Modulation of Retinoic Acid Receptor Alpha Activity by Lysine

Sep 10, 2008 - Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455. Received May 23, 2008. Metabolic ...
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Modulation of Retinoic Acid Receptor Alpha Activity by Lysine Methylation in the DNA Binding Domain M. D. Mostaqul Huq, Sung Gil Ha,* and Li-Na Wei* Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received May 23, 2008

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys347 trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys109 and Lys171 identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys109, but not Lys171, reduces RAdependent activation. Methylation at Lys109 plays a more dominant role than trimethylation at Lys347 in terms of RA activation of the full-length receptor. Lys109 is located in a homologous sequence (CEGCKGFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys109 in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha. Keywords: liquid chromatography-tandem mass spectrometry • retinoic acid receptor • lysine methylation

Introduction Lysine methylation of core histone proteins, one important contributing factor to the “histone code”, is of potential interest in studying chromatin structure and epigenetic regulation of gene transcription.1 Studies have reported several histone lysine methyltransferases (HKMTs) specific to lysine methylation of histone tails.2 HKMTs containing SET domains constitute a family of at least four groups based on structure or sequence similarity. Among these HKMTs, SET9 (also known as SET7) exerts specific histone methyltransferase (HMTase) activity for histone H3-K4, and catalyzes mono- and dimethylation of the substrates.3 SUV39H1, a mammalian homologue of Drosophila position-effect variegation modifier Su(var.)3-9, has H3-K9 methyltransferase activity.4 SET1 and SET2 exert H3-K4 and H3-K36 methyltransferase activity, respectively,5,6 PR-SET7 targets H4-K20,7 and G9a has a ‘dual’ methyltransferase activity for histone H3-K9 and H3-K27.8 Recent studies have also revealed lysine methylation of nonhistone proteins such as p53 and TAF10.8,9 SET7/9 methylates p53, recognizing a conserved K/R-S/T/A motif preceding the lysine substrate.10 In vitro, it also methylates TAF7 at Lys5.10 Additional examples of lysine methylation of nonhistone proteins include trimethylation of retinoic acid receptor alpha (RAR alpha) in its LBD (see below) and automethylation of histone lysine methyltransferase G9a.11,12 * To whom correspondence should be addressed. Li-Na Wei, Professor, Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455-0217. Tel: 1-612625-9402. Fax: 612-625-8408. E-mail: [email protected].

4538 Journal of Proteome Research 2008, 7, 4538–4545 Published on Web 09/10/2008

RARs belong to the nuclear receptor (NR) superfamily and can be activated by retinoic acid (RA) to regulate many genes involved in growth, development, differentiation, and apoptosis.13 RARs are capable of forming homodimer, or heterodimer with retinoid X receptor (RXR, Rxr), to bind to target DNA sequences known as RA response elements (RAREs) and regulate transcription in a ligand dependent fashion. Extensive studies have examined ligand dependent activation of RARs and other NRs.14,15 On the contrary, it is less clear as to how post-translational modification (PTM) affects NR’s ligand sensitivity. Recently, we have reported a mass spectrometric (MS) study that revealed trimethylation at Lys347 located in the ligand binding domain (LBD) of mouse RAR alpha.11 Trimethylation of this residue enhances RA sensitivity of the dissected LBD of RAR alpha, primarily by modulating its protein-protein interaction with coregulators without changing its ligand binding affinity. We extended this MS study and uncovered two monomethylated lysine residues, Lys109 and Lys171, located in the DNA binding domain (DBD) and the hinge region of the receptor, respectively. The current study focused on the role of these two newly found monomethylated residues in modulating the activities of the full-length receptor. We employed a reconstituted culture system to induce hypermethylation by forced expression of two HMTases, SUV39H1 and G9a, to further illustrate the biological significance. The result showed that only monomethylation at Lys109 was important for receptor function with respect to its ligand dependent activation and interaction with coregulators. Sequence alignments of mouse RAR alpha with other RAR subtypes, as well as other NRs in the NR superfamily, revealed that Lys109 is conserved in the 10.1021/pr800375z CCC: $40.75

 2008 American Chemical Society

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Modulation of Retinoic Acid Receptor Alpha Activity DBD of all members of the NR superfamily across the species boundary. This alignment also led us to discover that Lys109 resides in a conserved sequence (CEGCKGFFBRX) where B is Lys/Arg, and X, is either Ser/Thr or Ala within the DBD of NR. Together, this study demonstrates a potential conservation of PTM at a specific residue of the NR superfamily, which may be enforced by genetically conserving a specific amino acid that can be modified and is critical to the function of NR during evolution.

Materials and Methods Plasmids. Full-length Gal4BD-RAR, Gal4V16-RIP140, Gal4V16RXR, and Gal4VP16-PCAF plasmids were as described.11,16,17 The Gal4 fused LBD deleted constructs (Gal4RAR-D-LBD) were generated by single restriction enzyme deletion (between 612 bp and 945 bp) of the full length Gal4-RARs using PvuII. The LBD deleted the fragments were purified and ligated. Mouse wild type G9a and its catalytically inactive mutant G9a,18 and SUV39H1 and its catalytically inactive mutant19 constructs were obtained as generous gift, from Dr. Michael Stallcup (University of Southern California, Los Angeles, CA) and Dr. Janet Stavnezer (University of Massachusetts, Worcester, MA), respectively. Expression, Purification, and Mass Spectrometry of Mouse RAR Alpha. Mouse RAR alpha expression in Sf21 using Baculovirus, purification by affinity chromatography, in-gel trypsin digestion and LC-ESI-MS/MS were as described.11,20,21 The LC system was online with QSTAR Pulsar quadruple time-offlight (TOF) mass spectrometer (MS) (Applied Biosystems, Inc., Foster City, CA), equipped with the Protana’s nanoelectrospray source. We applied an electrospray voltage of 2250 V to the analytical column. The TOF region acceleration voltage was 4 kV and the injection pulse repetition rate was 6.0 kHz. The [M + 3H]3+ monoisotopic peak at 586.9830 m/z and [M + 2H]2+ monoisotopic peak at m/z 879.9705 from human renin substrate tetradecapeptide (Sigma-Aldrich, St. Louis, MO) were for external calibration. As peptides were eluted from the column they were focused into the mass spectrometer. We used information-dependent acquisition (IDA) to acquire MS/MS data with experiments designed such that the three most abundant peptides were subjected to collision-induced dissociation, using argon as the collision gas in every 15 s. We varied collision energies as a function of the m/z and the charge state of each peptide. To avoid continued MS/MS of peptides that had already undergone collision induced dissociation, we incorporated a dynamic exclusion for a further 45 s. IDA mode settings included continuous cycles of the full scan TOF MS from 400-1200 m/z (1.5 s) plus three product ion scans from 50-4000 m/z (3 s each). We selected precursor m/z values from a peak list automatically generated (default) by Analyst QS software v1.0 (ABI) from the TOF MS scans during acquisition, starting with the most intense ion. Protein Sequence Database Search and Manual Verification. We search all MS/MS spectra generated from IDA experiments using MASCOT version 2.2 against the National Center for Biotechnology Information nonredundant (NCBInr) protein sequence database (version 20071110 with 5,622,235 entries) specifying the taxonomy to Mus musculus with 139,509 entries). We set enzyme specificity to trypsin and allowed two missed cleavages. We searched carbamidomethylation as a fixed modification, and considered methionine oxidation and methylation of lysine for variable modifications. Mass tolerance for precursor peptides and MS/MS fragment ions was (1.2 and (0.6 Da, respectively. Search peptide sequence with an expec-

tation value (Mascot search) of less than 0.05 indicated the identity, which generally showed a MASCOT score of greater than 45 against the NCBI nr database (M. musculus). We selected a low cutoff peptide score of 20 to maximize the identification of methylated lysine residues and checked all peptide MS/MS spectra identified with a MASCOT score greater than 20 to verify sequence assignment and to identify the modification sites. We considered peaks with a minimum height of 3% relative to the base peak and used a 100 ppm tolerance to establish matches with the theoretical b and y ions predicted with Bioanalyst software v1.0 (Applied Biosystems). Site-Directed Mutagenesis. We used Quick Change XL sitedirected mutagenesis kit (Stratagene) for site directed mutagenesis, and employing mutagenic primers for replacement of lysine residues with alanine (A) and phenylalanine (F). The mutagenic primers (uppercase letters indicating mutations) were: K109A: sense: 5′-gcc tgt gag ggc tgt GCA ggc ttc ttc cga cga-3′ and antisense: 5′-tcg tcg gaa gaa gcc TGC aca gcc ctc aca ggc-3′; K109F: sense: 5′-gcc tgt gag ggc tgt TTC ggc ttc ttc cga cga-3′ and antisense: 5′-tcg tcg gaa gaa gcc GAA aca gcc ctc aca ggc-3′; K171A: sense: 5′-aag aaa gag gca ccc GCA ccc gag tgc tca gag-3′ and antisense: 5′-ctc tga gca ctc ggg TGC ggg tgc ctc ttt ctt-3′; K171F: sense: 5′ aag aaa gag gca ccc TTC ccc gag tgc tca gag-3′ and antisense: 5′- ctc tga gca ctc ggg GAA ggg tgc ctc ttt ctt-3. We conducted DNA sequencing to confirm the accuracy of mutations. Cell Culture, Transfection, and Reporter Assay. We maintained COS-1 cells in DMEM supplemented with 10% FBS and used Lipofectamine-2000 (Invitrogen) in transient-transfection in 24-well plates with 0.1 µg of Gal4BD-RARs or the control plasmid (Gal4BD empty vector), Gal4-tk-luciferase (0.5 µg) reporter and a CMV-lacZ as an internal control (0.05 µg) per well. Twenty-four h post-transfection, we replaced the medium with a fresh medium containing dextran-charcoal treated (DCC) FBS and treated cultures with either all trans-RA or 9-cisRA (2 µM) for 12 h. We conducted mammalian two hybrid tests in COS-1 cells with a Gal4 reporter, Gal4BD-fused RAR and Gal4VP16-fused PACF, RIP140, or RXR. Thirty-six hours posttransfection, we prepared total cell extracts by freeze-thaw and determined luciferase and lacZ activities, as well as the foldrelative luciferase activity by normalizing RLU (relative luciferase unit) activity of the experimental groups to the RLU activity of the empty vector control group. Metabolic Labeling, IP, and Western Blot. We conducted metabolic labeling of ectopically expressed GFP-tagged RAR alpha with or without methyltransferases in COS-1 cells using (3H)S-adenosyl methionine (SAM, 25 mCi/mL, Sigma). Briefly, we washed the confluent cultures with methionine free DMEM (Gibco) twice, added 3H-SAM (Sigma) directly to the medium and incubated the cultures for 6 h. We then washed the cultures twice with PBS and harvested cell lysates in a co- immunoprecipitation (Co-IP) buffer (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 0.1% NP-40, pH 8.0) by freeze-thaw cycles. To detect the lysine methylation status of RAR alpha in mammalian cells, we immunoprecipitated (IP) GFP-RAR alpha or Gal4-RAR alpha using anti-GFP (BD Sciences), anti-Gal4 (Santa Cruz) or anti-RAR alpha antibody (Affinity Bioreagents), followed by detection with an antimethylated lysine antibody (Stressgen Bioreagents) on the Western blot.

Results Mapping of Methylation Sites by MS/MS Analysis. We expressed and purified the Flag-tagged mouse RAR alpha-full Journal of Proteome Research • Vol. 7, No. 10, 2008 4539

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Table 1. LC-ESI-MS Profile of the Methylated Tryptic Peptide of RAR

m/z (z); M+; RT (min)c Sequence (Score)b

unmodified

modified

∆MS

SSGYHYGVSACEGCK109 (76/54) 168 EAPKPECSESYTLTPEVGELIEK190 (57/44) 340 QDLEQPDKVDMLQEPLLEALK360 (44/34)

554.57 (3), 831.35 (2), 1660.68, 34.07 869.44 (3), 2605.29, 50.34 823.44 (3), 2467.29, 54.51

559.24 (3), 1674.70, 34.40 874.12 (3), 2619.31, 50.58 628.09 (4), 837.12 (3), 2509.30, 51.03

14 14 42

Residue 109

Lsy Lys171 Lys347

95

a Tryptic digests of RAR alpha was subjected to LC-ESI-MS/MS. A full-scan ion chromatogram from m/z 0-1200 was recorded in an information-dependent acquisition (IDA) mode to acquire MS/MS data. The IDA data was searched online at MASCOT (http://www.matrixscience.com) MS/MS Ion search. The MS/MS data was analyzed manually to confirm the sequence of the modified and the unmodified forms of the same peptide identified by the data bank search. The full scan chromatograms were analyzed to assign the charged state, retention time, and intensities of the peptides; 86-156 aa: DNA binding domain (DBD), 230-388 aa: LBD. b Cysteine was modified by idoacetamide; methionine was oxidized; bold letters indicate methylated lysine; values in the parentheses indicate score for unmodified/modifies peptide; c RT, retention time in minute; M+, precursor ion mass.

length in Sf21 insect cells, a widely used eukaryotic culture system for mammalian protein expression. We then employed LC-ESI-MS/MS to analyze tryptic digests of this affinitypurified protein to map the lysine methylation sites (Table 1). We used information-dependent acquisition (IDA) to acquire MS/MS data and searched the data at MASCOT (http:// www.matrixscience.com). The MS/MS Ion search using the search parameters as described (see Methods) confirmed the identity of mouse RAR alpha (accession: gi|116734873, Mowse probability score 981, p < 0.05 for protein identity). Supplementary Table 1 (Supporting Information) provided a comparison to other mouse RARs. Since we expressed recombinant Flag-tagged mouse RAR alpha, we have excluded other variants from the manual MS/MS. Supplementary Table 2 (Supporting Information) provided the number of peptide matched, the sequences of the peptides of mouse RAR alpha, the precursor mass and the charge state. In the total ion chromatogram (TIC), we considered 14, 28, and 42 Da positive mass shifts to account for mono-, di-, and tri- methylation of lysine, respectively. On the basis of the mass shift, we predicted two monomethylated tryptic peptides, one spanning residues 95-109 in the DBD (86-156 aa) and the other spanning residues 168-190 in the hinge region, in addition to a trimethylated peptide spanning 340-360 aa residues located in the LBD (230-388 aa) as reported previously (Table 1). The LC-ESI-MS/MS analysis described before revealed only a 38% sequence coverage of mouse RAR alpha, due to a very low sequence coverage in the amino terminal domain (1-83 aa) of the receptor.11 No lysine residue was present in the uncovered sequence (1-83 aa), ruling out the possibility of missing other methylated lysine residues in the N-terminal domain. We manually analyzed the MS/MS data of the methylated tryptic peptides to assign the methylation sites, aiming to identify lysine methylation sites in other portions of the full length protein besides the reported LBD. We found two peptides in the DBD-hinge region that could be modified by monomethylation. This was based upon, first, a 14 Da positive mass shift, indicating covalent modification by lysine monomethylation, and then, the signature ions such as immonium ions for modified lysine residues. The modified peptide spanning 95-109 aa (Table 1) displayed a triply charged ([M + 3H]+3mono) precursor ion at m/z 559.24 (MWexp) 1674.70 Da, calcd. 1674.68 Da) at 34.40 min in the TIC. The precursor ions of the unmodified version appeared as a triply charged ([M+3H]+3 mono) ion at m/z 554.57 (MWexp ) 1660.68 Da, calcd. 1660.66 Da) and a doubly charged ion at m/z 831.35 (MWexp) 1660.69 Da, calcd. 1660.66 Da) at 34.07 ( 0.07 min. The mass of the precursor ion of the modified peptide indeed showed a + 14.02 Da shift as compared to the 4540

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unmodified peptide, suggesting monomethylation. The collision induced dissociation (CID) of the modified peptide (precursor ion m/z 559.24) provided a better amino acid sequence coverage contributed by both y- ions and b-ions (Figure 1A, top). The modified peptide displayed several singly charged b ions, in particular, b1/a1 through b8/a8 (Figure 1A, top), which were identical to those of the unmodified peptide (Figure 2, bottom), suggesting that the modified site was located in the sequence between b9 and b15. Each y ion such as y2* (y2-NH3, m/z 304.13), y3 (m/z 378.18) through y10 (m/z 1144.47), and a doubly charged y14 ion (m/z 794.82) of the modified peptide spectrum (Figure 1A, top), showed a + 14 Da shift relative to those derived from the unmodified peptide (Figure 1A, bottom). This narrowed down the site of modification to Cys108 or Lys109. Since Cys108 could be modified by carbamidomethylation, the only location for monomethylation was at Lys109. The modified peptide spanning 168-190 aa (Table 1) displayed a triply charged ([M + 3H]+3 mono) precursor ion at m/z 874.12 (MWexp) 2619.31 Da, calcd. 2619.26 Da) at 50.57 min in the TIC. The precursor ion of the unmodified peptide appeared as a triply charged ([M+3H]+3 mono) ion at m/z 869.44 (MWexp) 2605.29 Da, calcd. 2605.25 Da) at 50.34 min. The mass of the precursor ion of the modified peptide also showed a + 14.02 Da (calcd. 14.01 Da) mass shift as compared to the unmodified peptide, suggesting monomethylation. The MS/ MS analysis by CID of the precursor ion (m/z 874.12) of the modified peptide (Figure 1B, top) yielded y1 ion and y3 through y10, each of which corresponded to the unmodified peptide (Figure 1B, bottom). Further, several b/a ions, b1/a1 through b3/a3 were identical. The singly charged a8* (m/z 868.38) and b9 (m/z 1042.45) ions, and the doubly charged ([M + 2H]+2mono) b12 ion (m/z 697.3) each demonstrated a + 14 Da shift as compared to the unmodified peptide. This suggested that modification might be located within the internal peptide (171KPECS175) between b4 and b8. We considered the intense ion at m/z 115.086 as an immonium ion for monomethylated lysine. The C-terminal Lys190 was unmodified and Cys174 could be modified by carbamidomethylation; therefore, we concluded that monomethylation occurred at Lys171. Effects of Lysine Monomethylation on RAR Alpha Activity. With regards to biological effects, we first examined the role of monomethylation at Lys109 and Lys171 on RA-dependent activation of Gal4-fused full-length RAR alpha (Figure 2). We evaluated Lys/Ala mutated Gal4-RAR alpha full-length, K109A and K171A, in trans-activation assays using a standard COS-1 system. RA activated, to a similar extent, the wild type and the K171A mutant (Figure 2A), but failed to do so for the K109A mutant. Mutation of these lysine residues each into a glutamine (Lys/Gln) also produced similar results (not shown). This

Modulation of Retinoic Acid Receptor Alpha Activity

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Figure 1. Mapping of lysine monomethylation on mouse RAR alpha by LC-ESI-MS/MS. (A) The modified peptide (95-109 aa, top) showed precursor ion at m/z 559.24 (z ) 3) and the unmodified peptide (bottom) displayed precursor m/z 554.57 (z ) 3) and m/z 831.35 (z ) 2). The mass of the precursor ion of the modified peptide showed a +14.02 Da shift as compared to the unmodified peptide, suggesting a covalent modification by a single monomethylation. The MS/MS of modified peptide (m/z 559.24) showed b1/a1 through b8/a8 ions (top) identical to those of the unmodified peptide (bottom). The y ions such as y2* (y2-NH3, m/z 304.13), and y3 (m/z 378.18) through y10 (m/z 1144.47), and a doubly charged y14 ion (m/z 794.82) of the modified peptide spectrum (top), showed + 14 Da shift relative to those derived from the unmodified peptide (bottom), suggesting monomethylation of Lys109. (B) The modified peptide spanning 168-190 aa appeared at m/z 874.12 (z ) 3) and the precursor ion of the unmodified peptide appeared at ion at m/z 869.44 (z ) 3). There was + 14.02 Da difference between the mass of the precursor ion of the modified and unmodified peptides, suggesting modification by monomethylation. The collision induced dissociation (CID) of the precursor ion (m/z 874.12) of the modified peptide (top) showed y1 ion as well as y3 through y10 and several b/a ions, b1/a1 through b3/a3, which were identical to the unmodified peptide (bottom). The singly charged a8* (m/z 868.38) and b9 (m/z 1042.45) ions, and the doubly charged ([M + 2H]+2mono) b12 ion (m/z 697.3) demonstrated a + 14 Da shift, suggesting monomethylation of Lys171. Underlined mass values over the peptide sequence indicate that the fragment ions appeared as doubly charged ions.

suggested that monomethylated Lys109, but not Lys171 was important for the function of RAR alpha. Specifically, methylation at Lys109 positively modulated RA-dependent activation of RAR alpha. We then conducted sequence alignment of mouse RAR alpha to other mouse RAR subtypes, and to the entire NR super family across species (Supplementary Table 3, online). Intriguingly, Lys109 is conserved not only among the RAR subtypes, but also within the NR super family across species. In fact, Lys109 of RAR alpha is located in a highly conserved sequence CEGCKGFFBRX, where B is either K or R, and X is either S/T or A, of the DBD (Supplementary Table 3, Supporting Information). We previously reported that by increasing site specific hydrophobicity, such as replacing a target residue with a bulky hydrophobic amino acid residue phenylalanine (Phe), one could generate proteins mimicking trimethylation at lysine or monomethylation at arginine.11,22 In these earlier mutation studies, the Lys/Phe (K347F) mutated mouse RAR alpha LBD behaved like a wild type LBD trimethylated at Lys347, whereas the Lys/Ala or Lys/Gln mutants each behaved like a constitutive negative mutant. We then used the same strategy to generate a methylmimetic protein by replacing these two lysine residues (Lys109 and Lys171) each with a phenylalanine in the full-length

receptor. Surprisingly, the Lys/Phe (K109F) mutant receptor remained as a constitutive negative receptor (Figure 2A), suggesting that while site-specific hydrophobicity can mimic trimethylation at lysine or monomethylation at arginine, it may not mimic monomethylation at lysine. The fact that this mutant retains its constitutive trans-repressive activity suggests that this point mutation has not drastically altered its conformation such that it rendered the mutant a nonfunctional receptor. However, there might be complication from its effects on certain properties of the receptor such as DNA- or chromatin binding (see Discussion). We then attempted to examine the possible coordination between lysine mono-methylation at Lys109 and lysine trimethylation at Lys347 (Figure 2B) in modulating the activity of RAR alpha. We incorporated the constitutive negative mutation K109A into the methylmimetic mutant K347F, or the constitutive negative mutant K347A, to generate double mutants. If the effect of lysine monomethylation at Lys109 (in the DBD) was independent of the effect of lysine trimethylation at Lys347 (in the LBD), the incorporation of the constitutive negative lysine mutation (K109A) should not alter the biological activity of the methylmimetic receptor (K347F mutant). On the contrary, if there was any synergy or interdependency between lysine Journal of Proteome Research • Vol. 7, No. 10, 2008 4541

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Figure 2. Role of Lys109 methylation on RAR alpha activity. (A) Transactivation assay using Gal4-RAR-full length on a Gal4BDLuc reporter was conducted in COS-1 cells. Protein expression level of the wild type and the mutant receptors detected by Western blot using anti-Gal4 antibody was shown below. (B) Transactivation assay of Lys109 mutation cpmpared to that of the negative methylated LBD mutant (K347A) and hypermethylationmimetic mutant (K347F).

monomethylation of DBD and lysine trimethylation of LBD, the activity of the double mutant will be affected. The data showed that mutation at Lys109 rendered RAR alpha totally insensitive to RA, regardless of the trimethylation status of LBD at Lys347. This result suggests that while lysine trimethylation at Lys347 could enhance the ligand sensitivity of the isolated LBD domain, lysine monomethylation at Lys109 plays a significant, or even more dominant, role in the activation of the fulllength receptor. In vivo Methylation of RAR Alpha. To confirm the methylation status of RAR-full length protein in mammalian cells, we conducted metabolic labeling experiments in COS-1 cells using tritium labeled S-adenosyl methionine (3H-SAM) (Figure 3A). Metabolic labeling of ectopically expressed GFP-tagged RAR-full length protein followed by immunoprecipitation (IP) with anti-GFP antibody and autoradiography revealed that RAR alpha indeed was methylated in mammalian cells (Figure 3A). Since the LBD of RAR alpha could be trimethylated in mammalian cells,11 we then compared in vivo methylation of fulllength wild type and mutant receptors. Indeed, Lys347 mutant receptor could still be methylated, but at a much lower level as compared to the wild type protein (Figure 3B, lane 2 and 3), suggesting the existence of methylated sites other than Lys347 on the full-length protein. In fact, the level of methylation on the double mutant (K109A/K347F) was slightly lower than the single mutant (Figure 3B, lanes 2-4), supporting Lys109 methylation in mammalian cells. Based upon the signals on 4542

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Figure 3. In vivo methylation of RAR-full length protein. (A) Metabolic labeling of ectopically expressed GFP-tagged wild type RAR alpha followed by immunoprecipitation (IP) using anti-GFP in COS-1 cells. The Western blot with methylated lysine specific antibody demonstrated lysine methylation of RAR in mammalian cells. (B) Immuno-detection of methylated lysine on wild type and mutated receptor. Input controls were shown at the bottome of both panels.

the immunoblot, a very weak residual methylated fraction remained detectable for the double mutant protein, suggesting, perhaps, the existence of other residues that could be methylated on RAR alpha in mammalian cells. This remains to be confirmed. Effect of Lysine Monomethylation of RAR Alpha on Its Interaction with Coregulators. We also assessed the effect of Lys109 monomethylation on ligand-dependent full-length receptor interaction with its coregulators, p300/CBP-associated factor (PCAF) and receptor interacting protein 140 (RIP140, gene: Nrip1) in mammalian two-hybrid tests. As shown in Figure 4, K109A mutation drastically reduced RA-dependent interaction of receptor with both PCAF (Figure 4A) and RIP140 (Figure 4B), regardless of its methylation status at Lys347. This observation indicates that methylation at Lys109 is essential for ligand-dependent interaction of the full-length receptor protein with its coregulators, and it plays a more dominant role than trimethylated Lys347 in the context of the full-length protein. Lysine Methylation and Heterodimerization. RAR can form heterodimer with RXR to regulate RA-target gene expression. Therefore, we tested whether monomethylation on RAR alpha could affect its heterodimerization with RXR (Figure 5). In a

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Modulation of Retinoic Acid Receptor Alpha Activity

Figure 4. Effect of methylation at Lys109 on cofactor interaction. Ligand-dependent interaction with PCAF (A) and RIP140 (B) was each tested in mammalian two hybrid assays in COS-1 cells using Gal4BD-RAR as bait and Gal4VP16-PACF (AD-PCAF) or Gal4VPRIP140 (AD-RIP140) as prey.

and TAF7, each on a conserved sequence. Since the sites of lysine methylation on RAR alpha do not resemble the conserved consensus target sequence of SET9/7, we then tested two other SET domain-containing HKMTases, mouse G9a and SUV39H1, by simply asking if these two enzymes could change the methylation status of RAR alpha in vivo (in mammalian cells) (Figure S1, Supporting Information). We used anti-Gal4 antibody to precipitate Gal4-RAR alpha and detected the precipitated protein with the methylated lysine-specific antibody. The results showed that G9a indeed enhanced the level of lysine methylation of RAR alpha by ∼ 2 fold, and SUV39H1 only slightly enhanced the level of methylation, after normalization to the control (MeK-RAR vs total RAR) as shown in the densitometric analysis (Figure S1, right, Supporting Information). Further, to confirm the enhancement of lysine methylation of RAR alpha by G9a, metabolic labeling of Gal4-RAR with 3H-S-adenosyl methionine (3H-SAM) was conducted in the presence or absence of wild type G9a or its catalytically inactive mutant, as well as SUV39H1 or its catalytically inactive mutant (Figure S1, online). IP with anti-Gal4 antibody followed by autoradiography showed that only the wild type G9a (lane 3), but not the catalytically inactive mutant G9a (lane 4), could moderately enhance the methylation level of wild type RAR alpha. On the other hand, SUV39H1 exerted little effect on RAR alpha lysine methylation (lane 4). This suggests that G9a could possibly play a role, at least partially, in lysine methylation of RAR alpha. However, it remains to be confirmed whether RAR alpha is a direct substrate for lysine methylation by G9a. We then further examined the effects of these two enzymes (G9a and SUV39H1) on RA activation of the receptor (Figure S2, Supporting Information). We coexpressed the wild type, or the K109A mutant, with either SUV39H1 or G9a, and monitored RA activation of the reporter. It appeared that G9a could increase the basal transcription (without RA) of both wild type and K109A mutant RAR alpha to some extent, which probably was due to reported intrinsic activity of G9a as a coactivator for certain nuclear receptors.18 It appeared that G9a enhanced RA activation of the wild type RAR alpha, but not the K109A mutant. However, due to complication from the enhancement of the basal transcriptional activity, it remains to be determined whether there was a specific enhancement on RA activation triggered by G9a in this reporter system. Very differently, SUV39H1 exerted little effect on RA activation (Figure S2, Supporting Information). Further investigation is needed to differentiate the potential contribution of G9a as a coactivator or as a moderate lysine methyltransferase for RAR alpha.

Discussion

Figure 5. Effect of lysine methylation on RA-independent heterodimerization with RXR detected in a mammalian two-hybrid assay in COS-1 cells.

mammalian two hybrid protein interaction test, all mutants containing K109A mutation (K109A, K109A/K347A, and K109A/ K347F) had much reduced levels of 9-cisRA dependent heterodimerization with RXR. This suggests that monomethylation at Lys109 of RAR alpha is also important for promoting its heterodimerization process. Effect of HKMTase on RAR Alpha Activity. Set7/9 could methylate lysine residues of transcription factors p53, TAF10,

The primary sequence of a protein is predictable by the genetic code. However, the number of protein variants from the same gene is unpredictable because of combinatorial effects of different PTMs. This expands the functional diversity of a single protein dictated from a single gene. PTMs, such as phosphorylation, acetylation, methylation, and glycosylation, can all regulate protein functions and play important roles in multiple cellular processes including DNA repair, protein stability, nuclear translocation, protein-protein interactions, cellular proliferation, differentiation, and apoptosis.23-25 However, it remains a challenge to identify and confirm PTMs occurring in vivo. In this study, we applied LC-MS/MS to map potential lysine methylation sites on mouse RAR alpha expressed and purified from Sf21 cells. While studies have shown stoichiometric variation of PTMs between insect and mamJournal of Proteome Research • Vol. 7, No. 10, 2008 4543

research articles malian cells, the number of potential PTM sites appeared to be similar for the same protein in most cases.26-28 Therefore, we expected that lysine methylation sites on RAR alpha expressed in insect cells could also occur in mammalian cells. MS analysis revealed lysine methylation at Lys109, Lys171, and Lys347, and site directed mutagenesis validated the functionality of Lys109 and Lys347 with respect to RA activation of the receptor. Although mutation at Lys171 did not have a significant effect in this regard, it is still possible that it might have affected other properties of the receptor. This awaits further examination. Among the three lysine methylation sites on RAR alpha, monomethylated Lys109 and Lys171 are in the DBD and the hinge region, respectively, whereas trimethylated Lys347 is located in the LBD. In our previous report, we used the dissected LBD to address the functional role of methylated Lys347 in the dissected LBD to avoid the potential interference from the N-terminal domain and the DBD. Interestingly, in studying the effect in the context of the full-length protein as shown in this current study, we found that Lys109 in fact played an even more dominant role than Lys347 (Figure 2). While ligand sensitivity of NRs attributes, primarily, to the LBD, the N-terminal domain and/or the DBD can also modulate ligand sensitivity in the context of the full-length receptor as supported by the results shown here. It is quite interesting that Lys109 is conserved not only among RAR subtypes, but also within the NR super family and across the species. Lys109 of mouse RAR alpha is located in a highly conserved sequence CEGCKGFFBRX, where B is either K or R, and X is either S/T or A, of the DBD (Supplementary Table 3 online). It would be important to determine whether methylation also occurs at this conserved lysine in other NRs, and if it similarly modulates other NRs’ activation by other ligands. A previous study showed that mutation at the DBD of glucocorticoid receptor affected its transcriptional activity.29 Another study showed that mutation destabilizing dimer interface of the DBD of steroid receptors also affected their transcriptional activity of the receptors.30 Whether mutation at Lys109 of RAR affects the activity more directly associated with its DNA- or chromatin-binding, due to its close proximity to the DBD, will be examined in the future. Another interesting observation is the different effect of the two HKMTases on basal reporter and ligand-induced transactivating activity, which indicates that these two enzymes may differentially affect transcriptional machinery or the chromatin. Nevertheless, both enzymes exert little effect on transactivation of the Lys109 mutant, further supporting that methylation at Lys109 does modulate the transactivating activity of receptor. Studies have reported phosphorylation and intramolecular stabilization of the LBD in steroidogenic factor 1 (SF1).29 Phosphorylation at Ser203 located in the N-terminus to the LBD of SF-1 enhanced cofactor recruitment, which was analogous to ligand-induced recruitment of cofactors. The study showed that the SF-1 LBD adopted an active conformation with helices 1 and 12 packed against the predicted R-helical bundle in the apparent absence of ligand. Limited proteolyzes demonstrated that phosphorylation of Ser203 in the hinge region mimicked the stabilizing effects of ligand on the LBD. In addition, activation domains AF-1 and AF-2 cooperated in RA-induced proteosomal degradation of RAR gamma 2.30 In this cast, the AF-2 of RA-liganded holo-receptor acted through the recruitment of the proteasomal SUG-1 subunit when the AF-1 was phosphorylated at Ser66/Ser68 by p38MAPK. Possibly, methylation on the DBD and LBD as shown in mouse RAR alpha 4544

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Huq et al. might also stabilize the holo-form of the receptor. More studies would help to evaluate this possibility. Abbreviations: RAR, retinoic acid (RA) receptor; RXR, retinoid X receptor; LBD, ligand binding domain; DBD, DNA binding domain; RIP140, receptor interacting protein 140; PCAF, p300/CBP-associated factor; PKMT, protein lysine methyltransferase; HKMT, histone lysine methyltransferase; SF1, steroidogenic factor 1.

Acknowledgment. This work was supported by NIH grants DA11190, DA11806, DK54733, DK60521, K02-DA13926 to L.-N. Wei. The authors wish to thank Dr. LeeAnn Higgins at the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for recording the mass spectra and cordial assistance in database search. We also thank Dr. Michael Stallcup and Dr. Janet Stavnezer for the generous gifts of the lysine methyltransferases constructs. Supporting Information Available: Database search for MS/MS spectra, protein identity, and sequence alignments for nuclear receptors are included in the Supplementary Tables. Supplemental Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Mikkelsen, T. S.; Ku, M.; Jaffe, D. B.; Issac, B.; Lieberman, E.; Giannoukos, G.; Alvarez, P.; Brockman, W.; Kim, T. K.; Koche, R. P.; Lee, W.; Mendenhall, E.; O’Donovan, A.; Presser, A.; Russ, C.; Xie, X.; Meissner, A.; Wernig, M.; Jaenisch, R.; Nusbaum, C.; Lander, E. S.; and Bernstein, B. E. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 2007, 448, 553– 560. (2) Kouzarides, T. Histone methylation in transcriptional control. Curr. Opin. Genet. Dev. 2002, 12, 198–209. (3) Kwon, T.; Chang, J. H.; Kwak, E.; Lee, C. W.; Joachimiak, A.; Kim, Y. C.; Lee, J.; Cho, Y. Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet. EMBO J. 2003, 22, 292–303. (4) Mal, A. K. Histone methyltransferase Suv39h1 represses MyoDstimulated myogenic differentiation. EMBO J. 2006, 25, 3323–3334. (5) Schlichter, A.; Cairns, B. R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains. EMBO J. 2005, 24, 1222–1231. (6) Rao, B.; Shibata, Y.; Strahl, B. D.; Lieb, J. D. Dimethylation of histone H3 at lysine 36 demarcates regulatory and nonregulatory chromatin genome-wide. Mol. Cell. Biol. 2005, 25, 9447–9459. (7) Karachentsev, D.; Sarma, K.; Reinberg, D.; Steward, R. PR-Set7dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis. Genes Dev. 2005, 19, 431–435. (8) Kouskouti, A.; Scheer, E.; Staub, A.; Tora, L.; Talianidis, I. Genespecific modulation of TAF10 function by SET9-mediated methylation. Mol. Cell 2004, 14, 175–182. (9) Chuikov, S.; Kurash, J. K.; Wilson, J. R.; Xiao, B.; Justin, N.; Ivanov, G. S.; McKinney, K.; Tempst, P.; Prives, C.; Gamblin, S. J.; Barlev, N. A.; Reinberg, D. Regulation of p53 activity through lysine methylation. Nature 2004, 432, 353–360. (10) Couture, J. F.; Collazo, E.; Hauk, G.; Trievel, R. C. Structural basis for the methylation site specificity of SET7/9. Nat. Struct. Mol. Biol. 2006, 13, 140–146. (11) Huq, M. D.; Tsai, N. P.; Khan, S. A.; Wei, L. N. Lysine trimethylation of retinoic acid receptor-alpha: a novel means to regulate receptor function. Mol. Cell. Proteomics 2007, 6, 677–688. (12) Chin, H. G.; Este`ve, P. O.; Pradhan, M.; Benner, J.; Patnaik, D.; Carey, M. F.; Pradhan, S. Automethylation of G9a and its implication in wider substrate specificity and HP1 binding. Nucleic Acids Res. 2007, 35, 7313–7323. (13) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz, G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. The nuclear receptor superfamily: the second decade. Cell 1995, 83, 835–839. (14) Chambon, P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996, 10, 940–954.

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(25) McBride, A. E.; Silver, P. A. State of the arg: protein methylation at arginine comes of age. Cell 2001, 106, 5–8. (26) Cheng, X.; Hart, G. W. Glycosylation of the murine estrogen receptor-R. J. Steroid Biochem. Mol. Biol. 2000, 75, 147–148. (27) Wu, R. C.; Qin, J.; Yi, P.; Wong, J.; Tsai, S. Y.; Tsai, M. J.; O’Malley, B. W. Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic responses to multiple cellular signaling pathways. Mol. Cell 2004, 15, 937–949. (28) Gupta, P.; Huq, M. D.; Khan, S. A.; Tsai, N. P.; Wei, L. N. Regulation of co-repressive activity of and HDAC recruitment to RIP140 by site-specific phosphorylation. Mol. Cell. Proteomics 2005, 4, 1776– 1784. (29) Ramos, R. A.; Meilandt, W. J.; Wang, AE. C.; Firestone, G. L. Dysfunctional glucocorticoid receptor with a singlepoint mutation ablates the CCAAT/enhancer binding protein-dependent growth suppression response in a steroid-resistant rat hepatoma cell variant. FASEB J. 1999, 13, 169–180. (30) Liu, W.; Wang, J.; Yu, G.; Pearce, D. Steroid Receptor Transcriptional Synergy Is Potentiated by Disruption of the DNA-Binding domain dimer interface. Mol. Endocrinol. 1996, 10, 1399–1406. (31) Desclozeaux, M.; Krylova, I. N.; Horn, F.; Fletterick, R. J.; Ingraham, H. A. Phosphorylation and intramolecular stabilization of the ligand binding domain in the nuclear receptor steroidogenic factor 1. Mol. Cell. Biol. 2002, 22, 7193–7203. (32) Giannı`, M.; Bauer, A.; Garattini, E.; Chambon, P.; Rochette-Egly, C. Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RAR gamma degradation and transactivation. EMBO J. 2002, 21, 3760–3769.

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