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3029 Journal of Cell Science 107, 3029-3036 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 Nuclear import of serum response factor (SRF) requires a short aminoterminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site Jocelyne Rech, Isabelle Barlat, Jean Luc Veyrune, Annick Vie and Jean Marie Blanchard Institut de Génétique Moléculaire de Montpellier, UMR 9942, CNRS BP 5051, 1919 route de Mende, 34033 Montpellier cedex 1, France SUMMARY Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133-235 and 168-235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein. INTRODUCTION vivo SRE occupancy (Herrera et al., 1989) can be demonstrated whatever the state of the cell. SRF is extensively phosphorylated following its synthesis in serum-stimulated fibroblasts (Prywes et al., 1988; Gauthier et al., 1991a; Misra et al., 1991). The same sites that are phosphorylated in vivo (Janknecht et al., 1992; Marais et al., 1992) are phosphorylated in vitro by casein kinase II (CKII; Manak et al., 1990; Marais et al., 1992). This phosphorylation event was first thought to enhance SRF binding to DNA (Prywes et al., 1988; Manak et al., 1990), thus activating its function (Gauthier et al., 1991a). It has been subsequently demonstrated that CKII phosphorylation profoundly affected both on and off rates for SRF binding to its target without affecting the overall equilibrium dissociation constant (Marais et al., 1992). More recently, Manak and Prywes (1993) have even presented evidence against a role in growth factor regulation of c-fos expression via phosphorylation of SRF by CKII. To understand the role of CKII phosphorylation of SRF we expressed a series of SRF-β-galactosidase fusion proteins by transient transfection in rodent fibroblasts. This has allowed us to characterize a nuclear localization signal next to the CKII phosphorylation site, a situation encountered in several other nuclear proteins. However, this CK II site does not seem to control nuclear import, at variance with what has been proposed for SV40 T. Serum response factor (SRF) is a ubiquitous transcription factor that binds to the DNA sequence CC(A/T)6GG, which has been identified as an essential regulatory serum response element (SRE) of the c-fos proto-oncogene promoter (for reviews see Rivera and Greenberg, 1990; Treisman, 1990, 1992; Piechaczyk and Blanchard, 1994). Numerous experiments suggest that SRF is directly involved in signal transduction through transcriptional activation: (i) overexpression of SRF transactivates a cotransfected SRE in certain cell types (Gutman et al., 1991); (ii) activity of a mutated, uninducible SRE can be rescued by an SRF mutant with altered specificity (Hill et al., 1993); (iii) a GAL4-SRF fusion containing the carboxy-terminal part of SRF is sufficient to render a GAL4 site-containing reporter gene growth factor responsive (Johansen and Prywes, 1993); (iv) microinjection of titrating amounts of double-stranded SRE oligonucleotides or immunodepletion of SRF from cell nuclei prevents the chromosomal c-fos gene from being activated by serum or the ras oncogene (Lamb et al., 1990; Gauthier et al., 1991a,b). Regulatory SRFDNA-binding activity does not seem to be a necessary step in SRE-mediated serum or growth factor gene regulation, since both in vitro DNA-binding activity (Treisman, 1990) and in Key words: SRF, nuclear localization signal, nuclear import kinetics, casein kinase 3030 J. Rech and others MATERIALS AND METHODS Vectors and cloning strategies The β-galactosidase-containing pCH110 (Hall et al., 1983) and the human SRF cDNA-containing T7∆ATG (generous gift from R.Treisman) plasmids were used as starting materials for all constructs described in this paper. Various portions of SRF were fused in-frame with the bacterial enzyme by insertion between the HindIII and KpnI sites of pCH110. SRF cDNA was fragmented using either existing restriction sites or selective amplification by PCR with the following oligonucleotides: (1) 5′-GGAAGCTTAGAATGGCGCCCACCGCGGGG; (2) 5′-GGGAAGCTTAGAATGAGCCTGAGCGAGATG; (3) 5′-GGGAAGCTTAGAATGGGCGCCGAGCGGCGC; (4) 5′-GGAAGCTTCTAGAATGGTGGTCGGTGGGCCC; (5) 5′-CCCGGTACCGGCTTCAGTGTGTCCTTGG; (6) 5′-CCCGGTACCTCGGGCCCACCGACCAC; (7) 5′-CCCGGTACCTCCTCCTCCTCGCC; (8) 5′-GCGAAGCTTAGAATGGACACACTGAAGCCG; (9) 5′-CGCGGTACCCATTCACTCTTGGTGCT. The pSV-NLSlacZ vector was obtained by insertion within the same sites of the double stranded oligonucleotide: linked anti-mouse antibody and signals were visualized using ECL protocol from Amersham. Experiements were carried out at least three times for each construct. SRF and SV40-β-galactosidase fusion proteins: purification and microinjection Recombinant proteins were purified after in vivo biotinylation (Cronan, 1990; Germino et al., 1993). pCH110-derived vectors expressing the SRF domain corresponding to amino acids 69 to 115 linked to β-galactosidase, under the wild-type or mutated casein kinase site configuration, were restricted with HindIII and BamHI. The inserts were cloned into the same sites of the Pin Point Xa3 vector (Promega), which gives rise to the expression in E. coli of a fusion protein linked to a peptide specifically biotinylated in vivo, thus allowing a rapid purification through binding to an avidin-containing resin (Softlink, Promega). Protein purity was monitored by SDS-polyacrylamide gel electrophoresis and Coomasie Blue staining, and estimated to be more than 95%. Rat embryo fibroblasts (REF-52) cells were injected in the cytoplasm with 50-100 µg/ml solutions of pure SRF-β-galactosidase fusion proteins in 50 mM HEPES pH 8, 1 mM MgCl2. Cells were fixed at different times after injection and β-galactosidase activity was monitored as described above. 5′-AAGCTTTCTAGAATGGCTCCAAAAAAGAGAAAGGTACC. The region of SV40 DNA between map coordinates 4414 and 4521, containing the CKII site and the NLS, was amplified by PCR using the following oligonucleotides: 5′-GGGAAGCTTGAGGAAAACCTGTTTTGC; 5′-CGCGGTACCGGGTCTTCTACCTTTCTC. Site-directed mutagenesis was carried out on double-stranded DNA according to Deng and Nickoloff (1992) with the Pharmacia USE kit. Briefly, two oligonucleotides were used: one introduces the desired mutation(s) and the second mutates a unique non-essential restriction site (ScaI) into another one (MluI). Elimination of this site renders the mutated plasmid DNA resistant to restriction, thus providing a selection based on the differential transforming efficiency of linear vs circular DNA in a mutS Escherichia coli strain. Two successive rounds of transformation-recutting were carried out prior to plating the cells. The casein kinase II site (CKII), the putative nuclear localization signal (NLS) and the ScaI restriction site of pUC19 were, respectively, mutated using the following oligonucleotides: 5′-GGGAAGCTTAGAATGGCCGTCGGCGTCGAGGGCGACGTGGAGGTGGGCGAGGAGGAGGAG (CKII); 5′-CTCAGGCTCCCCATCAGGCCGCCCAGCTCGGCGCC (NLS); 5′-CTGTGACTGGTGACGCGTCAACCAAGTC (ScaI, Pharmacia). All mutants and fusion constructs were confirmed by sequencing. Cell culture, transfection and β-galactosidase detection HeLa and 293 human cells, or rat embryo (REF52), mouse (Ltk−) and hamster (CCL39) fibroblasts, were cultured in DMEM (Gibco) containing 5% fetal calf serum under a 5% CO2-containing atmosphere. A total of 105 cells in 3 cm2 plastic dishes were transiently transfected with 4 µg DNA by the standard calcium phosphate procedure. At 20 hours later, cells were washed thrice with ice-cold PBS, fixed for 5 minutes with 2% formaldehyde/0.2% glutaraldehyde in PBS and stained with 1 mg/ml X-Gal, 2 mM MgCl2, in the presence of 5 mM each of potassium ferrocyanide and ferricyanide for at least one hour at 37°C. The sizes of the fusion proteins expressed in 293 cells were estimated by western blot analysis. Nitrocellulose membranes were saturated for 1 hour in 10 mM Tris-HCl, pH 8 (25°C), 150 mM NaCl, 0.2% Tween-20 (TBST), 6% non-fat milk, and then incubated for 2 hours in TBST containing mouse anti-β-galactosidase antibody. After 4 washes in TBST, filters were probed with horseradish peroxidase- RESULTS Expression of SRF-β-galactosidase fusion proteins in transfected cells We have shown previously that the portion of SRF containing its DNA-binding and dimerization domains, SRF-DB (spanning from amino acid 133 to 264, see Fig. 1), worked as a negative dominant effector of c-fos expression when microinjected into serum-stimulated fibroblasts (Gauthier-Rouvière et al., 1993). To discriminate between endogenous SRF and recombinant protein we resorted to gene tagging using β-galactosidase as an easily detectable marker. We used as controls for cytoplasmic and nuclear localization, respectively, vectors expressing either β-galactosidase alone (pCH110, Fig. 2A) or a chimera containing the SV40-T NLS, CPKKRKV, linked at the amino terminus of the same enzyme (pSV-NLSLacZ, Fig. 2B). To our surprise, the SRF-DB-β-galactosidase fusion protein did not localize to the nucleus (Fig. 2C) despite the presence of several stretches of basic amino acids reminiscent of known nuclear localization sequences (NLS; for reviews see Garcia-Bustos et al., 1991; Silver, 1991; Laskey and Dingwall, 1993). This result clearly did not depend upon the level at which the recombinant proteins were expressed: the same localizations were obtained whether a SV40 or a rat β-actin promoter was used, or whether X-gal staining was carried out 6 hours or 24 hours post-washing of calcium phosphate precipitates (results not shown). Moreover, the same results were also invariant, whether human HeLa, Chinese hamster CCL39 or mouse Ltk− cells were used for transfection (not shown). This prompted us to design a set of deletion mutants containing various portions of human SRF fused in-frame to β-galactosidase. Vector construction is described in Materials and Methods, and β-galactosidase activity was monitored as above after transient transfection into REF cells. Characterization of recombinant proteins was carried out in parallel by western blotting on total cellular extracts after transient transfection in human 293 cells (not shown). Nuclear import of SRF 3031 Fig. 1. Summary of the various mutants analyzed in this work. (A) The global structure of human serum response factor (SRF) is schematized at top of the figure. The sequence of the casein kinase II phosphorylation site (CK II, rectangle with dots) and of the nuclear localization signal (NLS, filled rectangle) are shown below. DNA stands for ‘DNA-binding domain’, which is shaded in the various deletion mutants shown below. Numbers refer to the last amino acid removed by the deletion. β-Galactosidase, which is fused in-frame at the carboxy terminus of each fragment of SRF, is not represented. (B) Point mutations generated in the domain spanning from amino acid 69 to 115, fused at the amino terminus of β-galactosidase. A portion of the sequence of the wild-type protein is shown line a+b. - in the sequence refers to an invariant amino acid; and a and b refer to small peptides containing, respectively, CK II and NLS; + and − stand, respectively, for a nuclear or cytoplasmic localization of the fusion protein. While neither carboxy-terminal (amino acids 262 to 508, Fig. 3F) nor DNA-binding (amino acids 133 to 264, Fig. 2C) domain-containing fusion proteins were found specifically in the nucleus, the amino-terminal half (amino acids 1 to 264, Fig. 3A) was very efficient in targeting β-galactosidase to the nucleus. A series of progressive amino-terminal deletions was carried out: amino acids 1 to 68 (Fig. 3B), 1 to 91 (Fig. 3C), 1 to 100 (Fig. 3D), and 1 to 115 (not shown), which suggested that amino acids spanning from 91 to 100 were essential in this phenomenom. This was confirmed by the internal deletion of residues 69 to 115, which gave rise to a cytoplasmic locale (Fig. 3E). We then generated a series of point mutations starting with the construct expressing residues 69 to 115. When fused to βgalactosidase this domain is able to direct the protein to the nucleus (Fig. 4A). It contains the SRF casein kinase II (CK-II) phosphorylation site, SGSEGDSESGEEEE, and a stretch of basic amino acids, RRGLKR, which is a good candidate for a NLS. For sake of simplicity these two motifs were named, respectively, a and b and the various combinations of mutations summarized in Fig. 1, where am and bm stand for the mutated counterpart of each motif. Mutation of the a motif did not affect the nuclear localization of the fusion protein (Fig. 4B) while mutation of b alone (Fig. 4C) or in conjunction with a (Fig. 4D) changed it dramatically. When fused alone a was insufficient to drive the protein to the nucleus (Fig. 4E), while b was very potent in doing so (Fig. 4F). Finally, when we expressed an influenza hemagglutinin epitope-tagged entire SRF, mutation of the RRGLKR stretch was sufficient to abolish its nuclear location (not shown), thus strongly suggesting the uniqueness of the NLS. The casein kinase phosphorylation site next to the NLS is not important for the modulation of nuclear entry kinetics It had previously been demonstrated that the rate at which recombinant SV40 T antigen/β-galactosidase fusion proteins are transported to the nucleus is dependent on the presence of a phosphorylation site next to the NLS (Rihs and Peters, 1989; Rihs et al., 1991). An analysis carried out both in vitro and in vivo identified the CK-II site S111/S112 to be the determining factor in the efficiency of the cytonuclear transport. Many proteins harbor a NLS that is close to amino acids known to be phosphorylated either in the cytoplasm or in the nucleus and which are putative CK II phosphorylation sites (reviewed by Rihs et al., 1991). This invited us to speculate that phosphorylation at this site in the SRF is similarly involved in regulating nuclear import. We generated recombinant proteins containing 3032 J. Rech and others Fig. 2. Cytoplasmic localization of an SRF DNA-binding domain/β-galactosidase fusion protein in REF cells. REF 52 cells were transiently transfected and stained with Xgal as described in Materials and Methods. Vectors expressed β-galactosidase alone (A); SV40 NLS linked to the amino terminus of β-galactosidase (B); and the SRF DBD (amino acids 133 to 264) fused to the same enzyme (C). Bar, 10 µm. Fig. 3. Subcellular localization of fusion proteins containing various portions of human SRF linked to βgalactosidase. REF 52 cells were transiently transfected with βgalactosidase fusion protein expressing vectors, which contained: SRF residues 1 to 264 (A); 69 to 264 (B); 91 to 264 (C); 101 to 264 (D); 1 to 264 with the internal deletion of residues 69 to 115 (E); and 262 to 508 (F). While several hundreds of positive cells were obtained for each transfection only a few representative examples are shown. Arrowheads point to some negative cells for comparison. Bar, 10 µm. Nuclear import of SRF 3033 Fig. 4. Point mutagenesis carried out on the SRF domain spanning from amino acid 69 to 115. Mutagenesis was carried out as described in Materials and Methods and the fusion proteins were analyzed as shown in the preceding figures. (A) Wild-type protein, ab; (B) residues 77SGSEGDSES85 mutated into VGVEGDVEV, mutant a b; (C) residues 95RRGLKR100 mutated into LGGLMG, mutant ab ; (D) combined m m mutations, mutant ambm; (E) fusion protein containing amino acids spanning from residue 75 to 90, mutant a; (F) fusion protein containing residues 91 to 111, mutant b. As mentioned in Fig. 2 only representative examples are shown. Arrowheads, see Fig. 3. Bar, 10 µm. SRF sequences spanning from amino acid 69 to 115 fused to β-galactosidase in the Pin Point Xa vector (Promega), which encodes a peptide biotinylated in E. coli, thus functioning as a purification tag (Cronan, 1990; Germino et al., 1993). Biotinylated proteins produced in this system were affinity-purified using a monomeric avidin resin that allows an elution of the fusion protein under non-denaturing conditions, providing us with native proteins that were readily used through microinFig. 5. SDS-polyacrylamide gel electrophoresis of purified recombinant proteins. Purified proteins (2 µg) were labeled in vitro with pure CKII from rabbit reticulocytes (generous gift from C. Gauthier-Rouvière) and [γ-32P]ATP in a standard kinase reaction mix. Proteins were then separated through a 7% SDS-polyacrylamide gel, stained with Coomassie Blue R250 (lanes 1, 2, 3, 4) and dried before autoradiography. Lanes 1, total bacterial extract after induction (only the wild-type fusion protein is shown); 2 and 5, pure wild-type ab protein; 3 and 6, pure mutant amb protein; 4, protein standards (myosin, 200 kDa; β-galactosidase, arrow, 116 kDa; phosphorylase b, 97 kDa; serum albumin, 66 kDa; glutamic dehydrogenase, 55 kDa); 5 and 6, autoradiogram of the in vitro phosphorylated proteins. jection in REF cells. Protein purity was assessed by SDS-gel electrophoresis and the ability of the wild type to function, at least in vitro, as a substrate for CKII determined (Fig. 5). Recombinant proteins were injected in the cytoplasm and βgalactosidase activity was assayed as described above. Injected kDa 3034 J. Rech and others Fig. 6. Transport kinetics of fusion proteins containing SRF amino acids 69 to 115. REF 52 cells were microinjected with either a wild-type: ab (A,B,C,D) or a mutated: amb (E,F,G,H) bacterially expressed fusion protein, fixed at different time intervals and stained with X-gal; 20-30 cells were microinjected for each time point and cells were fixed either immediately (A and E), or 30 (B and F), 60 (C and G) and 120 (D and H) minutes after injection. Only representative examples are shown. Arrowheads point to uninjected cells for comparison. proteins relocalized rapidly into the the nucleus, where more than 90% of the β-galactosidase activity was found within 6090 minutes (Fig. 6). This result did not depend upon the presence of the tag and a fusion protein containing a mutated NLS (abm) remained cytoplasmic (not shown). Within the limits of the technique no gross difference was observed between the wild type (Fig. 6A,B,C,D) and the mutated form (Fig. 6E,F,G,H) of the fusion protein, suggesting that the CK II site is not regulatory for nuclear import kinetics, at least in rodent fibroblasts. When the same experiment was carried out with a β-galactosidase fusion protein containing only the SV40 T NLS, the protein migrated much more slowly to the nucleus (Fig. 7A,B,D). In contrast, the control protein containing both CKII and NLS sequences was readily found in the nucleus two hours after microinjection (Fig. 7E,F). Nuclear import of SRF 3035 Fig. 7. Transport kinetics of fusion proteins containing SV40 T NLS. REF 52 cells were microinjected with fusion proteins containing either SV40 T NLS alone (A,B,C,D) or a CKII phosphorylation site next to the NLS (E,F), and processed as described for Fig. 5. Cells were fixed either immediately (A and E) or 2 (B,F), 4 (C) and 24 hours (D) after injection. DISCUSSION The switch from quiescence to proliferation is characterized by the induction of several waves of genes coding for proteins that will be required for the onset of DNA synthesis. One of these early response genes involved in the G0-G1 transition is the proto-oncogene c-fos. Its promoter contains a DNA regulatory sequence: the serum response element (SRE), which plays a major role in the transcriptional induction of c-fos in response to extracellular stimuli. The SRE is the target for the binding of several proteins among which is a family of 62-67 kDa dimeric proteins (Pollock and Treisman, 1991). Its generic element p67SRF is associated with chromatin throughout the cell cycle (Gauthier-Rouvière et al., 1991a) and is extensively modified both by phosphorylation, immediately after its synthesis (Prywes et al., 1988; Manak et al., 1990; Gauthier et al., 1991a; Misra et al., 1991; Janknecht et al., 1992; Marais et al., 1992), and by glycosylation (Schröter et al., 1990). Phosphorylation of SRF by CK II was first proposed to be necessary for its binding to SRE (Prywes et al., 1988; Manak et al., 1990) and involved in c-fos induction by serum (Gauthier et al., 1991a), although this point is debated (Manak and Prywes, 1993). The role(s) of this phosphorylation is not known but it clearly increases the in vitro rate of exchange of SRF without affecting its affinity very much (Janknecht et al., 1992; Marais et al., 1992). However, there is still the possibility that phosphorylation modulates the interactions of SRF with other proteins like p62TCF/ Elk-1 (Schröter et al., 1990; Hill et al., 1993; Marais et al., 1993). We have mapped close to the CK II site a stretch of basic amino acids, RRGLKR, which is necessary and sufficient to target SRF very efficiently to the nucleus. This NLS sequence is at least as potent as that of SV40 T (PKKKRKV), which is often taken as a reference (Kalderon et al., 1984; Landford et al., 1986). A similar result has been recently obtained by chemically coupling a peptide containing this sequence to rabbit immunoglobulin G (Gauthier-Rouvière, personal communication). The SRF NLS is present in the amino-terminal part of the molecule, outside its DNA-binding domain, which surprisingly contains several stretches of basic amino acids that are totally dispensable as far as nuclear transport is concerned. Comparison of human SRF with SRF from Xenopus as well as with other SRF-related proteins shows that, while the NLS per se is grossly conserved between human and Xenopus, the amino terminus of the SRF family is poorly conserved (Mohun et al., 1991; Pollock and Treisman, 1991; Chambers et al., 1992; Treisman and Ammerer, 1992). This suggests that even though clear homologies are encountered within SRF-related proteins with respect to their DNA-binding properties, SRF harbors some specific properties like, for example, its nuclear import. Interestingly, SV40 T NLS is also close to a CK II site that is phosphorylated both in vitro and in vivo, and which has been shown to be instrumental in the control of the rate of nuclear import (Rihs and Peters, 1989; Rihs et al., 1991). Using recombinant fusion proteins expressed in bacteria and microinjected into growing REF cells, we have shown that the rate of nuclear entry is unchanged whether the CK II site is intact or mutated. This latter result again makes the SRF conspicuous with regard to cyto-nuclear transport and is consistent with its constitutive chromatin location (Gauthier et al., 1991b). More recently, p90rsk kinase, a growth factor- 3036 J. Rech and others inducible kinase, has been proposed to phosphorylate SRF in vitro at serine 103, which is transiently phosphorylated in vivo upon serum stimulation (Rivera et al., 1993). Whether this has anything to do with transport or transcriptional modulation of SRF remains to be investigated. We thank R. 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