Analysis of Phosphorylation-Dependent ProteinâProtein Interactions

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Analysis of Phosphorylation-Dependent Protein−Protein Interactions of Histone H3 Rebecca Klingberg,† Jan Oliver Jost,†,‡ Michael Schümann,§ Kathy Ann Gelato,∥ Wolfgang Fischle,∥ Eberhard Krause,§ and Dirk Schwarzer*,†,‡ †

Laboratory of Protein Chemistry and §Mass Spectrometry, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany ‡ Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany ∥ Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany S Supporting Information *

ABSTRACT: Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from L- and D-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylenalanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.

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methylation have been intensively studied, histone phosphorylation is still poorly understood.1−3,8 The N-terminal tail of histone H3 possesses five phosphorylation sites (T3, T6, S10, T11, and S28) and H3S10ph is the most abundant of these PTM marks (Figure 1a).9,10 H3S10ph occurs globally along the chromosomes during mitosis, but it is also associated with restricted chromatin regions of active transcription during interphase.10,11 In agreement with the histone code theory, binding proteins of H3S10ph have been identified: three members of the 14-3-3 family of proteins were found to bind H3S10ph preferentially in combination with acetylation of H3K9ac or H3K14ac.12−14 It was further shown that this interaction is important for active transcription of the histone deacetylase 1 (HDAC1) gene.13 In addition to constituting binding sites for regulatory proteins, H3S10ph might also suppress interactions between binding proteins and histone tails. Such inhibitory function has been reported for the cross-talk of H3S10ph and the heterochromatin binding protein 1 (HP1). HP1 binds

osttranslational modifications (PTMs) are central means of regulating structure, function, and activity of proteins. Histones package eukaryotic genomes into chromatin and represent prime examples of extensively modified proteins.1−4 The four core histones (H2A, H2B, H3, and H4) are basic proteins. These form an octamer complex with 147 bp of DNA wrapped around.5 The structure is referred to as nucleosome core particle and constitutes the basic repetitive element of chromatin. PTMs are primarily found on the N-terminal tail regions of all four core histones. These are not part of the protein scaffold but protrude from the nucleosomal cores outward into the nucleus. Histone PTMs are known to regulate the activity of genes. A “histone code theory” has been proposed that interprets the histone tails as programming platforms where specialized enzymes index defined chromatin areas by restricted modification.2,6 The histone code is “read” by recruitment of regulatory proteins that bind patterns of PTM marks and direct the function of DNA associated with specifically modified areas of chromatin.7 Finally, enzymes that remove PTM marks reestablish the unmodified state of the histone tails and serve as erasers of the histone code. Among the early discovered histone PTMs is phosphorylation of the amino acids serine and threonine, which have been known since the 1960s.8 While histone acetylation and © XXXX American Chemical Society

Received: July 16, 2014 Accepted: October 20, 2014

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dx.doi.org/10.1021/cb500563n | ACS Chem. Biol. XXXX, XXX, XXX−XXX

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control probes will greatly facilitate the identification of specific binding proteins of unmodified histone tails. Here we report the development of a strategy for simultaneous analysis of proteins that are recruited and excluded from histone tails by PTMs based on the SILAC approach. We have applied this strategy to study the effects of H3S10ph. The H3 tail synthesized from D-amino acids was introduced as control for unspecific binders of unmodified H3. Differential pull-down experiments with histone tails synthesized from L- and D-amino acids, as well as with a nonhydrolyzable mimic of phosphorylated serine residues at H3S10ph, identified six isoforms of 14-3-3 as specific binders of H3S10ph. Histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) were uncovered as specific binders of unmodified H3. Both proteins were excluded from L-H3 by phosphorylation. Based on these findings, we propose a model for possible functions of the cross-talk between the H3 tail and HAT1/RBBP7.

Figure 1. Phosphorylation of histone H3. (a) Histone H3 is composed of a structured globular domain and a disordered N-terminal tail that possesses five phosphorylation sites at T3, T6, S10, T11, and S28. (b) Histone H3 derived peptide baits. Phosphonomethylene alanine (Pma) mimics the biophysical properties of phospho-serine but cannot be hydrolyzed by PPs. BP, binding protein; PP, protein phosphatase.

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RESULTS AND DISCUSSION Strategy. We aimed to establish an improved strategy to study the effects of H3S10ph on the binding properties of the N-terminal tail of histone H3 based on the SILAC approach. This approach should include proteome-wide screens for specific binding proteins of H3S10ph as well as specific binding proteins of unmodified H3 that are blocked by H3S10ph. To this end, we resorted to synthetic histone tails as baits and controls.8,20−22 We have previously encountered that changes of the net charge of histone tails caused by PTMs have major effects on the interaction profiles of unspecific binders.25 Since phosphorylation alters the net charge of H3-tail peptides, we considered that a simple differential pull-down of an H3S10ph bait versus an unmodified H3 control peptide will not discriminate between specific binding of H3 that is blocked by H3S10ph and unspecific interactions that are modulated by different net charges of bait and control. Hence, a new control peptide needs to be established that validates the specificity of the interaction between any protein and the unmodified H3tail. To this end, we considered that specific interactions should depend on the native L-configuration of amino acids constituting the binding sites. D-Peptides possess the same net charge and charge distribution as the corresponding Lpeptides, and therefore D-peptides should represent stringent controls for the specificity of interactions between proteins and L-peptide baits. Hence, we introduced H3 tails synthesized from D-amino acids as control for unspecific binders. In addition, we intended to analyze whether this approach can be extended to the erasers of the phosphorylation code, in this case the protein phosphatases (PPs) that remove H3S10ph. Phosphorylated peptides cannot be used as baits for PPs because PPs will hydrolyze the phosphate groups. However, nonhydrolyzable mimics of phosphorylated serine, phosphonates, have been reported as probes for phosphorylationspecific binding proteins and as PP inhibitors (Figure 1b and 2).26−33 Collectively, our strategy is based on three synthetic H3derived peptide baits: the histone tail synthesized from L-amino acids (L-H3) and D-amino acids (D-H3), which should allow the identification of specific binders of the H3 tail in unmodified state in differential pull-down experiments. The third bait is also synthesized from L-amino acids and contains the nonhydrolyzable phospho-serine mimic phosphonomethylenealanine (Pma) instead of S10 (H3Pma10). H3Pma10 should

methylated lysine 9 of H3 (H3K9me3) via its chromodomain, and this interaction is inhibited by simultaneous, neighboring H3S10ph.15−17 Recently, H3S10ph was also shown to block writers of the histone code by inhibiting the kinases that phosphorylate H3T6 and H3T11.18 Mass spectrometry is among the most widely used tools for investigating the interactions between proteins and PTMs.19 Due to their small size, histone tails can be synthesized by solidphase peptide synthesis in the desired modification state. These are used as baits for specific enrichment of interacting factors in complex mixtures such as cellular lysates.8,20−22 Captured proteins copurify with the baits and can be analyzed by capillary liquid chromatography−tandem MS after tryptic digestion. In contrast to Western blotting, MS-based approaches can easily uncover multiple binding proteins in a single experiment. However, the high sensitivity of mass spectrometry makes it difficult to discriminate between background proteins and those of physiological relevance. Quantitative mass spectrometry using “stable isotopic labeling with amino acids in cell culture” (SILAC) can be applied to solve this problem.23,24 The SILAC approach allows a quantitative comparison of proteins pulleddown by modified histone tails and unmodified control peptide that does not facilitate specific binding. Several PTMdependent binding proteins of histone tails have already been identified by the SILAC approach.20 The synthetic histone tail peptides used in these approaches commonly differ in the modification of interest only, which is important because the biophysical properties of bait and control are similar. Histone tails are highly charged and attract several unspecific binding proteins by electrostatic interactions. When the net charge of bait and control is similar, unspecific binders can be distinguished from specific PTM interaction partners by similar enrichment rates on both peptides. However, when we adapt this strategy for identifying specific binding proteins of unmodified histone tails, the unmodified histone tail peptides are used as baits. Consequently, new control peptides need to be established that possess similar charge and charge distribution as the unmodified bait but do not enable specific interactions. The development of such B



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3-3ζ bound efficiently to H3Pma10 and H3S10ph but was recovered on L-H3 to a much lesser extent (Figure 2). These observations indicated that H3Pma10 is a functional substitute of H3S10ph for phospho-serine binding 14-3-3 proteins. Proteomics. In order to study the consequences of H3Pma10 on the binding properties of the H3 tail on a proteomic scale, we used quantitative mass spectrometry based on SILAC.23 Swiss 3T3 cells were cultivated in “heavy”, “medium”, or “light” media, supplied with the corresponding isotopically labeled amino acids lysine and arginine (Figure 3a). H3Pma10, L-H3, and D-H3 were separately incubated with equal amounts of nuclear extracts (H3Pma10 → “heavy”, L-H3 → “medium”, and D-H3 → “light”). Afterward bound proteins were eluted, combined, and separated on 1D SDS-PAGE. After tryptic in-gel digestion, peptides were analyzed and quantified by nLC-ESI-MS/MS (Figure 3a). The experiment was performed twice (SILAC1 and SILAC2) with different batches and amounts of Swiss 3T3 nuclear extract (3T3 NE) (Supplementary Tables 1 and 2, Supporting Information). Selected proteins that show high heavy (H)/medium (M) (specific binders of H3Pma10) and medium (M)/light (L) (specific binders of L-H3) isotope ratios are summarized in Table 2. We considered only proteins that were quantified on the basis of at least three unique peptides in both experiments. Binding Proteins of H3Pma10. Six proteins showed enrichments on H3Pma10 with high H/M isotope ratios in SILAC1 and SILAC2, while all other proteins displayed a significantly lower enrichment (Figure 3b). These six proteins belong to the 14-3-3 family of phospho-serine/threoninebinding proteins including the previously reported H3S10phbinders 14-3-3γ, 14-3-3ε, and 14-3-3ζ. Three other members of the 14-3-3 family, which had not been described in this context, were also identified: 14-3-3θ, 14-3-3η, and 14-3-3β (Table 2).12−14 We confirmed these findings with selected 14-3-3 isoforms and HeLa nuclear extract (HeLa NE) as input. Immobilized H3S10ph, H3T3ph, H3T6ph, H3T11ph, and H3S28ph, as well as L-H3 and D-H3 served as baits. Efficient pull-down of tested 14-3-3 isoforms (14-3-3β, 14-3-3γ, and 14-3-3ζ) by H3S10ph and H3S28ph was observed by Western blot analysis (Figure 4a). Pull-downs with H3S28ph showed higher enrichments of 14-3-3 proteins than H3S10ph, which is consistent with previous reports.12−14 None of the phosphorylated threonine residues mediated binding of the 14-3-3 proteins, although 143-3 proteins are known to bind such phosphorylation sites as well. The second amino acid downstream of the phosphorylation site (P + 2) is critical in most 14-3-3 binding sites, and proline residues are strongly favored at this position.34 In the

Figure 2. Phosphonomethylene alanine (Pma) mimics phosphorylated serine residues in the context of the H3 tail. The known binding protein of H3S10ph 14-3-3ζ is efficiently pulled down by the H3Pma10 bait but not by L-H3 (input, 1.8 μM recombinant 14-3-3ζ and 500 μM BSA in 20 μL). H3Pma10 also binds endogenous 14-3-3ζ from Swiss 3T3 nuclear extracts. Coomassie Brilliant Blue stained SDS-PAGE of pull-down experiment with recombinant 14-3-3ζ (top). Western blot analysis of endogenous 14-3-3 pulled-down from 3T3 NE (bottom).

trap binding proteins of H3S10ph and potentially the corresponding PPs. Substrate Synthesis and Validation. Synthetic H3 tails covering residues 1−33 of H3 were synthesized from conventional building blocks of L- and D-amino acids (L-H3 and D-H3) by solid-phase peptides synthesis. Commercially available L-Pma was Fmoc-protected and subsequently incorporated into H3Pma10. In addition, we synthesized H3 tails containing each of the known phosphorylation sites in modified form: H3S10ph, H3T3ph, H3T6ph, H3T11ph, and H3S28ph (see Table 1). All peptide baits were immobilized on iodoacetamide-conjugated agarose matrix via a C-terminal cysteine. First, we validated the ability of H3Pma10 to mimic the natural phosphorylation state. Recombinant 14-3-3ζ was efficiently enriched on H3Pma10 and H3S10ph but not on LH3 (Figure 2). Encouraged by these findings, we also performed pull-down experiments with nuclear extracts from a murine fibroblast cell line (Swiss 3T3 cells) as input. Western blot analyses showed a similar binding pattern: endogenous 14Table 1. Amino Acid Sequence of All Peptide Baits Useda name D-H3 L-H3

H3Pma10 H3S10ph H3T3ph H3T6ph H3T11ph H3S28ph

modification

sequence

D amino acids none Pma phospho-serine phospho-threonine phospho-threonine phospho-threonine phospho-serine

artkqtarkstGGkaprkqlATkaarksapatGGc ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGC ARTKQTARKPmaTGGKAPRKQLATKAARKSAPATGGC ARTKQTARKpSTGGKAPRKQLATKAARKSAPATGGC ARpTKQTARKSTGGKAPRKQLATKAARKSAPATGGC ARTKQpTARKSTGGKAPRKQLATKAARKSAPATGGC ARTKQTARKSpTGGKAPRKQLATKAARKSAPATGGC ARTKQTARKSTGGKAPRKQLATKAARKpSAPATGGC

a D-Amino acids are written with lowercase letters. Two amino acids (A21 and T22) of D-H3 were introduced as L-amino acids because they needed to be introduced as Fmoc-Ala-Thr(ΨMe,Mepro)-OH pseudoproline dipeptide building block that was only available in L-configuration.

C



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We also analyzed the usability of phosphonates as proteomic probes for the PPs of H3S10ph. Protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) have been implicated in the dephosphorylation of H3S10ph.9,10 We identified several phosphatases in SILAC1 and SILAC2; however none of them were enriched on H3Pma10 (Supporting Tables 1 and 2, Supporting Information). It is conceivable that phosphonates do not mimic all necessary characteristics required for tight binding of phosphatase or that structures larger than the H3-tail are required for efficient substrate recognition. Probably more important is the fact that Pma mimics the ground-state and not the transition-state of the dephosphorylation reaction, which should mediate the strongest PP-binding. Furthermore, the highly abundant 14-3-3 proteins compete with PPs for the binding site of H3Pma10. Therefore, future investigations may require the development of peptide-based probes that target PPs more efficiently than phospho-serine binding proteins or depletion of 14-3-3 proteins from nuclear extracts prior to the pull-down experiments. Binding Proteins of Unmodified H3. Specific binding proteins of the unmodified H3-tail should be enriched on L-H3 compared with D-H3 (Table 2). We observed that the M/L ratios were lower than the H/M ratios (Figure 3c). The WD repeat-containing protein 5 (WDR5) is a confirmed binder of the unmodified H3-tail and was found enriched on L-H3 (Table 2).35 Initial studies suggested that WDR5 is a specific binder of H3K4me3, but this notion was later revised, and WDR5 was shown to bind unmodified H3 and H3R2me2, as well as the SET domain of mixed-lineage leukemia (MLL) complex.35−40 Structural and biochemical data showed that the first three residues of H3 are critical for this interaction as indicated by the observation the H3T3ph blocks binding of WDR5. 35 Endogenous WDR5 was pulled-down (input, 30 μg of HeLa NE) by L-H3 and only to a much lesser extent by D-H3 (Figure 4b). In addition, we confirmed that phosphorylation of T3 impairs this interaction. However, none of the other phosphorylated residues modulated the interaction between the H3 tail and WDR5. The same binding pattern was observed when recombinant WDR5 was used as input (Figure 4b). Fluorescence polarization titration (FP) experiments were used to quantify these interactions. We determined a Kd of 1.6 μM for the interaction of WDR5 with L-H3, which is in good agreement with previous reports.35,38 Under the same experimental conditions, we could not detect any interaction with D-H3 (Figure 4c). The heat shock protein HSC70 was reported to bind fulllength H3.41−43 HSC70 and related proteins were enriched on L-H3, and we confirmed the binding preferences in pull-down experiments followed by Western blot analysis with HeLa NE (30 μg) as input (Figure 4b). Phosphorylation of the H3-tail had no significant effect on this interaction (Figure 4b). Only H3S10ph showed a slightly reduced enrichment of endogenous HSC70 on L-H3, but this effect was not observed with recombinant protein. This finding might indicate that the binding sites of HSC70 and 14-3-3 proteins overlap. Kd values of 0.5 and 6.4 μM were determined for L-H3 and D-H3, respectively (Figure 4c). Two LIM-domain containing proteins, paxillin and Fhl3, were reproducibly enriched on L-H3 in the SILAC experiments (Table 2). Physiological functions of these interactions are not evident. A possible nuclear localization of paxillin is discussed, but this protein is primarily recruited to focal adhesions where it serves as an adapter protein.44 The function of the four-and-

Figure 3. SILAC pull-down. (a) General strategy of triple SILAC pulldown. Nuclear extracts (NE) prepared from differentially labeled cells: heavy (H), medium (M), and light (L). Labeled NEs are used as input for pull-down experiments with H3Pma10, L-H3, and D-H3 as baits. Bound proteins are combined and analyzed by LC-MS/MS. Analysis of H/M ratio (b) and M/L ratio (c) of SILAC1 pull-down. In total, we identified 2751 proteins in this experiment, and 2599 of these proteins could be quantified. Proteins that do not fulfill the quality criteria (identification in SILAC1 and SILAC2 pull-downs and quantification with greater than or equal to four peptides) were not considered for further validation experiments.

case of H3S28ph and H3S10ph proline and glycine occupy the P + 2 site, respectively, which explains why 14-3-3 proteins bind more strongly to H3S28ph than to H3S10ph.34 H3T3ph and H3T6ph do not possess proline or glycine residues at this site (Figure 1a). In case of H3T11ph, the putative 14-3-3 recognition site is similar to that of H3S10ph with a P + 2 glycine residue (Figure 1a). However, in contrast to H3S10ph, H3T11ph is not recognized by 14-3-3 proteins, indicating that the binding mode is more complex and not determined by the phosphorylation site and the P + 2 residue alone. D



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Table 2. Ten Proteins with the Highest Isotope Ratios in the H/M and M/L Screena no.

gene name

uniprot ID

1 2 3 4 5 6 7 8 9 10

Ywhab Ywhah Ywhag Ywhae Ywhaz Ywhaq Rbm25 Puf60 Wdr41 Fbp11

Q9CQV8 P68510 P61982 P62259 P63101 P68254 B2RY56 Q3UEB3 Q3UDP0 Q9R1C7

1 2 3 4 5 6 7 8 9 10

Pxn Hspa4 Wdr5 Fhl3 Hat1 Hspa1b Rbbp4 Rbbp7 Hspa9 Hspa8

Q8VI36 Q61316 P61965 Q9R059 Q8BY71 P17879 Q60972 Q60973 P38647 P63017

a

SILAC1 H/M ratio

protein name

Proteins enriched on H3-Pma10 14-3-3 protein beta/alpha 76.0 14-3-3 protein eta 66.8 14-3-3 protein gamma 50.7 14-3-3 protein epsilon 49.2 14-3-3 protein zeta 28.5 14-3-3 protein tau 21.4 RNA-binding protein 25 5.2 poly(U)-binding-splicing factor PUF60 4.8 WD repeat-containing protein 41 3.7 pre-mRNA-processing factor 40 homolog A 3.2 Proteins enriched on L-H3 paxillin 0.6 heat shock 70 kDa protein 4 (HSP110) 1.1 WD repeat-containing protein 5 (WDR5) 0.9 four-and-a-half-LIM domains 3 (Fhl3) 1.1 histone aminotransferase 1 (HAT1) 0.6 heat shock 70 kDa protein 1B 1.7 retinoblastoma binding protein 4 (RBBP4) 0.7 retinoblastoma binding protein 7 (RBBP7) 0.6 heat shock 70 kDa protein 9 1.6 heat shock cognate 71 kDa protein (HSC70) 1.5

SILAC1 M/L ratio

SILAC2 H/M ratio

SILAC2 M/L ratio

0.6 0.8 0.6 0.6 0.6 0.7 1.1 1.0 1.2 1.2

64.3 56.0 55.1 42.6 31.2 14.4 4.8 5.4 3.5 3.2

0.6 0.6 0.6 0.7 0.6 0.7 1.1 0.9 1.3 1.2

17.1 7.7 7.6 6.6 6.1 6.0 5.8 5.4 4.7 4.6

0.7 1.1 1.1 1.4 0.6 1.7 0.9 0.9 1.0 1.7

18.4 8.2 8.0 8.1 7.3 6.5 5.9 4.3 4.6 6.1

Proteins were sorted by the H/M or M/L ratios of SILAC 1.

specific modification state that cannot be established in bacteria. Interestingly, similar effects have been reported for the interaction between HAT1/RBBP7 and the H4-tail: recombinant HAT1/RBBP7 bind to the first α-helix of histone H4 but not to the H4-tail alone.46 However, endogenous HAT/RBBP7 are readily pulled-down from HeLa cell lysates by the H4 tail alone.47 The initial finding that HAT1 catalyzes acetylation of H4 and remains bound to its substrate has led to different models for this unusual binding behavior.42 At first it was considered that HAT1 remains bound to H4 to ensure that the H4K5acK12ac diacetylation pattern is maintained until newly synthesized H4 is deposited into chromatin.42 However, since the diacetylation pattern of histone H4 was found to be less important for chromatin assembly, there have been doubts about the physiological relevance of this model.42 Our findings support this skepticism because H3 is not acetylated by HAT1. It has been further suggested that the association of HAT1 with H4 may prevent aberrant modification of residues other than H4K5 and H4K12.42 In addition a possible function of HAT1/RBBP7 as histone chaperones is discussed.42 Both of these models can be adapted to our findings assuming that prevention of aberrant PTMs or chaperone activity of HAT1/RBBP7 is not limited to H4 but affects H4 and H3 alike.

a-half-LIM protein 3 (Fhl3) is only poorly understood, and little is known about its localization. There are reports indicating that Fhl3 is recruited to integrin receptors like paxillin.45 Further investigations are necessary in order to determine whether paxillin and Fhl3 interact with H3 and whether such an interaction is of physiological relevance. Histone acetyl transferase 1 (HAT1) and the retinoblastomabinding protein 7 (RBBP7), also referred to as RbAp46, were also enriched on L-H3. Both proteins form a complex with newly synthesized histones H3 and H4 in the cytoplasm and the nucleus. However, histone H4 has been considered as the primary interaction site because HAT1 acetylates H4K5 and H4K12.42 Western blot analyses uncovered that HAT1 and RBBP7 bound preferentially to L-H3 (Figure 5). Interestingly, phosphorylation of all known H3-phosphorylation sites inhibited this interaction (Figure 5). This effect was more pronounced than expected from the SILAC experiments, probably due to the high amounts of input used in SILAC1 and SILAC2. We further probed the H3 baits with recombinant HAT1 and RBBP7 expressed in bacteria (Supporting Figure S1, Supporting Information). However, under these conditions, we did not observe specific binding of HAT1 or RBBP7 to L-H3. This experiment is consistent with the previous finding that recombinant HAT1 does not bind to GST-tagged full-length histone H3.46 To ensure that the interaction pattern of endogenous HAT1/RBBP7 and the H3 baits is not limited to HeLa cells, we performed pull-down experiments of endogenous HAT1 from unlabeled 3T3 NE and essentially observed the same interaction profiles (Figure 5). These experiments indicate that the binding properties of HAT1 and RBBP7 are more complex than those observed for WDR5 and HSC70. HAT1 and RBBP7 might represent indirect binding proteins of the H3-tail that are recruited by a direct adapter protein. Alternatively, specific binding of HAT1 and RBBP7 may require mammalian signaling molecules or a

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CONCLUSIONS We have established an efficient strategy for identifying interaction partners of unmodified histone tails by proteomewide analysis of pull-down experiments with histone tails synthesized from L- and D-amino acids. Identifying two known interaction partners of histone H3 (WDR5 and HSC70) gives confidence in the applicability of this method. Possible limitations may arise from limited interactions of specific binding proteins and the D-peptide probes that are facilitated by the unstructured and flexible nature of the peptide baits. Future E



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Figure 4. Validation of interactions between proteins and phosphorylated and unmodified histone H3 tails. (a) Pull-down experiments of endogenous 14-3-3β (input, 300 μg of total protein extract), 14-3-3γ, and 14-3-3ζ from HeLa NE (input, 30 μg of total protein extract). D-H3, L-H3, and peptides derived from all known phosphorylation sites of the H3 tail are used as baits. (b) Pull-down experiments of endogenous and recombinant WDR5 and HSC70. Baits were probed with HeLa NE (input, 30 μg of total protein extract) and analyzed by Western blot analyses after pull-down of endogenous proteins (upper lanes labeled HeLa NE). Recombinant WDR5 and HSC70 were used as input at a concentration of 5 μM in the presence of 500 μM BSA and in a volume of 20 μL. Experiments were analyzed by Coomassie Brilliant Blue stained SDS-PAGE (bottom lines labeled recombinant). (c) Fluorescence polarization (FP) experiments with recombinant WDR5 and HSC70 and L-H3 and D-H3 were used to determine the dissociation constants.

particularly useful when probing the interaction profiles of proteins and highly charged peptides, which are prone to mediate unspecific electrostatic interactions. We further studied the application of phosphonates as phospho-serine substitutes as potential probes for protein phosphatases. While H3Pma10 served as efficient H3S10ph substitute for phospho-serine binding 14-3-3 proteins, the corresponding phosphatases were not enriched on this bait. Since H3Pma10 also mediates binding of high-abundance 14-3-3 proteins, further studies will be required in order to determine whether phosphatases can be targeted by phosphonates in different sequence contexts. An important finding is that HAT1 and RBBP7 are part of the protein−protein interaction network that binds to the unmodified tail of histone H3. The nature of this interaction behavior remains unknown since recombinant proteins do not

Figure 5. Analyzing the interaction profiles of HAT1 and RBBP7 with phosphorylated and unmodified histone H3 tails. Pull-down experiments of endogenous HAT1 and RBBP7 from HeLa NE and Swiss 3T3 NE (input, 30 μg of total extract).

investigations will uncover the full application range of this strategy. However, it appears likely that this method will be F



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reflect the binding properties of endogenous proteins in cellular lysates. In this regard, the observed binding properties are similar to the interaction profile of HAT1/RBBP7 and the H4tail. This finding provides further insights into possible roles of HAT1/RBBP7 in processes such as nuclear import of histones and chromatin assembly.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details, recombinant HAT1 and RBBP7 pull-down experiments, and complete profiles from SILAC1 and SILAC2 experiments (Excel format). This material is available free of charge via the Internet at http://pubs.acs.org.

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METHODS

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Synthesis of Building Blocks, Peptides, Recombinant Production of Proteins, Pull-Down Experiments, and Western Blot Analysis. Detailed information is provided in the Supporting Information. SILAC Cell Culture. For isotope labeling, the cells were transferred into DMEM medium deficient in arginine and lysine (SILAC protein quantitation kit, Pierce, Thermo Scienctific GmbH). The SILAC DMEM media were supplemented with either 0.13 g L−1 L-Lys·2HCl and 0.08 g L−1 L-Arg·HCl (Pierce) for the “light” population, 0.13 g L−1 D4-L-Lys·2HCl (Silantes) and 0.08 g L−1 13C6-L-Arg·HCl (Cambridge Isotope Laboratories, Andover, MA) for the “medium” isotopic coding, or 0.10 g L−1 13C6,15N2-L-Lys·NH3 (Silantes) and 0.08 g L−1 13C6,15N4-L-Arg·HCl (Cambridge Isotope Laboratories) for the “heavy” population. Cells were cultured in “light”, “medium”, or “heavy” media for 8 days in the presence of 10% dialyzed FBS (Invitrogen). For harvest, the PBS washed cells were detached from the culture flask with a cell scraper. The labeling efficiency was determined as >95% by LC-MS/MS analysis before the cells were harvested. Unlabeled HeLa cells were cultivated by fermentation as described before.25 Preparation of MS Samples. For MS analysis, the whole gel lanes were cut into 40 slices. Each gel slice was washed with ultrapure water, 25 mM ammonium bicarbonate in acetonitrile/water (1:1), and 50 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile and dried in a speed-vacuum centrifuge. The dry gel pieces were reswollen in 10 μL of 50 mM ammonium bicarbonate containing 50 ng of trypsin (sequencing grade modified, Promega). After overnight incubation at 37 °C, the enzymatic reaction was terminated by addition of 10 μL of 0.5% (v/v) trifluoroacetic acid in acetonitrile, samples were sonicated for 10 min, and the liquid was separated. The gel pieces were shrunk for a second time with 50 μL of acetonitrile. The combined liquids were evaporated to dryness under vacuum, and the residue was dissolved in 6 μL of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water. Liquid Chromatography-Tandem Mass Spectrometry. LCMS/MS analyses were performed on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher) equipped with an Eksigent 2D nanoflow LC system (Axel Semrau GmbH) as previously described.25 In brief, LC separations were performed on a capillary column (PepMap C18, 3 μm, 100 Å, 250 mm × 75 μm i.d., Dionex) at an eluent flow rate of 200 nL/min using a gradient of 3−50% B in 90 min. Mobile phase A was 0.1% formic acid (v/v) in water, and B was 0.1% formic acid in ACN. Mass spectra were acquired in a datadependent mode with one MS survey scan (with a resolution of 30000) in the Orbitrap and MS/MS scans of the four most intense precursor ions in the LTQ. The MS survey range was m/z 350−1500. The dynamic exclusion time (for precursor ions) was set to 120 s, and automatic gain control was set to 1 × 106 and 2 × 104 for Orbitrap-MS and LTQ-MS/MS scans, respectively. Data Processing and Quantification. Identification and quantification of proteins were carried out with version 1.1.1.36 of the MaxQuant software package as described.48 Generated peak lists were submitted to the Andromeda search engine using an IPI mouse protein database (version 3.68).49 The mass tolerance of precursor and sequence ions was set to 20 ppm and 0.35 Da, respectively. Methionine oxidation and the acrylamide modification of cysteine were used as variable modifications. False discovery rates were

Analysis of Phosphorylation-Dependent ProteinâProtein Interactions

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