 53, 687-693. 5. Staufenbiel, M. & Deppert, W. (1984) J. Cell Biol. 98, 18861894. 6. Lanford, R. E., Kanda, P. & Kennedy, R. C. (1986) Cell 46, 575-582. 7. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 8. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 9. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239, 487-491. 10. Bos, T. J., Rauscher, F. J., III, Curran, T. & Vogt, P. K. (1989) Oncogene 4, 123-126. 11. Havarstein, L. S., Morgan, I. M., Wong, W.-Y. & Vogt, P. K. (1992) Proc. Natl. Acad. Sci. USA 89, 618-622. 12. Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J. & Sutrave, P. (1977) J. Virol. 61, 3004-3012. 13. Kawai, S. & Nishizawa, M. (1984) Mol. Cell. Biol. 4, 11721174. 14. Nishimura, T. & Vogt, P. K. (1988) Oncogene 3, 659-663. 15. Bos, T. J., Monteclaro, F. S., Mitsunobu, F., Ball, A. R., Jr., Chang, C. H. W., Nishimura, T. & Vogt, P. K. (1990) Genes Dev. 4, 1677-1687. 16. Garcia-Bustos, J., Heitman, J. & Hall, M. N. (1991) Biochim. Biophys. Acta 1071, 83-101. 17. Silver, P. A. (1991) Cell 64, 489-497. 18. Nigg, E. A., Baeuerle, P. A. & Luhrmann, R. (1991) Cell 66, 15-22. 19. Nasmyth, K., Adolf, G., Lydall, D. & Seddon, A. (1990) Cell 62, 631-647. 20. Moll, T., Tebb, G., Surana, U., Robitsch, H. & Nasmyth, K.
(1991) Cell 66, 743-758.
21. Rihs, H.-P., Jans, D. A., Fan, H. & Peters, R. (1991) EMBO J. 10, 633-639. 22. Sanchez, E. R., Toft, D. O., Schlesinger, M. J. & Pratt, W. B.
(1985) J. Biol. Chem. 260, 12398-12401.
23. Ghosh, S. & Baltimore, D. (1990) Nature (London) 344, 678682. 24. Abate, C., Patel, L., Rauscher, F. J., III, & Curran, T. (1990) Science 249, 1157-1161.
