extfieldsofstudy: Biology Medicine

text:
Lipopolysaccharide-induced methylation of HuR, an mRNA-stabilizing protein, by CARM1. Coactivator-associated arginine methyltransferase.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein. Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg(217) was identified as the major HuR methylation site. Arg(217) is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor alpha mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most commonly methylated proteins in mammalian cells, but other RNA-binding proteins are also substrates for methylation in vitro and in vivo (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Protein arginine methyltransferases (PRMTs) transfer the methyl group from S-adenosylmethionine (AdoMet) to specific arginines in substrate proteins (26,36). Thus far, cDNA clones encoding six genetically distinct but related mammalian PRMTs have been isolated. PRMT1 is the major arginine methyltransferase in mammals (37) and is essential for early development of mouse embryos (38). Besides methylating hnRNPs, PRMT1 can methylate other nucleic acid-binding proteins, such as poly(A)-binding protein 2, fibrillarin, nucleolin, and histone H4 in vivo (26 -28, 37, 39 -41). Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4), which was originally discovered by its interaction with glucocorticoid receptor-interacting protein 1 in a yeast two-hybrid screen (42), methylates histone H3 (42)(43)(44) and p300/CBP (45) in vivo. Specific protein substrates have also been identified for PRMT3, JBP1 (PRMT5), and PRMT6, but no substrates have been reported yet for PRMT2 (see Refs. 46 and 47 and references therein). Thus far, arginine methylation of proteins has been implicated in the regulation of gene transcription, signal transduction, and nuclear-cytoplasmic protein transport (36,47). Recent studies have revealed changes in cellular levels of protein arginine methylation after a variety of stimuli. For example, the increased methylation of STAT1 parallels its enhanced transcription activation function in response to interferon ␣/␤ (48). CARM1-directed arginine methylation of histone H3 in the promoters of steroid hormone-responsive genes is induced by steroid hormone treatment of cells (43). CARM1 methylation also appears to inhibit cAMP-mediated gene expression, preventing the association between transcription factor CREB and cofactor CBP/p300 by methylation of the p300 domain required for CREB binding (45). Thus, it appears that, like phosphorylation and acetylation, methylation may play an important role in the transduction of extracellular signals to the transcription machinery.
Here, we present evidence that CARM1-directed methylation may also be involved in gene regulation at the post-transcriptional level. We show that HuR associates with and is specifically methylated in vitro by CARM1. Using antibodies that specifically recognize the methylated form of HuR, we demonstrate that HuR is methylated in vivo. The level of HuR methylation is altered by overexpression of CARM1 or by stimulation of macrophages with LPS, which is known to cause HuR-mediated mRNA stabilization (18). Our data suggest that methylation by CARM1 plays a role in mRNA stabilization by HuR.

EXPERIMENTAL PROCEDURES
Cell Culture-COS-7 and RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium, and Jurkat cells were maintained in RPMI 1640, each supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum. LPS (Sigma) was added into the medium at 10 g/ml for 1 h to stimulate RAW 264.7 cells.
Plasmids, Peptides, and Antibody Production-Construction of plasmids pSG5-HA-CARM1 and pGEX-4T1-CARM1 has been previously reported (42). To construct plasmid pET3d-HuR for the expression of full-length recombinant human HuR protein, the coding region was amplified from pGST-HuR (3) by high fidelity PCR using oligonucleotides to engineer an NcoI-compatible BspHI site at the ATG and an NotI site immediately following the last codon of the HuR cDNA. The BspHI-NotI HuR cDNA fragment was inserted into an NcoI-NotI-digested derivative of the pET3d vector (Novagen) encoding a C-terminal hexahistidine and c-myc epitope tag (49), yielding pET3d-HuR. The HuR hinge region mutants were made by first creating a BssHII site (GCG/ CGC at alanine 204/arginine 205) and an ApaI site (GGG/CCC at glycine 209/proline 210) with silent mutations, resulting in plasmid H918. Bold indicates which nucleotide was mutated to generate the restriction site. The HuR hinge deletion mutant was then made by digestion of H918 with BssHII and NcoI (at methionine 223), blunting, and religation, resulting in deletion of amino acids Arg 206 -Pro 222 . The individual arginine-to-lysine hinge region mutants were made by digestion of H918 at preexisting sites PvuII (at glutamine 198/leucine 199) or NcoI (at methionine 223) or the engineered BssHII or ApaI sites (see above), and replacing the intervening sequences with double-stranded oligonucleotides encoding the desired mutation. This approach yielded HuR arginine-to-lysine mutants R205K, R206K, R217K, and R219K in the pET3d derivative. The eukaryotic HuR expression plasmids were created from the pET3d constructs by excising the coding region using an EcoRI site just upstream of the ATG and the NotI site just downstream of the last codon and inserting this HuR fragment into a modified vector derived from pSG5-HA (42), so that an N-terminal hemagglutinin tag and a C-terminal hexahistidine tag were fused on either end. The resulting HA-HuR protein carried the following additional amino acids: N-terminal to HuR methionine 1, MGYPYDVPDYAEF; C-terminal to HuR Lys 326 , AAAHHHHHH-stop. GST-PRMT1 (41), GST-PRMT3 (50), GST-GAR (41), and GST-hnRNP K (51) expression vectors have been previously described. GST-PRMT2 was kindly provided by Dr. Steven Clarke (UCLA). Peptide P1 (HYHSPARRFGGPVH-HQAQRFRFSPMGV-NH 2 ) (AnaSpec Inc.) encoding the HuR hinge do-main was generated for in vitro methylation and radiosequencing analysis. To raise an antibody that could specifically recognize methylated HuR (anti-me(R217)HuR antibody), methylated peptide HHQAQ(DMA)FRFSPGC (where DMA represents asymmetric dimethylarginine) was synthesized and conjugated to keyhole limpet hemocyanin before injection into rabbits. Specificity of the antiserum was confirmed by enzyme-linked immunosorbent assays with the DMAcontaining peptide and a corresponding unmethylated peptide.
Immunoprecipitation and Immunoblot-Cell lysates, prepared as described above (one 100-mm dish of cells/immunoprecipitation sample), were cleared with protein A/G beads (Santa Cruz Biotechnology, Inc.) for 1 h at 4°C. 2 g of anti-met(R217)HuR or anti-mono/dimethyl arginine (Ab412; Abcam Ltd.) were added to the cell lysates and incubated overnight at 4°C on a rotator. Protein A/G beads were added and incubated for another 3 h. Beads were washed three times with radioimmune precipitation buffer and subjected to SDS-PAGE. Blots were probed with mouse monoclonal anti-HuR (Santa Cruz Biotechnology) at 1 g/20 ml of blocking buffer (5% (w/v) nonfat milk in TBST: 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.1% (v/v) Tween 20). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used at 1 g/10 ml of blocking buffer and ECL reagents (Amersham Biosciences) were used for detection.
In Vitro Protein-Protein Interaction-GST fusion proteins were produced in Escherichia coli BL21 as described previously (52). Hexahistidine fusion proteins were purified with His-Bind Buffer Kit (Novagen) according to the manufacturer's protocol. Pull-down assays were conducted as described previously (53). CARM1 protein was translated in vitro in the presence of [ 35 S]methionine using the TNT-T7-coupled reticulocyte lysate system (Promega). The binding assay was conducted by incubating beads containing 2 g of His-HuR with slow rotation overnight at 4°C with 10 l of the translation reaction in a 500-l total volume of NETN-0.01% (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.0, and 0.01% (v/v) Nonidet P-40). Beads were washed four times with NETN-0.01% and analyzed by SDS-PAGE and autofluorography.
Methylation Assay-Methyltransferases were prepared as recombinant GST fusion proteins and eluted from glutathione-agarose beads (Sigma) with 20 mM glutathione. 0.5-3 g of commercially obtained histone H3 (Roche Molecular Biochemicals) or recombinant proteins were incubated with 1 g of methyltransferase in the presence of 6 M S-adenosyl-[methyl- 3 (42). SDS-loading buffer or radioimmune precipitation buffer was used to stop the reaction. Labeled proteins were identified by SDS-PAGE and autofluorography for 12 h. To prepare the in vitro methylated samples for immunoprecipitation, nonradiolabeled AdoMet was used instead at 0.1 mM.
Radiosequencing-Approximately 5 g (1.6 nmol) of [ 3 H]methyl-HuR hinge peptide P1 was purified from a CARM1-catalyzed methylation reaction by reversed phase high performance liquid chromatography on a 2.1 ϫ 30-mm RP-300 (C8) column (Brownlee Laboratories). Elution was carried out at 1.0 ml/min using a linear gradient of 5-40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid, and the peptide was detected by monitoring absorbance at 214 nm. The purified [ 3 H]methyl peptide was found to contain ϳ0.07 mol of [ 3 H]methyl/mol of peptide. Two samples (650 pmol each) of the purified peptide were sequenced for 22 cycles by automated Edman degradation on a Hewlett-Packard G1105A sequenator. The first sample was sequenced by standard procedures to verify the correct amino acid sequence and to establish the repetitive yield. For the second sample, the phenylthiohydantoin-derivatized amino acids from each cycle were collected for measurement of 3 H by liquid scintillation counting.

HuR Is Specifically Methylated by CARM1 in Vitro-Because
it is unclear how extracellular signals mediate HuR-induced stabilization of labile ARE-containing mRNAs, we sought to establish whether post-translational modification of HuR might play a role. No modifications of HuR (including phosphorylation) have been found as yet. Because arginine methylation is a common modification of RNA-binding proteins, we tested whether HuR could be methylated by one of the PRMTs. Various recombinant PRMTs were incubated with recombinant hexahistidine-tagged HuR (His-HuR) in the presence of [ 3 H]AdoMet, and the products were examined by SDS-PAGE and autofluorography. His-HuR was methylated by CARM1 but not by PRMT1, PRMT2, or PRMT3 (Fig. 1A, lanes 4 -7). Staining of the gel demonstrated that His-HuR and PRMT proteins were present in the appropriate reactions (Fig. 1B,  lanes 4 -7), and methylation of known substrates for PRMT1, PRMT3, and CARM1 (hnRNPK, GAR, and histone H3, respectively) verified the specificity and activity of these PRMTs (Fig.  1A, lane 1-3). (No known substrate for PRMT2 is currently available as a control.) Methylation of degradation products of GST-hnRNP K and His-HuR and an aggregation product of histone H3 was also observed (Fig. 1A, lanes 1, 2, and 7). CARM1 and HuR Associate in Vitro-To further test the substrate specificity of CARM1 for HuR, we investigated whether CARM1 could bind HuR directly in vitro. Either CARM1 or PRMT1 was translated in vitro in the presence of [ 35 S]methionine (Fig. 2, lane 1) and incubated with protein-free Ni 2ϩ beads, Ni 2ϩ beads to which His-HuR had been bound, glutathione-agarose beads with bound GST, or glutathioneagarose beads to which GST-hnRNP K had been bound (Fig. 2,  lanes 2-5). Strong interactions between CARM1 and His-HuR (lane 3) and between PRMT1 and hnRNP K (lane 5) were observed, whereas no association between the other combinations was detected. Thus, the substrate specificity observed in the in vitro methylation assay is supported by the selective association between the two PRMTs and their respective substrates.
The HuR Hinge Region Harbors the Methylation Site(s)-Whereas PRMT1 methylation sites are frequently located in Arg-Gly-Gly (RGG) repeats (26), neither histone H3, the first identified in vivo substrate for CARM1 (42), nor HuR contains RGG repeats. Based upon two CARM1 methylation sites in histone H3, a potential consensus motif, KAXRK, has been proposed (44). Whereas HuR does not contain KAXRK sequences, we noted that two arginines in the hinge region were preceded by alanine at the Ϫ2 position (PARRF and QAQRF) ( Fig. 3A). We therefore tested whether an HuR mutant from which a 16-amino acid tract containing the two possible methylation sites had been deleted (Fig. 3A) could still be methylated. In contrast to intact HuR, which was strongly methylated (Fig. 3B, left panel, lane 1), no in vitro methylation of this deletion mutant by CARM1 was seen (Fig. 3B, left panel, lane 2), although similar quantities of mutant and wild type HuR proteins were used as substrates (Fig. 3B, right panel). This result suggests that the hinge region of HuR contains the methylation site(s) or is important for substrate recognition by CARM1.
To determine which of the four arginines located in hinge region (Fig. 3A) might be methylated, we generated four point mutants in the context of full-length HuR, each one containing a single arginine-to-lysine substitution. Mutation of arginine 217 to lysine (R217K) completely abolished in vitro methylation of HuR by CARM1, whereas each of the other three HuR mutants (R205K, R206K, and R219K) was methylated to a level comparable with that of wild type HuR (Fig. 3C). This suggests an essential role of Arg 217 as the methylation site and/or as a residue that is important for recognition of HuR by CARM1.
We next synthesized a HuR hinge peptide, P1, containing all four arginines (Fig. 3A). CARM1 efficiently methylated the P1 peptide in vitro, whereas PRMT1 did not (Fig. 4A). Thus, the determinants that mark HuR as a CARM1 substrate are retained in the 27-amino acid P1 sequence. In order to establish directly the location of the methylation site(s), a sample of the peptide, [ 3 H]methyl-labeled by CARM1, was subjected to radiosequencing by automated Edman degradation. Approximately 25% of the 3 H was recovered in cycles 7-9, whereas the remaining 75% was recovered in cycles 18 -22 (Fig. 4B). The first peak indicates selective methylation of Arg 206 . 3

CARM1-mediated Methylation of HuR
ration, respectively. Thus, Arg 217 is the major methylation site in the HuR hinge peptide when modified by CARM1 in vitro.
HuR Is Methylated by CARM1 in Vivo-To test whether HuR is methylated in vivo, we developed antibodies directed against HuR methylated at Arg 217 (anti-me(R217)HuR). We also used an antibody that generally recognizes proteins containing mono-or dimethylarginine (anti-M/DMA), which has been used successfully to demonstrate the in vivo methylation of STAT1 (48) and hepatitis C virus NS3 protein (55). Both antibodies were characterized for discrimination between HuR methylated in vitro by CARM1 (using unlabeled AdoMet) and unmethylated HuR (Fig. 5). (The in vitro methylation was confirmed in a parallel reaction with [ 3 H]AdoMet (data not shown).) Following immunoprecipitation by antime(R217)HuR or anti-M/DMA antibodies, the precipitated HuR protein was detected by immunoblot using antiserum raised against unmodified HuR protein. Both the antime(R217)HuR and anti-M/DMA antibodies specifically immunoprecipitated methylated HuR (Fig. 5). Although no HuR was detected in immunoprecipitates obtained with appropriate isotype control antibodies (data not shown), a small amount of HuR was precipitated from the unmethylated reaction with anti-me(R217)HuR antiserum (Fig. 5B, lane 1). This weak signal might be due to low levels of antibodies directed against parts of the peptide flanking the methylation site. Nevertheless, both anti-me(R217)HuR and anti-M/DMA antibodies were highly specific for methylated versus unmethylated HuR. Neither antibody could detect the in vitro methylated HuR on immunoblots (data not shown).
In order to determine whether HuR can be methylated by CARM1 in vivo, we co-transfected COS-7 cells with expression vectors encoding HA-tagged CARM1 and either HA-HuR (Fig. 6A, lane 2) or HA-HuR(R217K) (lane 4). COS-7 cells transfected with HA-HuR alone (lane 1) or HA-HuR(R217K) alone (lane 3) were used as controls. Cell lysates were then subjected to immunoprecipitation with either anti-M/DMA antibody or anti-me(R217)HuR antibody, and immunoprecipitated HA-HuR was detected by immunoblotting using an anti-HA antibody. Whereas the expression level of HA-HuR was similar in all four samples (Fig. 6A), expression of HA-CARM1 substantially increased the amount of HA-HuR immunoprecipitated by both methyl-directed antibodies (Fig.  6, B and C, compare lanes 1 and 2) but had no effect on the amount of HA-HuR(R217K) immunoprecipitated (compare lanes 3 and 4). These results demonstrate that HuR can be methylated in vivo by CARM1 and support the notion that Arg 217 is the major methylation site in vivo as well as in vitro.
HuR Methylation Is Enhanced in RAW 264.7 Cells Treated with LPS-Having demonstrated that HuR methylation occurs in vivo, we next asked whether this post-translational modification could play any role in the regulation of gene expression by HuR. HuR can bind to AREs present in a large variety of mRNAs, including those encoding mediators of inflammation (13,17,18,23). This binding of HuR has been linked to stabilization of mRNAs encoding interleukin-3, nitric-oxide synthase II, and TNF-␣, which are induced by ionophores, 12-Otetradecanoylphorbol 13-acetate, cytokines, or LPS, depending on the cell type (13,17,18,23). Recently, interferon (IFN) signaling, another component of the inflammatory response, was shown to be mediated at least in part through increased methylation of STAT1, an IFN-activated transcription factor (48). A role for arginine methylation in IFN signaling was supported by the observation that the IFN-␣ receptor interacts with PRMT1 and that antisense PRMT1 oligonucleotides mitigated the IFN response (56). Whereas CARM1 appears to play a role in steroid hormone and cAMP-mediated signaling, there has as yet been no evidence for its involvement in the inflammatory response. However, its ability to methylate HuR, combined with the observed role of HuR in the mRNA stabilization following inflammatory stimuli in a variety of cells, suggested that CARM1 methylation might play a role in the inflamma-

CARM1-mediated Methylation of HuR
tory response as well. We therefore tested whether the in vivo methylation status of HuR might change in LPS-stimulated RAW 264.7 cells (a murine macrophage-like cell line). RAW 264.7 cells respond to LPS by strongly inducing TNF-␣ expression, mediated in part through TNF-␣ mRNA stabilization by HuR binding (18).
We first determined by immunoblot that RAW 264.7 cells contain CARM1 protein (data not shown). Then cell lysates from either untreated or LPS-treated RAW 264.7 cells were analyzed by immunoprecipitation with anti-me(R217)HuR antibody (Fig. 7A) followed by immunoblotting with anti-HuR. The total level of HuR protein in the cells was not increased by LPS treatment (Fig. 7A, input samples). Whereas HuR was barely detectable when lysates from unstimulated RAW 264.7 cells were used for immunoprecipitation, a strong HuR band became visible upon LPS stimulation (Fig. 7A, lanes 3-5). These observations suggest that methylation of HuR occurs in response to LPS stimulation. A similar observation was made when Jurkat cells, a human T cell leukemia cell line that responds to a wide variety of agents, including LPS (57), was examined for induction of HuR methylation (Fig. 7B). Since the anti-me(R217)HuR antibodies specifically recognize methylated HuR and CARM1 is the only one of the four PRMTs tested that can utilize HuR as a substrate, our results strongly suggest that CARM1 methylates HuR in response to LPS and indicate that CARM1 might play a role in LPS-mediated mRNA stabilization. DISCUSSION We have identified a new target for arginine methylation by CARM1: the RNA-binding protein HuR. HuR is methylated by   FIG. 4. Mapping of the major HuR methylation site to Arg 217 . A, 5 g of synthetic peptide P1 (Fig. 3A) or recombinant GST-hnRNP K were tested for in vitro methylation by either GST-CARM1 or GST-PRMT1 and analyzed by autofluorography (left panel) and Coomassie staining (right panel). Minor contaminants in P1 or degradation products of GST-hnRNP K were methylated as well. B, radiosequencing of in vitro methylated P1 was carried out as described under "Experimental Procedures." C, a refinement of data from B in which corrections were made for the ragged N terminus, for sequencing lag, and for a measured repetitive yield of 95%. Numbers above the bars refer to amino acid residue numbers in HuR.

CARM1-mediated Methylation of HuR
CARM1 in vitro but not by PRMT1, -2, or -3 (Fig. 1). Analysis of arginine-to-lysine mutants of HuR indicated that Arg 217 is the major methylation target site (Fig. 3). Radiosequencing of a HuR hinge peptide methylated in vitro supported this conclusion but also identified Arg 206 as a possible minor methylation site (Fig. 4). However, since Arg 206 was not methylated in full-length HuR carrying the R217K mutation (Fig. 3), methylation at Arg 206 is either dependent on the presence of Arg 217 (in the methylated or unmethylated form) or does not happen in the context of the full-length protein, perhaps because it is not accessible. Major and minor methylation sites have been observed in other CARM1 substrates (44,45,58), but their significance is presently unclear.
Whereas PRMT1 methylates many RNA-binding proteins at arginine residues in so-called RGG repeats (26,47), the sequence specificity of CARM1 is not clear at present. However, a sequence or structural context that affects specificity must exist, because Arg 205 and Arg 219 in the same peptide are not methylated (Fig. 4), and CARM1 methylated only a few proteins in a large membrane-bound array of polypeptides (58). Recently, CBP/p300 and poly(A)-binding protein 1 (PABP1) were also identified as CARM1 substrates (45,58). Comparison of all known major CARM1 target sites with those of HuR does not yield any obvious consensus, apart from the alanine at position Ϫ2, which is present in four of six previously identified major CARM1 methylation sites (Table I) and the fact that two major methylation sites are found in each protein, separated by 23 amino acids or fewer.
Since antibodies against a HuR peptide methylated at Arg 217 (Fig. 5) immunoprecipitated increased amounts of HuR, but not HuR(R217K) from COS-7 cells overexpressing CARM1 (Fig. 6), we conclude that Arg 217 is the major CARM1 methylation site in HuR, both in vitro and in vivo. To determine whether HuR methylation levels might be modulated in response to certain signals, we studied HuR methylation in RAW 264.7 cells stimulated with LPS. HuR has been shown to mediate stabilization of TNF-␣ mRNA in macrophages following LPS stimulation. The mechanism by which this occurs appears to be competition of HuR with the destabilizing protein tristetraproline for binding to the TNF-␣ mRNA ARE (18,23,59). Using our antimethyl-HuR antibody, we showed that HuR methylation was increased in RAW 264.7 cells following LPS treatment (Fig. 7). This observation suggests that methylation by CARM1 might play a role in post-transcriptional gene regulation following inflammatory stimuli.
What could the effect of HuR methylation be, and how could this lead to mRNA stabilization? Immunostaining experiments had previously suggested that HuR was mainly nuclear, but heterokaryon analyses and cell fractionation experiments have demonstrated that the protein shuttles between the nucleus and cytoplasm (8 -11). Only cytoplasmic HuR would be able to stabilize mRNA. Thus, one possible function of HuR methylation might be to raise its cytoplasmic concentration by increasing HuR export from the nucleus. Changes in intracellular localization caused by methylation have been observed for hnRNP A2 in mammalian cells and the yeast protein Np13p, which are substrates of mammalian PRMT1 and its yeast homologue RMT1, respectively. Intriguingly, methylation can have opposite effects on the intracellular location of these proteins, promoting a nuclear localization for hnRNPA2 (60) but stimulating a cytoplasmic location for Np13p (61). Methylation does not affect the charge of arginine residues but could cause steric changes and prevent hydrogen bond formation. Thus, it is assumed that changes in intracellular location caused by methylation result from altered protein/protein interactions.
Shuttling of HuR is mediated by sequences in the hinge region between RRMs 2 and 3 (amino acids 205-237 (Fig. 3), referred to as the HuR nucleocytoplasmic shuttling sequence, or HNS (8)). Interestingly, Arg 206 and Arg 217 are located within  (44); PABP1 (58), p300 (45), and HuR (3) from man. The methylated arginine is indicated in bold, the alanine at Ϫ2 is highlighted in gray, and residues flanking the arginines, which are identical within one protein, are underlined.  1 and 4) or were treated with LPS at 10 g/ml in cell culture medium for 1 h (lanes 2, 3, and 5). Cell extracts were incubated overnight at 4°C with 1 g of either normal mouse IgG or anti-me(R217)HuR antibodies as indicated, resolved by SDS-PAGE, and probed with anti-HuR. 5% of each cell extract used for immunoprecipitation was loaded for comparison (input lanes; a shorter exposure confirms that signal in both input lanes is very similar). B, Jurkat cells were untreated or treated with LPS at 10 g/ml in cell culture medium for 1 h. Cell extracts were incubated overnight at 4°C with 1 g of anti-me(R217)HuR antibodies, resolved by SDS-PAGE, and probed with anti-HuR. 5% of each cell extract used for immunoprecipitation was loaded for comparison (input lanes).
the HNS. Shuttling of HuR is thought to be influenced by proteins that interact with the HNS (such as pp32 and APRIL), thereby linking HuR to one or more nuclear export pathways (16,62,63). Methylation of the hinge domain could modify the interaction between HuR and the export machinery. In this respect, it is of great interest that the HuR export pathway appears to change following exposure of cells to stress, which increases export of HuR by the CRM1 pathway (62)(63)(64). The molecular mechanism for this switch has not yet been identified and may be related to methylation.
Whereas it is attractive to propose that methylation might increase nuclear export, an increase in cytoplasmic RNA-bound HuR could also be explained by alternative mechanisms. For example, methylation could prevent reimport of cytoplasmic HuR into the nucleus, lead to stabilization of cytoplasmic HuR protein, or alter HuR RNA-binding properties to favor binding to AU-rich sequences, thereby indirectly contributing to cytoplasmic accumulation of HuR. Whether methylation and shuttling are directly linked may be difficult to determine; distinguishing methylation effects from shuttling effects will be challenging, because the Arg 217 methylation site is located in the shuttling sequence.
Whether methylation of proteins can affect their nucleic acid-binding properties remains unclear. Methylation of hnRNP A1 appears to have a moderate effect on its ability to bind to homopolymers (65). The RGG domains methylated in many RNA-binding proteins can participate in RNA binding, and their methylation may therefore influence the RNA/protein interaction (66). In the case of HuR, Arg 217 lies outside the three RNA-binding domains, and a prominent role for the hinge region in RNA binding seems unlikely. Nevertheless, methylation could indirectly influence RNA binding, by affecting the interaction with hinge-binding proteins, which could in turn modulate RNA binding, perhaps by controlling the access of RRM2 and/or RRM3 to RNA (67).
One of the most intriguing questions that remains unanswered is whether methylation is dynamic. To date, no demethylase has been identified, and if such an enzyme does not exist, a signal achieved through increased methylation must be silenced by protein degradation or sequestration of the methylated protein. It has also been suggested that methylation could be an incremental signal, accumulating slowly on proteins that carry multiple methylation sites (36). This is not likely to be the case for HuR, since the response to LPS is rapid (within 1 h) and there appears to be only one or two methylation sites.
In conclusion, CARM1, which was previously identified as a transcriptional coactivator for nuclear receptors (42,43), now appears to extend its role into the post-transcriptional domain of gene regulation. The potential for CARM1 involvement in post-transcriptional regulation of genes in response to a variety of stimuli is considerable, since one of its targets (HuR) not only mediates the stabilization of TNF-␣ mRNA upon stimulation with LPS (18) but also the stabilization of p21 mRNA upon exposure of cells to UV light (19), of vascular endothelial growth factor mRNA by hypoxia (15), and of nitric-oxide synthase II mRNA upon cytokine stimulation (13). How broad the regulatory network modulated by CARM1 might be will depend on how many target proteins it methylates in vivo. Thus far, CARM1 appears to have markedly fewer substrates than PRMT1 (36,47,58), but its recently demonstrated ability to methylate poly(A)-binding protein 1 (58) further supports its potential role in post-transcriptional gene regulation. Our results suggest that methylation by CARM1 might create a link between transcriptional and post-transcriptional events, affecting RNA synthesis as well as RNA transport and/or stability, and that methylation of the shuttling RNA-binding protein HuR may be one way in which nuclear and cytoplasmic regulation is coupled.


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A yeast model system for functional analysis of β-catenin signaling

We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of β-catenin (β-cat) signaling. Coexpression of mammalian β-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both β-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with β-cat, reduces β-cat binding to TCF4, and leads to increased cytoplasmic localization of β-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to β-cat and restores reporter gene transcription. Additional β-cat signaling components, Axin and glycogen synthase kinase 3β, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein β-transducin repeat-containing protein reduces the stability of β-cat and decreases reporter activation. Thus, we have reconstituted a functional β-cat signal transduction pathway in yeast and show that β-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

Introduction
The ␤ -catenin ( ␤ -cat)* signal transduction pathway plays an important role in a variety of developmental and differentiation processes in many organisms (Wodarz and Nusse, 1998) and is aberrantly activated in a majority of human colorectal cancers as well as in other tumor types (Bienz and Clevers, 2000;Peifer and Polakis, 2000;Polakis, 2000). Cell culture and animal studies have validated a key role for active ␤ -cat signal transduction in promoting critical aspects of malignant transformation (Bienz and Clevers, 2000;Peifer and Polakis, 2000).
Inhibition of ␤ -cat degradation serves an important role in providing a pool of signaling-competent ␤ -cat. Furthermore, the cytoplasmic and cadherin-bound pools of ␤ -cat may be in equilibrium (Fagotto et al., 1996;Orsulic and Peifer, 1996;Sanson et al., 1996) and the cadherin-associated population of ␤ -cat may serve as a reservoir of ␤ -cat that can be liberated for nuclear translocation and signal transduction (Birchmeier, 1995;Gumbiner, 1995). However, although a free pool of ␤ -cat is a prerequisite for subsequent association with TCF/LEF and transcriptional modulation, it is not sufficient for nuclear localization. This is evidenced by the observed cytoplasmic as well as nuclear distribution of ␤ -cat under circumstances, such as Wnt-1 signaling or APC mutation, where ␤ -cat degradation is inhibited (Munemitsu et al., 1995;Kawahara et al., 2000;Barker and Clevers, 2001;Brabletz et al., 2001). Nuclear accumulation of ␤ -cat and consequently ␤ -cat-TCF/LEF signaling must therefore involve a regulated balance between nuclear import and export.
The mechanisms of ␤ -cat nuclear transport are unclear because ␤ -cat does not have a classic NLS and can enter the nucleus by binding directly to the nuclear pore machinery in the absence of other known factors, in a manner similar to importin ␤ (Fagotto et al., 1997;Yokoya et al., 1999). Furthermore, ␤ -cat export is not blocked by leptomycin B, a specific inhibitor of nuclear export signal (NES)-mediated export (Eleftheriou et al., 2001;Wiechens and Fagotto, 2001). APC contains two NLS sequences in its central domain (Zhang et al., 2001) and can be found in the nucleus (Neufeld and White, 1997). APC also contains several consensus NES sequences, two at its NH 2 terminus (Neufeld and White, 1997;Henderson, 2000) and at least three within the 20-aa repeat region (Rosin-Arbesfeld et al., 2000). Mutation of these sequences results in accumulation of APC in the nucleus and correlates with increased ␤ -cat nuclear accumulation, leading to the hypothesis that nucleocytoplasmic shuttling of APC participates in the regulation of the nuclear localization of ␤ -cat (Henderson, 2000;Neufeld et al., 2000;Rosin-Arbesfeld et al., 2000;Zhang et al., 2001).
As a novel approach to study ␤ -cat signal transduction in a simplified context, we developed a model system where a significant portion of the ␤ -cat signal transduction pathway has been reconstructed in yeast. Individual known components of the mammalian ␤ -cat signal transduction pathway were systematically introduced into yeast cells and protein complex formation, subcellular localization, and function in regulation of ␤ -cat-TCF/LEF-mediated transcription were measured. The data suggest that ␤ -cat signaling can be regulated at multiple levels, including modulation of subcellular localization, complex formation, and stability of key proteins that participate in the ␤ -cat pathway.

Expression of ␤ -cat signaling components in yeast
Plasmids for regulated expression of epitope-tagged human ␤ -cat, LEF1, and TCF4 and corresponding NH 2 -terminal deletion mutants were introduced into wild-type yeast as a first step toward reconstituting a basic ␤ -cat signaling network. Full-length recombinant proteins ( Fig. 1 A, lane 2) and deletion mutants ( Fig. 1 A, lane 3) were specifically detected at their predicted molecular weights by Western blot analysis ( Fig. 1 A). ␤ -cat-⌬ N90, an NH 2 -terminal deletion mutant of ␤ -cat that is stabilized in mammalian cells, reproducibly accumulated to significantly higher levels as compared with the full-length ␤ -cat protein ( Fig. 1 A, ␤ -cat, compare lanes 2 and 3).
Yeast cells expressing ␤ -cat and TCF4, alone or in combination, were subjected to indirect immunofluorescence microscopy (IF) to ascertain the subcellular localization of these proteins ( Fig. 1 B). Either ␤ -cat or TCF4, when expressed alone, accumulated in the nucleus ( Fig. 1 B, panel A). Coexpression of ␤ -cat with TCF4 had no effect on the localization of either protein ( Fig. 1 B, panel D). Thus, translocation of either protein into the nucleus occurs independently. LEF1 localization was indistinguishable from that of TCF4, and that of NH 2 -terminal deletion mutants of ␤ -cat, TCF4, Full-length and NH 2 -terminal deletion mutants of epitopetagged human ␤-cat, LEF1, and TCF4 were expressed in yeast and equivalent amounts of protein from cells containing vector only (lane 1), full-length protein (lane 2), or NH 2 -terminal deletion mutant protein (lane 3) were subjected to Western blot analysis using antibodies against the FLAG epitope (top and middle) or ␤-cat (bottom). (B) Yeast cells containing expression plasmids for ␤-cat only (panel 1, A-C), TCF4 only (panel 2, A-C), or ␤-cat together with TCF4 (panels 1 and 2, D-F and G-I) were grown under conditions where protein expression was induced (panels 1 and 2, A-F) or repressed (panels 1 and 2, G-I). Cells were subjected to IF with an antibody against ␤-cat (panel 1) or the FLAG epitope to detect TCF4 (panel 2). Proteins (A, D, and G), DNA (B, E, and H), and cells (C, F, and I) were photographed. and LEF1 was identical to their full-length counterparts (unpublished data).

␤ -Cat cooperates with LEF1 and TCF4 to activate yeast reporter gene transcription
To determine if ␤ -cat and TCF4 or LEF1 expressed in yeast function in regulating transcription, we generated reporter plasmids containing wild-type (WT 5X LEF) or mutated (Mut 5X LEF) LEF/TCF-dependent transcriptional elements upstream of a yeast CYC1 minimal promoter driving expression of either Escherichia coli LacZ or yeast HIS3 reporter genes (Fig. 2 A). WT 5X LEF and Mut 5X LEF LacZ reporters were introduced into yeast expressing full-length or NH 2 -terminal-deleted forms of ␤ -cat, TCF4, or LEF1 and cultures were assayed for ␤ -galactosidase ( ␤ -gal) enzymatic activity (Fig. 2, B and C). Wild-type ␤ -cat or LEF1 alone induced little ␤ -gal activity (Fig. 2 B). Conversely, coexpression of ␤ -cat with LEF1 increased reporter gene expression (Fig. 2  B). Similar results were obtained with TCF4 (unpublished data; Fig. 2 C; Fig. 3). No induction of ␤ -gal activity was observed with Mut 5X LEF LacZ (Figs. 2, B and C). Furthermore, deletion of the NH 2 -terminal DNA binding domain of either TCF4 or LEF1 ( ⌬ N30 and ⌬ N67, respectively) resulted in significant reduction of reporter gene expression in the presence of ␤ -cat ( Fig. 2 C). The ⌬ N90 mutant of ␤-cat retained full ability to activate reporter gene expression as expected ( Fig. 2 C). All of the ␤-cat and LEF or TCF proteins were efficiently expressed (Fig. 2 D;unpublished data).
Expression of both ␤-cat and TCF4 (Fig. 3 B) along with the WT 5X LEF HIS3 reporter (Fig. 2 A) enabled yeast strains harboring mutations in the HIS3 gene to grow on medium lacking histidine ( Fig. 3 A, upper right quadrant). Cells containing the HIS3 reporter with mutated LEF/TCF sites ( Fig. 3 A, lower quadrants), or cells grown under conditions where ␤-cat and TCF4 were not expressed ( Fig. 3 A, left quadrants) were unable to grow. Similar results were obtained with LEF1 in conjunction with ␤-cat (unpublished data). Thus, ␤-cat and TCF/LEF are capable of using the conserved yeast nuclear import and transcriptional machinery to function in the specific activation of reporter gene expression.
E-cad and APC-25 bind to ␤-cat and inhibit ␤-cat/TCF4 transcriptional activation ␤-cat expressed in our system is constitutively active as a transcriptional coactivator, due at least in part to its localization in the nucleus. To recreate the ␤-cat-E-cad interaction, an expression plasmid encoding the plasma membrane teth- reporter constructs. (B) Cells expressing ␤-cat and LEF1, ␤-cat only, or LEF1 only along with either WT or Mut 5X LEF LacZ reporter plasmids were assayed for ␤-gal activity. (C) Cells with either WT or Mut 5X LEF LacZ reporter plasmids were induced to express either full-length or NH 2 -terminal-deleted forms of the indicated proteins and ␤-gal activity was determined. (D) Equivalent amounts of total protein from cells used for the assay in C expressing ␤-cat alone (lanes 3), ␤-cat ⌬N90 alone (lanes 6), ␤-cat with LEF1 or LEF1⌬N67 (lanes 4 and 5, respectively), or ␤-cat ⌬N90 with LEF1 or LEF1⌬N67 (lanes 7 and 8, respectively) from strains also containing WT 5X LEF LacZ (top two panels) or Mut 5X LEF LacZ (bottom two panels) were subjected to Western blot analysis. ering domain of human regulator of G protein signaling 4 (RGS4) fused to the NH 2 terminus of the cytoplasmic tail of mouse E-cad was introduced. RGS-E-cad localized to the cytoplasm and plasma membrane ( Fig. 4 A, left, panel J). Nuclear ␤-cat redistributed to the cytoplasm in cells coexpressing RGS-E-cad ( Fig. 4 A, left, panels A and D). In cells expressing only ␤-cat and TCF4, TCF4 coimmunoprecipitated with ␤-cat ( Fig. 4 A, right, lane 3). However, when RGS-E-cad complexed with ␤-cat, the amount of TCF4 in ␤-cat immunoprecipitates was reduced ( Fig. 4 A, right, lane 4). Furthermore, RGS-E-cad expression reduced the ability of ␤-cat-TCF4 to induce both HIS3 and LacZ reporter genes by 30% and 50%, respectively (Fig. 4 B). Although there was no detectable ␤-cat in the nucleus in the presence of RGS-E-cad, ␤-gal expression was only reduced by 50%, illustrating the relative sensitivity of reporter gene expression compared with IF. Thus, binding of ␤-cat to RGS-E-cad results in its redistribution to the cytoplasm, reduction in ␤-cat-TCF4 complex formation, and concomitant reduction in LEF/TCF-dependent reporter gene expression.
In mammalian cells, APC complexes with cytoplasmic ␤-cat, resulting in its Axin-dependent phosphorylation by GSK3␤ and targeting to the ubiquitin-mediated degradation pathway. The central 733 aa of APC (APC-25) are sufficient to bind ␤-cat and promote its degradation (Munemitsu et al., 1995). We expressed APC-25 in yeast containing stably integrated ␤-cat and TCF4 along with either HIS3 or LacZ reporter genes. APC-25 localized throughout the cell, mainly in the cytoplasm (Fig. 5 A, left, panel J). In the presence of APC-25, ␤-cat redistributed from the nucleus to the cytoplasm ( Fig. 5 A, left, compare panels D and A). In the absence of APC-25, ␤-cat associated with TCF4 ( Fig. 5 A, right, lane 2). APC-25 coimmunoprecipitated with ␤-cat ( Fig. 5 A, right, lane 4), correlating with a loss of TCF4 in the ␤-cat immunoprecipitates ( Fig. 5 A, right, lane 4). Furthermore, the ability of strains containing ␤-cat and TCF4 along with APC-25 to grow on media stringently selecting for HIS3 expression was dramatically reduced (Fig. 5 B). ␤-cat plus TCF4-induced LacZ reporter activity was also inhibited in the presence of APC-25 ( Fig. 5 B). Thus, similar to results with RGS-E-cad, expression of APC-25 led to a dramatic reduction in ␤-cat/TCF4-dependent reporter gene expression accompanied by redistribution of ␤-cat from the nucleus to the cytoplasm. Steady-state ␤-cat levels, examined by Western blot, were similar with or without APC-25, despite the association of ␤-cat with APC-25 and its redistribution to the cytoplasm ( Fig. 5 A, right).

APC-25, Axin, and GSK3␤ associate with ␤-cat
In mammalian cells, a complex containing APC, Axin, ␤-cat, and GSK3␤ forms in the cytoplasm, and negatively regulates the transcriptional activity of ␤-cat by targeting it to the ubiquitin-mediated degradation machinery. To systematically define the role of each of these components in regulation of ␤-cat signaling, various combinations of APC-25, Axin, and GSK3␤ were introduced into yeast containing stably integrated ␤-cat, TCF4, and either HIS3 or LacZ reporter plasmids, followed by evaluation of their ability to associate with ␤-cat and to modulate LEF/TCF-dependent transcription. As described above, TCF4 efficiently coimmunoprecipitated with ␤-cat ( Fig. 6 A, anti-FLAG, IP, lanes 5-8), but not in the presence of APC-25 ( Fig. 6 A, an-tiFLAG, IP, lanes 1-4). APC-25 and Axin, either alone or in combination, complexed with ␤-cat ( Fig. 6 A, anti-HA, IP, lanes 1, 2, and 6). GSK3␤ was not found in ␤-cat immunoprecipitates either alone or in combination with APC-25 ( Fig. 6 A, GSK3␤, IP, lanes 3 and 7), despite being present in total cell lysates (GSK3␤, T, lanes 3 and 7). GSK3␤ associated with ␤-cat only when coexpressed with Axin ( Fig. 6 A, GSK3␤, IP, lanes 4 and 8), consistent with reports indicating a role for Axin as a scaffolding protein in the ␤-cat complex (Hart et al., 1998). Of note is that none of the protein combinations described reduced steady-state levels of ␤-cat in total protein extracts ( Fig. 6 A, ␤-cat, T, lanes 1-8).
Wherever APC-25 was present, ␤-cat localized predominantly to the cytoplasm (Fig. 6 B, panels M, P, S, and V). Ex- Figure 3. ␤-catand TCF4-dependent transcription of the HIS3 reporter gene allows for yeast cell growth on medium lacking histidine. His Ϫ yeast containing ␤-cat and TCF4 plasmids with WT or Mut 5X LEF HIS3 reporter plasmids were grown on media lacking histidine with either glucose or galactose. (A) Photograph of yeast growth after 4 d. Yeast grown on glucose do not express ␤-cat or TCF4 and therefore cannot grow on medium lacking histidine (left). Yeast cells grown on galactose are induced to express ␤-cat and TCF4. Cells containing a WT 5X LEF HIS3 reporter are able to grow, whereas those containing a Mut 5X LEF HIS3 reporter cannot (right). (B) Western blot demonstrating expression of TCF4 and ␤-cat proteins under induced (lane 2), but not uninduced (lane 1), conditions. Blots were probed with antibodies directed against the FLAG epitope (top) and against ␤-cat (bottom).
pression of Axin or GSK3␤ had no detectable effect on the localization of ␤-cat (Fig. 6 B,    reporter were induced to express ␤-cat and TCF4 with (10 6 cells) or without (10 3 cells) constitutively expressed APC-25, were plated under stringent HIS3 selection conditions, and the number of surviving colonies was quantitated. (B, right) Cells induced to express ␤-cat and TCF4 with or without constitutively expressed APC-25 along with the 5X LEF LacZ reporter were assayed for ␤-gal activity.
on ␤-gal activity (Fig. 5 B; Fig. 6 D). These data indicate that of the proteins tested, only APC-25 can inhibit ␤-cat signaling, and this correlates with diminished complex formation between ␤-cat and TCF4 as well as a redistribution of ␤-cat from the nucleus to the cytoplasm, where it binds to APC-25.
␤-cat is constitutively phosphorylated on serine/threonine In mammalian cells, ␤TRCP recognizes phosphorylated ␤-cat and targets it to the ubiquitin degradation machinery. Consequently, we examined phosphorylation of ␤-cat by Western blot using antibodies specific for ␤-cat phosphorylated at serine/threonine residues 33, 37, 41, and 45. Phosphorylation of ␤-cat was detectable in yeast with or without expression of exogenous GSK3␤ (Fig. 7 A), indicating that it must be phosphorylated by an endogenous yeast kinase and could serve as a substrate for ubiquitin-mediated degra-dation. Densitometric scanning and normalization of the data indicated that coexpression of APC-25 or Axin in the presence or absence of exogenous GSK3␤ led to a slight increase in ␤-cat phosphorylation (Fig. 7 A).
␤TRCP decreases both the level of ␤-cat and ␤-cat-mediated transcriptional activation Because significant differences in the steady-state levels of ␤-cat were not detected, the stability of ␤-cat was examined by pulse-chase analysis. Because the yeast system may lack a specific F-box protein required for targeting ␤-cat degradation, the effect of ␤TRCP expression on ␤-cat levels was examined. Yeast containing stably integrated ␤-cat and TCF4 with combinations of ␤TRCP and APC-25 were induced to express ␤-cat by growth in galactose-containing medium (pulse) followed by repression of ␤-cat synthesis by transfer into medium containing glucose (chase). Coexpression of ␤TRCP, with ␤-cat and TCF4 in the absence of APC-25, Figure 6. Analysis of complex formation, subcellular localization, and effects on transcriptional activity using combinations of APC-25, Axin, and GSK3␤ with ␤-cat/TCF4. (A) Expression vectors for APC-25, Axin, and GSK3␤ were introduced into yeast cells containing stably integrated ␤-cat and TCF4. ␤-cat was immunoprecipitated from protein extracts followed by Western blot with antisera against the HA tag (to detect APC-25 and Axin), human GSK3␤, the FLAG tag (to detect TCF4), or ␤-cat in total extracts (T) and ␤-cat immunoprecipitates (IP). (B and C) Cells containing integrated ␤-cat and TCF4 alone (A-C and M-O) or with Axin (D-F and P-R), GSK3␤ (G-I and S-U), or Axin plus GSK3␤ (J-L and V-X) and with either a control vector (Control) or an APC-25 expression vector (APC-25) were subjected to IF with an antibody against ␤-cat (B) or the HA tag (to detect Axin or APC-25) (C). Proteins (columns 1and 4), DNA (columns 2 and 5), and cells (columns 3 and 6) were photographed. (D) Yeast cells with integrated ␤-cat and TCF4 and combinations of APC-25, Axin, and GSK3␤ as well as the 5X LEF LacZ reporter were assayed for ␤-gal activity.
reduced the amount of ␤-cat protein (Fig. 7 B, 4-h chase, compare lanes 1 and 2). Alternatively, expression of APC-25 with ␤-cat and TCF4 resulted in increased ␤-cat protein (Fig. 7 B, 4 h, compare lanes 1 and 3). However, with the addition of ␤TRCP, the amount of ␤-cat was again diminished ( Fig. 7 B, compare lanes 3 and 4). Thus, in the yeast system, ␤-cat is constitutively phosphorylated on serine/ threonine and, in the presence of ␤TRCP, displays decreased levels, suggesting a reconstitution of some aspects of the ubiquitin-mediated degradation of ␤-cat. Various gene combinations including Axin or exogenous GSK3␤ did not reveal further changes in the levels of ␤-cat beyond those demonstrated in Fig. 7 B (unpublished data).
␤TRCP reduced the ability of ␤-cat plus TCF4 to induce LacZ reporter expression by %04ف (Fig. 7 C). Combination of ␤TRCP and APC-25 decreased reporter activity beyond that seen with APC-25 alone (Fig. 7 C). These data indicate that ␤TRCP can reduce the capability of ␤-cat to activate transcription, presumably due to its facilitation of ␤-cat degradation; however, other mechanisms are possible.
Putative NES sequences of APC-25 are required for down-modulating ␤-cat signaling APC contains NESs and is reported to enter and exit the nucleus in mammalian cells. APC-25 has six of seven 20-aa repeats (20R2-7) found in the central region of APC and several functional NES sequences have been localized to these 20-aa repeats (20R3, 4, and 7) (Fig. 8 A). Sequence alignment reveals additional potential NES sequences in repeats 2, 5, and 6 ( Fig. 8 A). To explore the role of previously identified and putative NES sequences, we systematically mutated each of three hydrophobic core amino acids in the six potential NES sequences to alanines (Fig. 8 B). APC-25 with mutation of previously identified NES sequences in 20aa repeats 3, 4, and 7 (Fig. 8 B, 20R2, 5, 6-⌬3, 4, 7) showed only a slight loss of its ability to suppress ␤-cat-dependent reporter gene expression (Fig. 8, C and D). Mutation of additional putative NES sequences in 20-aa repeats 2, 5, and 6, in combination with ⌬3, 4, and 7 ( Fig. 8 B), resulted in incremental loss of the ability of APC-25 to suppress ␤-catdependent transcription (Fig. 8, C and D). Mutation of all putative NES sequences (Fig. 8 B, 20R-⌬2, 3, 4, 5, 6, 7) resulted in complete ablation of APC-25 suppression of ␤-cat-dependent reporter gene activation, restoring expression to levels comparable to the absence of APC-25 (Fig. 8,  C and D). These results indicate that at least one intact putative NES sequence within APC-25 is capable of conferring partial suppression of reporter activation. Interestingly, putative NES sequences in 20-aa repeat 2 are the least homologous to the consensus sequence for leucine-rich NESs (Fig. 8  A) and are alone incapable of conferring partial suppression (Fig. 8, C and D). APC, as well as the central domain of APC analogous to APC-25 (Rosin-Arbesfeld et al., 2000), rapidly shuttle between nucleus and cytoplasm in mammalian cells. In yeast, APC-25 appears to be almost exclusively cytoplasmic at steady-state (Figs. 5 and 6), raising the possibility that APC-25 enters the nucleus and is very rapidly exported. This suggests that mutation of NES sequences should result in nuclear accumulation of APC-25. Surprisingly, mutation of the known and putative NES sequences within APC-25 did not result in accumulation of APC-25 in the nucleus when examined by IF (Fig. 9 A, panels D, G, F, and I). Although mutants of APC-25 remained localized to the cytoplasm, most of them failed to retain ␤-cat (Fig. 9 B). ␤-cat was primarily cytoplasmic in yeast with wild-type APC-25, whereas mutation of the three previously defined APC NES sequences (20R2, 5, 6-⌬3, 4, 7) resulted in an increase in nuclear ␤-cat ( Fig. 9 B, compare panels B and C). Mutation of additional putative NES sequences resulted in a further increase in nuclear ␤-cat, with restoration of the predominantly nuclear localization by mutants lacking all hydrophobic NES sequences (20R-⌬2, 3, 4, 5, 6, 7) or containing only the sequences in 20R2 (20R2-⌬3, 4, 5, 6, 7) ( Fig. 9 B, panels O and N, respectively). Nuclear location of ␤-cat in cells with NES mutants of APC correlated with reporter gene activity (Fig. 8, C and D).
Mutation of previously identified as well as putative NES sequences in APC-25 resulted in decreased binding to ␤-cat  1 and 3) in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of stably integrated constitutively expressed APC-25 were induced to express regulated proteins (␤-cat, TCF4, and ␤TRCP) by growth in medium containing galactose (0 h, top). Cells were then washed into medium containing glucose to repress expression of regulated proteins for 4 h (bottom). Cell extracts prepared at 0 or 4 h were subjected to Western blot analysis for ␤-cat protein levels (top panel exposed 10 s and bottom panel exposed 1 min). (C) Cells with integrated ␤-cat and TCF4 along with various combinations of stably integrated APC-25 or plasmids encoding ␤TRCP and 5X LEF LacZ reporter were assayed for ␤-gal activity. and ablated binding altogether in mutants lacking NES sequences 3, 4, 5, 6, and 7 ( Fig. 9 C, lanes 9 and 10). Decreased binding of NES mutants correlated with an increase in TCF4 binding to ␤-cat (Fig. 9 C). Of note is that putative NES sequences in 20-aa repeat 2 are alone unable to confer binding of APC-25 to ␤-cat ( Fig. 9 C, lane 9), consistent with the inability of this APC-25 mutant to suppress reporter gene activation and to redistribute ␤-cat to the cytoplasm. Thus, the putative NES sequences in APC-25 also appear to be required for binding of APC-25 to ␤-cat, and the ability of APC-25 to modulate ␤-cat signaling may be defined by the ability to bind and retain ␤-cat in the cytoplasm.

Discussion
We introduced genes encoding key players in the mammalian ␤-cat signaling pathway, along with a pathway-specific transcriptional reporter gene, into the yeast Saccharomyces cerevisiae. Using this simplified system, it was possible to sys-tematically define aspects of protein complex formation, subcellular localization, and signaling regulation in the ␤-cat pathway in the absence of the signaling network complexities that exist in mammalian cells.

Protein complex formation in yeast
Similar to other cellular systems, ␤-cat and TCF/LEF proteins readily formed a transcriptionally competent complex. Both APC-25 and RGS-E-cad displaced TCF/LEF upon binding to ␤-cat, confirming previous reports that APC, E-cad, and LEF1 compete for binding to the armadillo repeats of ␤-cat (Rubinfeld et al., 1993;Hulsken et al., 1994;Sadot et al., 1998;Orsulic et al., 1999). We show that mutation of putative NES sequences within 20-aa repeats (20Rs) of APC-25 results in defective binding of APC-25 to ␤-cat and prevents displacement of ␤-cat from TCF4. This is the first report where all 20Rs have been mutated in the same APC protein.
Our results confirm that the 20R region is critical for ␤-cat binding and reveal differences in the capacity of different re-  (Control) or with plasmids expressing wild-type APC-25, APC-25 NES mutants, or RGS-E-cad as well as the 5X LEF LacZ reporter were assayed for ␤-gal activity. The x axis labels indicate the mutated NES sequence whereas a plus indicates the presence of a particular wild-type NES sequence in the indicated 20-aa repeat regions (20R2-7). (D) HIS Ϫ cells induced to express ␤-cat and TCF4 (Control) along with wild-type APC25 or APC-25 NES mutants in the presence of the 5X LEF HIS3 reporter were plated under stringent HIS3 selection conditions and surviving colonies were quantitated. The x axis labels are as in C.
peats for binding to ␤-cat. Residues comprising the 20R2 region alone are unable to sustain binding to ␤-cat, whereas those of the 20R5 and 20R6 regions are individually sufficient for interaction with ␤-cat. Other studies showed that Axin interacted directly with ␤-cat and GSK3␤ to bridge these two proteins (Hart et al., 1998;Ikeda et al., 1998;Itoh et al., 1998;Sakanaka et al., 1998). This result is borne out in yeast where Axin associated with ␤-cat, but we only observed GSK3␤ in complex with ␤-cat in the presence of Axin.
␤-cat and LEF/TCF must cooperate to activate transcription LEF/TCF transcription factors are known to require additional factors for transcriptional activation (Carlsson et al., 1993;Giese and Grosschedl, 1993). Consistent with these data, neither TCF4 nor LEF1 display transcriptional activity when expressed alone. Similarly, ␤-cat by itself is inactive and the combination of ␤-cat and either LEF1 or TCF4 is required for activation of transcription. Under these conditions, ␤-cat complexes with LEF1 or TCF4, confirming the necessity of cooperation between ␤-cat and LEF/TCF transcription factors for transcriptional activation. Furthermore, the ability of ␤-cat and LEF/TCF to function without additional introduced pathway components demonstrates that these proteins, in conjunction with the conserved yeast basal transcriptional machinery, are sufficient to activate pathwayspecific reporter gene transcription.

Nuclear localization of LEF/TCF and ␤-cat
In mammalian cells, LEF/TCF proteins are found predominately in the nucleus, whereas ␤-cat localizes to the plasma membrane as a component of adherens junctions as well as to the cytoplasm and nucleus (Gumbiner, 1995;Eastman and Grosschedl, 1999). ␤-cat enters the nucleus via an unconventional mechanism involving direct interaction with the nuclear pore complex in an NLS-independent manner (Fagotto et al., 1998;Yokoya et al., 1999). Both ␤-cat and TCF4 independently and constitutively localized to the nucleus when introduced into yeast cells, suggesting that they utilize the basic yeast nuclear transport machinery and that no other specific ␤-cat pathway components are required for ␤-cat entry and accumulation in the nucleus. In the yeast system, ␤-cat and LEF/TCF localize to the nucleus under conditions where reporter gene expression is activated. If ␤-cat is relocalized to the cytoplasm by RGS-E-cad or APC-25 coexpression, there is a concomitant decrease in reporter gene transcription. Restoration of ␤-cat nuclear accumulation by mutations in APC-25 that disrupt complex formation results in restoration of reporter gene expression. Thus, consistent with other systems, our data indicate that nuclear localization of ␤-cat is required Figure 9. Subcellular localization and complex formation with ␤-cat is altered in the presence of APC NES mutants. (A) Yeast cells with integrated ␤-cat and TCF4 in combination with integrated wild-type APC-25 (APC-25) or APC-25 NES mutants or expressing RGS-E-cad from a plasmid were subjected to IF with an antibody against the HA epitope (APC-25 proteins) or against E-cad. Proteins (Protein, panels A, D, G, and J) and DNA (DNA, panels B, E, H, and K) were photographed. False color images were captured and merged (Merge, panels C, F, I, and L), overlapping signals of protein (green) and DNA (red) are indicated by yellow color. (B) Yeast cells with integrated ␤-cat and TCF4 alone plus either a control vector (Control), wild-type APC-25 expression vector (APC-25), or expres-sion vectors for the indicated APC-25 NES mutants were subjected to IF with an antibody against ␤-cat. Proteins (panels A-E and K-O) and DNA (panels F-J and P-T) were photographed. (C) ␤-cat immunoprecipitates of protein extracts from cells expressing ␤-cat and TCF4 (Control) and either wild-type APC-25 or APC-25 NES mutants were analyzed by Western blot. Antisera against the HA tag (to detect APC-25 proteins), FLAG tag (to detect TCF4), or ␤-cat were used to identify the corresponding proteins in total cell extracts (T) as well as in complex with ␤-cat (IP). for its role in transcriptional activation (Funayama et al., 1995;Behrens et al., 1996;Molenaar et al., 1996;Orsulic and Peifer, 1996;Schneider et al., 1996). Interestingly, despite binding to ␤-cat, Axin had no effect either alone or in combination with other regulators on ␤-cat localization, transcriptional activation, or complex formation with TCF4. It is possible that the interaction is too weak to sequester ␤-cat in the cytoplasm or that additional components are required.

Regulation of ␤-cat signaling and nuclear localization
In normal mammalian cells, in the absence of a Wnt signal or an activating mutation, the steady-state localization of ␤-cat is cytoplasmic, mostly at the plasma membrane. Surprisingly, in the yeast system, ␤-cat and TCF/LEF are almost exclusively nuclear, and reporter gene transcription is constitutive. This supports a model where nuclear localization of ␤-cat represents a default state of the cell and active inhibition of ␤-cat signaling is necessary. Therefore, the "resting" state in a mammalian cell would require an active pathway to counteract the nuclear localization of ␤-cat. Mechanisms that could downregulate the nuclear accumulation of ␤-cat include inhibition of nuclear import, active nuclear export, cytoplasmic sequestration, and degradation. Inactivation of this inhibitory mechanism, via stimulation by Wnt or other growth factor signals, would lead to activated ␤-cat-modulated transcription.
It is likely that a regulated balance exists between active export of ␤-cat from the nucleus and its constitutive import. Export of ␤-cat from Xenopus nuclei in vitro is saturable, indicating a need for specific interactions for export (Wiechens and Fagotto, 2001). This hypothesis is further supported by studies suggesting that ␤-cat is exported from the nucleus by APC in a manner dependent upon the CRM-1 export receptor (Henderson, 2000;Rosin-Arbesfeld et al., 2000). To test this mechanism in the yeast model system, we expressed a set of APC-25 proteins with mutations in previously defined and additional putative NES sequences. Because wild-type APC-25 and APC-25-␤-cat complexes are cytoplasmic, and because APC is hypothesized to shuttle between the nucleus and cytoplasm, we expected to find that mutation of relevant NES sequences would lead to accumulation of APC-25 in the nucleus along with associated ␤-cat. Surprisingly, APC-25 mutants lacking all potential NES sequences were not detected accumulating in the yeast nucleus. Phosphorylation of a serine near the second NLS of APC-25 may be required for its efficient import into the nucleus (Zhang et al., 2001), and if this does not occur in yeast, APC-25 may inefficiently enter the nucleus and the effect of mutating NES sequences would not be evident. In this case, the observed redistribution of ␤-cat out of the nucleus is due to complex formation with cytoplasmic wild-type APC-25 and consequent sequestration. Alternatively, APC-25 may enter the yeast nucleus and be efficiently exported, independent of the NES sequences that were mutated. This could be due to the presence of cryptic NES sequences in APC-25, or NES-independent export.
An alternate mechanism to inhibit active nuclear localization of ␤-cat is sequestration via interaction with cytoplasmic proteins. This is supported by a report demonstrating that ␤-cat can exit the nucleus independently of CRM1 and RanGTP, suggesting passive export (Wiechens and Fagotto, 2001). In the yeast system, ␤-cat was redistributed from the nucleus to cytoplasm upon binding either cytoplasmic APC-25 or plasma membrane RGS-E-cad, supporting the model of regulation by cytoplasmic retention. Other studies with mammalian cells showed that ␤-cat nuclear localization and signaling can be inhibited by cadherin binding (Sadot et al., 1998;Orsulic et al., 1999;Gottardi et al., 2001). In tumor cells where E-cad expression is lost and ␤-cat signaling is activated, this may represent an important loss of regulatory function, underscoring the physiological relevance of an active inhibitory mechanism for attenuation of this signaling pathway.

Regulation of ␤-cat degradation
In the yeast system, complex formation between ␤-cat and either APC-25 or RGS-E-cad leads to cytoplasmic accumulation of ␤-cat and loss of transcriptional activation, however, there is no evidence for degradation of ␤-cat protein. In fact, complex formation with APC-25 appears to stabilize ␤-cat when analyzed in a pulse-chase experiment. This is similar to the stabilized ␤-cat-APC or ␤-cat-E-cad complexes observed in mammalian cells responding to Wnt-1, where ␤-cat degradation is inhibited (Hinck et al., 1994;Papkoff et al., 1996). In SW480 colon carcinoma cells with a truncating APC mutation, ␤-cat accumulates to high levels, yet much of the protein remains in the cytoplasm and associated with APC (Munemitsu et al., 1995;Kawahara et al., 2000;Barker and Clevers, 2001;Brabletz et al., 2001). These findings suggest, first, that complex formation between ␤-cat and proteins such as APC can be uncoupled from degradation and, second, that inhibition of degradation of ␤-cat is a prerequisite, but not sufficient, for nuclear accumulation.
In an effort to define the minimal requirements for ␤-cat degradation, we introduced additional genes into yeast containing ␤-cat and TCF4. APC-25, Axin, or GSK3␤, alone or in all possible combinations, did not lead to measurable differences in steady-state levels of ␤-cat, despite detection of the expected complexes between these proteins. In contrast, ␤TRCP led to decreased ␤-cat protein levels when expressed without APC-25, Axin, or GSK3␤. No further decrease in ␤-cat levels was observed when these proteins were included. In yeast, ␤-cat was constitutively phosphorylated on serine/threonine, potentially by one of the four known endogenous yeast GSK3 proteins (Andoh et al., 2000). Exogenous GSK3␤ does not lead to significant additional phosphorylation of ␤-cat, either because the protein is already maximally phosphorylated or because mammalian GSK3␤ is not active in our yeast system. However, a temperature-sensitive yeast mutant lacking all four GSK3 genes can be rescued by expression of mammalian GSK3␤, suggesting that it is functional in yeast (Andoh et al., 2000). Taken together, these data suggest that when ␤-cat is appropriately phosphorylated, potentially by a yeast GSK3 homologue, ␤TRCP expression is sufficient for its enhanced turnover. In mammalian cells, APC and Axin would be needed to enable phosphorylation of ␤-cat, presumably by the complexed GSK3␤, whereas in the yeast system, ␤-cat is phosphorylated without additional scaffolding proteins.
We have developed a novel, defined yeast system for the study of ␤-cat signaling. Our results support a general model where activated ␤-cat signaling is a default process, and in normal, unstimulated cells, this pathway must be actively in-hibited at several levels. Regulation of ␤-cat-TCF/LEF transcriptional activation can be accomplished through modulation of ␤-cat stability, protein complex formation, subcellular compartmentalization, and nuclear import/export. This yeast system will allow further study of the complex ␤-cat pathway in a simplified format. Furthermore, the dual reporter system described here will enable the design of genetic strategies for the selection of additional genes that regulate ␤-cat signaling.

Plasmids
Mammalian genes were amplified by PCR from plasmids or Marathon-ready cDNA libraries (CLONTECH Laboratories, Inc.) and subcloned into pRS shuttle vectors or vectors for high level-regulated or constitutive expression in yeast (American Type Culture Collection [ATCC] accession nos. 87538, 87669, and 87670). Full-length human ␤-cat (NH 2 -terminal HA tag) and human GSK3␤ were amplified from a human mammary library, the APC-25 fragment of APC (Munemitsu et al., 1995) from a human brain library, ␤TRCP from a human testis library, and human Axin form I (Ikeda et al., 1998; Genbank/EMBL/DDBJ accession no. AF009674) from a human fetal brain library. The ⌬N90 (NH 2 -terminal HA tag) mutant was generated by PCR amplification of ␤-cat lacking the NH 2 -terminal 90 aa. NH 2 -terminal FLAG-tagged human LEF1 and ⌬N67 mutant were gifts from Don Ayer (University of Utah, Salt Lake City, UT). Human TCF4 and ⌬N30 mutant were PCR amplified from IMAGE clones (ATCC accession nos. 1288178 and 184930). To generate yeast LEF/TCF-dependent reporter constructs, five tandem copies of the wild-type or mutated LEF/TCF binding site (5Ј-CCTTTGAA-3Ј or 5Ј-CAAT-TCAA-3Ј, respectively, provided by Dorre Gruenberg, Aventis Pharmaceuticals) were cloned into the polylinker of pNB404 (ATCC accession no. 87513) upstream of the CYC1 promoter to generate WT or Mut 5X LEF LacZ. The LacZ gene was replaced with a HIS3 gene from pRS423 to generate HIS3 reporter plasmids. Reporter constructs containing the LYS2 nutritional marker were generated by subcloning the entire reporter cassette from pNB404based vectors into pRS317. GAL-RGS-E-cad constructs were generated by fusing PCR products encoding the plasma membrane targeting NH 2 -terminal 33 aa of the human RGS4 gene (Srinivasa et al., 1998) and the COOH-terminal 150 aa of mouse E-cad, amplified from a mouse embryo cDNA library. For each APC-25 NES mutant, the three hydrophobic amino acids were substituted with alanine using a single oligonucleotide designed using the Quick-Change site-directed mutagenesis kit (Stratagene).

Yeast strains
Growth and maintenance of yeast strains, introduction of plasmids, and all genetic manipulations were performed as previously described (Lee et al., 1996). Wild-type yeast strains FY833 and FY834 were used as a basis for all strains generated (ATCC accession nos. 90844 and 90845) and were crossed with strain BY4704 containing designer deletion alleles (ATCC accession no. 200868) to generate strains containing additional selectable markers to accommodate the multiple genes expressed. Different plasmids with mammalian genes were generated such that a selection of different nutritional markers was available. Yeast strains with integrated copies of mammalian genes under the control of GAL1 (␤-cat and TCF4) or ADH1 (APC-25 constructs) promoters were generated by homologous recombination using standard genetic techniques (Abelson and Simon, 1991).

Assay for ␤-gal in liquid cultures
␤-gal assays and calculation of activity units were performed exactly as previously described (Ausubel et al., 1994). Triplicate yeast cultures of 50-100 cells were induced for protein expression in medium containing 2% galactose, and an OD 600 was determined for each sample.

Histidine reporter plating assay
Yeast strains containing the desired plasmid(s) were grown on appropriate selective medium. Aliquots of 10 6 or 10 3 cells were plated in triplicate on selective medium lacking histidine with 10 mM 3-amino-triazol. Aliquots were also plated on selective medium containing histidine to determine the number of cells plated that were capable of growth. After 4 d of growth, colonies were counted and the percentage of viable cells surviving was calculated.

Immunofluorescence microscopy
IF was performed as previously described (Lee et al., 1996). Mouse monoclonal anti-␤-cat (Transduction Laboratories), anti-E-cad (Transduction Laboratories), and anti-FLAG M5 (Sigma-Aldrich) antibodies were used at a 1:500 dilution. Mouse monoclonal anti-HA (BabCo) antiserum was used at a 1:200 dilution. Proteins were visualized with a 1:500 dilution of FITCconjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Nuclei were stained with 10 g/ml of DAPI. Cells were photographed using differential interference microscopy (DIC) optics (100ϫ) with a SPOT camera (Diagnostic Instruments) using SPOT and Adobe Photoshop ® software.

Western blot analysis and immunoprecipitation
Protein extracts from yeast cells were prepared as previously described (Lee et al., 1996). Yeast proteins (40 g total yeast protein) were separated on 10% SDS-PAGE (Bio-Rad Laboratories), transferred to nitrocellulose, and probed with mouse monoclonal anti-HA, polyclonal anti-GSK3␤ (Transduction Labs), and anti-␤-cat antibodies at a 1:2,000 dilution, anti-FLAG M5 at a 1:1,000 dilution, or a mixture of two antibodies recognizing phosphorylated ␤-cat (Cell Signaling Technology) at 1:1,000 each. HRPconjugated secondary antibodies were used at a 1:5,000 dilution (Santa Cruz Biotechnology, Inc.), and blots were developed with ECL detection reagents (Amersham Biosciences). For immunoprecipitations, 0.5-1 mg total yeast protein extract was incubated with 50 l of a 50% slurry of protein A-sepharose beads and 1 g of ␤-cat antibody per sample for 2 h at 4ЊC. Immune complexes were washed three times with lysis buffer and solubilized in SDS-PAGE sample buffer before gel electrophoresis. 12-25fold more total protein was used for immunoprecipitation than was analyzed in the comparable total protein lane of gels. Digital images were generated using Adobe Photoshop ® software.