MS Analysis Provides New Insights into the

of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen si...
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NanoLC-MS/MS Analysis Provides New Insights into the Phosphorylation Pattern of Cdc25B in Vivo: Full Overlap with Sites of Phosphorylation by Chk1 and Cdk1/cycB Kinases in Vitro Jean-Pierre Bouché,*,† Carine Froment,‡ Christine Dozier,† Charlotte Esmenjaud-Mailhat,† Matthieu Lemaire,† Bernard Monsarrat,‡ Odile Burlet-Schiltz,*,‡ and Bernard Ducommun† LBCMCP-CNRS-IFR109, Institut d’Exploration Fonctionnelle des Génomes, University of Toulouse III, France, and IPBS-CNRS-UMR5089, Institut de Pharmacologie et de Biologie Structurale, University of Toulouse III, France Received September 26, 2007

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule. Keywords: cell cycle • Cdc25B • Chk1 • Cdk1 • post-translational modification • phosphorylation • electrospray • mass spectrometry

1. Introduction Mass spectrometry has demonstrated over the past decade its efficiency in the characterization of posttranslational modifications of proteins including phosphorylations.1,2 The determination of complex phosphorylation patterns, associated to their dynamics and to the low abundance of particular sites in the cell, however, remains a challenging task. To overcome these difficulties, many approaches based on a combination of phosphoprotein and/or phosphopeptide enrichment methods and phosphorylation site identification by mass spectrometry have been developed.3–7 The main steps include sample preparation to preserve the protein phosphorylation status, detection of phosphorylated proteins, protein digestion into peptides, detection of phosphorylated peptides, and characterization of phosphorylation sites by mass spectrometry. The recently evolving field of phosphoproteomics aims at identifying the largest possible number of phosphorylation sites of a pool of phosphoproteins in a whole cell lysate. For this global analysis, enrichment of phosphoproteins associated to subsequent phosphopeptide enrichment is a critical step to handle the large number of phosphoproteins and phosphorylation dynamic range. More targeted studies focusing on one protein * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +33 561-556-542. Fax: +33 561-558-109. Or [email protected] (cocorresponding author). Phone: +33 561 175 547. Fax: +33 561 175 994. † LBCMCP, Bat 4R3B1, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse, France. ‡ IPBS, CNRS, 205 route de Narbonne, 31077 Toulouse, France.

1264 Journal of Proteome Research 2008, 7, 1264–1273 Published on Web 02/01/2008

of interest can also be envisaged. In this case, sequence coverage becomes a key element to determine phosphorylation patterns. The activation of cyclin-dependent kinase (CDK) complexes required for progression through the cell cycle is regulated by the dephosphorylation of specific tyrosine/threonine residues by Cdc25 phosphatases. In mammalian cells, three paralogous phosphatases, Cdc25A, B, and C, contribute to this regulation. Cdc25 phosphatases are made of a relatively short, ca. 170 amino acids long, catalytic domain, whose 3-D structure has been resolved, and of large, 250 to 400 amino acids long, regulatory sequences. Earlier studies concluded that cell cycle transition was controlled by one or another Cdc25 phosphatase. It is now admitted that the three phosphatases participate in G1/S8–10 and G2/M11–13 transitions. Phosphorylation is the major modification of Cdc25 phosphatases, affecting cellular location, stability, and activity. Table 1 summarizes the phosphorylation sites identified within the Cdc25 phosphatases mostly studied in vertebrates. It includes the sites identified independently from this study in CDC25B and an updated listing of the sites identified in other CDC25 phosphatases. A few critical sites common to the regulatory regions of the several phosphatases have been found to respond to regulatory kinases by similar mechanisms. For example, phosphorylation by stress kinase Chk1 of a position common to the C-terminal end of CDC25A, B, and C (lower line in Table 1) inhibits the interaction of each phosphatase with Cdk/cyclin complexes.22,30 Phosphorylation of CDC25C S216 by stress kinases Chk1,35 Chk2,39 C-TAK1,41 P-ERK1/2,46 and Eg326 10.1021/pr700623p CCC: $40.75

 2008 American Chemical Society

research articles

New Insights into the Phosphorylation Pattern of Cdc25B a

Table 1. Phosphorylation Sites Previously Identified in CDC25B, CDC25A, and CDC25C CDC25B3 H. sapiens

CDC25A H. sapiens

CDC25A X. laevis

CDC25C H. sapiens

CDC25C X. laevis

refs

accession # NP_068659 — AASS50PVTb GSET69PKSb — SSES103SES — CMDS115PSP — RFQS151MPVb LGHS160PVLb RNIT167NSQ b ITNS169QAP — GAA(SS)186-187SGE RPSS230APD EELS249PLA — — LFRS321PSM RSPS323MPCb — — — RRRS353VTPb RSKS375LCHb KTRS563WAG

accession # NP_001780 FACS18PPP GLLS39PVT — — RMGS76SES STDS82GFC CLDS88PGP — RIHS107LPQ LGCS116PAL RSHS124DSL HSDS126LDH — — RQNS178AQL — — — LFDS262PSL DSPS263LCS PERS279QEE — PGST288KRR RRKS293MSG QSLS317LAS KSRT507WAG

accession # NP_001081956 — TALS36PVT QCET57PTR — RTDS73SES — CLDS85PIK — RRNS120LPQ LGSS129PAF RNLS137YSL LSYS139LEC — — RQKS190APA — — — — — — — PERS295QEE RRKS299MSE RSKS321FSS KSRT504WAG

accession # NP_001781 — VPRT48PVG SGGT67PKC — LTTS90ADL LDET96GHL HLDS101SGL — — LCST130PNG — — — — — — YLGS169PIT MEFS198LKD LYRS214PSM RSPS216MPE — KDNT236IPD — — KTVS263LCD —

accession # NP_001081918 — QPLT48PVT SGET67PKR PLPT86ESP PTES88PDR RISS94GKV KVES99PKA GLFT112PDL — LCST138PSF — — LFKS163PNC — — — ILGS205PIS — LYRS285PSM RSPS287MPE — — — RRRS317TSS KTLS343LCD KCKT533SVG

14 15, 16 15 17 18 to 20 19 21 17 22 14 to 16, 23, 24 18–20, 22, 25, 26 27, 28 17 29 19, 26, 30, 31 27 15, 16 32 15, 33, 34 27, 33, 35 to 46 19, 26 47 22 19, 26, 27, 48, 49 27, 42 22, 30

a Previously identified phosphorylation sites (in boldface) and corresponding homologous putative phosphorylation sites (not bold) with surrounding sequence motifs are indicated for CDC25B3, CDC25A, and CDC25C in different species. The absence of a homologous S/T-containing motif is indicated by dashes. Accession numbers correspond to the RefSeq database. b Sites also identified in vivo in this study.

promote phosphatase inactivation via cytoplasmic retention and degradation,46,35 while phosphorylation of related site S323 of CDC25B3 appears to suppress activity through cytoplasmic retention and impediment to substrate access.50 Phosphorylation at S216 or S323 is inhibited by prior phosphorylation at a nearby site (S214 or S321) by Cdk1/cyclin B.33 In addition, it has been shown, in the case of Xenopus Cdc25C, that the nearby phosphorylation by Cdk1/cyclin B speeds up the dephosphorylation of the S216-related site by phosphatase PP1.34 Besides these and other examples of mechanisms affecting activity, stability, or cell localization, common to two or three phosphatases, each phosphatase is regulated by unique processes. For example, CDC25A is rapidly degraded upon response to DNA damage. This degradation is mediated by Chk1dependent formation of a phosphodegron at a DSG site (S82), which is absent in CDC25B and C.19,51 Another example is the specific role of CDC25B in resumption of division following DNA damage and in Chk1-dependent coordination of spindle formation with chromosome condensation during mitosis.52–55 Because the pattern of phosphorylation determining regulatory pathways that are either common to several phosphatases or unique is not fully established, we undertook an extended investigation of the phosphorylation sites of CDC25B phosphorylated in vivo using nanoLC-MS/MS analysis. We identified 18 sites of CDC25B that are phosphorylated in proliferating cells, including four previously known sites, four that are homologous to sites phosphorylated in other species, and ten entirely new sites. All these sites have then been shown to correspond to those phosphorylated in vitro by two kinases,

which are central in regulating the activity of CDC25 in the course of an unperturbed cell cycle: Cdk1 and Chk1.

2. Experimental Section 2.1. Plasmids and Cell Lines. A C-terminal extension containing a hexahistine tag, SGAWSHPQFEKSGHHHHHHSGA, was added by site-directed mutagenesis and linker addition to pCDNA3-HA-CDC25B2.56 CDC25B1 and B3 derivatives were generated by swapping BstEII-Bsu36I fragments, which contain each specific splice variant sequence. Fragments containing CDC25B were cloned into pUHD10-HA,29 generating pUHD10HA3-CDC25B1/2/3-His6. The B3 variant was also cloned into a pET vector for expression under control of the T7 phi10 promoter in Escherichia coli, yielding plasmid pJPB543. Details of this construction are available on request. U2OS-tTA cells expressing the tTA tetracycline repressor chimera (Clontech) were stably transfected with pUHD10-HA3CDC25B3-His6 as previously described.29 2.2. Purification of Histidine-Tagged CDC25B3 from Escherichia coli. E. coli strain BL21(DE3)/CodonPlus-RP (Stratagene) transformed with pJPB543 was grown at 37 °C in LB medium containing 15 µg/mL of chloramphenicol, 50 µg/mL of ampicillin, and 500 µg/mL of oxacillin. CDC25B3 synthesis was for 6 h in the presence of 10 µM IPTG. Cells were washed with 20 mM Tris-HCl (pH 8)/0.1 M NaCl/5 mM EDTA, resuspended in the same buffer, incubated with 200 µg/mL of lysozyme for 20 min at room temperature, and sonically disrupted. Journal of Proteome Research • Vol. 7, No. 3, 2008 1265

research articles Inclusion bodies were recovered (30 000 g, 10 min), washed with 20 mM Tris-HCl (pH 8)/0.5 M KCl/2% Triton X-100/2 M urea, and dissolved in solution D (20 mM Tris/0.2 M KCl/7.5 M urea/1.5 M thiourea/5 mM 2-mercaptoethanol/5 mM imidazole; pH 8.0). Protein was applied on a column of Ni-NTA agarose (Qiagen) equilibrated with solution D, washed with this solution, and eluted with solution D containing 300 mM imidazole. To renature CDC25B3, one volume of Ni-NTA eluate was flash-diluted by adding seven volumes of 20 mM Tris, 0.3 M KCl, 0.5 M arginine, and 50 mM 2-mercaptoethanol (pH 8.0). The resulting solution was dialyzed against 20 mM Tris-HCl (pH 8.0), 0.2 M KCl, 50 mM 2-mercaptoethanol, and 50% glycerol (v/v). The catalytic activity of CDC25B3 thus obtained, measured using fluorescein diphosphate as substrate, was at least equal to that of MBP-CDC25B.48 2.3. Expression and Purification of CDC25B3 from U2OS Cells. U2OS-tTA cells (Clontech) stably transfected with pUHD10HA3-CDC25B3-His6 were cultured in complete DMEM medium with 100 µg/mL of G418 and 2 µg/mL of tetracycline. After 24 h in tetracycline-free medium for transgene expression, plates were washed with cold PBS, and cells were scrapped off the plates in absolute ethanol, collected by centrifugation, and freeze-dried. Proteins were solubilized with gentle shaking in 50 mM Tris-HCl, pH 8.0, 7.5 M urea, 1.5 M thiourea, 4% CHAPS, 20 mM spermine, 5 mM 2-mercaptoethanol, and 5 mM imidazole. Cell debris was removed by centrifugation, and CDC25B was purified on a Ni-NTA agarose column as described above. After iodoacetamide treatment, the eluate was diluted in renaturation solution as described above, and CDC25B was bound to anti-HA sepharose beads (Roche) with gentle mixing. Beads were washed with binding buffer and taken up in SDSPAGE loading buffer, heated to 90 °C, and supernatant was loaded onto a NU-PAGE Bis-Tris 4–12% gel (Invitrogen). The Coomassie blue-stained band (71 kD) corresponding to CDC25B3 was excised, digested with trypsin, and analyzed by nanoLCMS/MS as described below. Cells treated by proteasome inhibitor acetyl-Leu-Leu-norleucinal (ALLN) received ALLN (50 µM) at the time of tetracycline removal and were cultured for another 18 h before harvest. Amounts of CDC25B protein in total extracts and partially purified fractions were estimated by Western blot analysis with monoclonal anti-HA antibody (Roche) using dilutions of the purified recombinant protein as standard. 2.4. 2-D Electrophoresis. For the first dimension, pH 4–7, 18 cm strips, and an IPG Phor electrofocusing unit (Amersham Bioscience) were used. 130 µg proteins from NI-NTA eluate (2.5 µg CDC25B3) were applied in 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 24 mM spermine, bromphenol blue, and ampholine. Rehydration was for 20 h, followed by 50 V (10 h), 200 V, 500 V, 2000 V (1 h each) gradient increases to 8000 V (total: 50 000 Vh). The second dimension was on a 20.5 × 25 cm 10% polyacrylamide precast gel (Ettan Dalt six, Amersham Biosciences). The strip was equilibrated with 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 1% SDS, containing 10 mg/ mL of DTT (20 min), then 45 mg/mL of iodoacetamide (20 min). Migration was with constant power (100 W). Proteins were revealed by Colloidal Blue staining. 2.5. Phosphorylation of CDC25B in Vitro. Phosphorylation by kinase Chk1 was carried out at 30 °C for 30 min with 100 ng of purified recombinant Chk1 kinase (Upstate) and recombinant MBP-CDC25B348 or HA3-CDC25B3-His6 (1 µg), in 20 µL of kinase buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 1 mM 1266

Journal of Proteome Research • Vol. 7, No. 3, 2008

Bouché et al. DTT; 100 µM ATP). Phosphorylation by kinase Cdk1/CyclinB was carried out at 30 °C for 2 h with 180 ng (0.11 units) of Cdk1cyclin B (Upstate) and HA3-CDC25B3-His6 (2 µg), in 25 µL of kinase buffer (20 mM MOPS-NaOH, pH 7,2, 25 mM 2-glycerophosphate, 0.5 mM EDTA, 1 mM EGTA, 15 mM MgCl2, 1 mM DTT, 50 µg/mL of ovalbumine, 100 µM ATP). Phosphorylated samples were separated by SDS-PAGE. The excised CDC25B3 bands were digested with trypsin or CNBr and analyzed by nanoLC-MS/MS as described below. 2.6. In-Gel Tryptic Digestion, Peptide Extraction, and CNBr Cleavage. The excised bands containing about 1 µg of CDC25B3 were rinsed and subjected to in-gel reduction and S-carbamidomethylation. In-gel tryptic digestion using modified porcine trypsin (Promega, Lyon, France) at 20 ng/µL and peptide extraction from the gel were performed as previously described.25 The CNBr cleavage was performed as previously described with minor modifications.57 Briefly, 30 µL of CNBr (Fluka) in 70% TFA (one small crystal was dissolved in 250 µL of 70% TFA) was added to the CDC25B3 dried band, and cleavage was performed overnight in the dark at room temperature. The CNBr cleavage was quenched by washing the gel piece with 1 mL of water and evaporating off the CNBr and TFA by SpeedVac centrifuge. 2.7. nanoLC-MS/MS Analysis. The dried peptide extracts were dissolved in 10 µL of 0.1% FA in 2% ACN and analyzed by online capillary HPLC (Dionex/LC Packings, USA) coupled to a nanospray Qq-Tof mass spectrometer (QSTAR Pulsar XL, Applied Biosystems, Foster City, USA). Peptides were separated on a 75 µm ID × 15 cm C18 PepMap column after loading onto a 300 µm ID × 5 mm PepMap C18 precolumn (Dionex/LC Packings, USA). The flow rate was set at 200 nL/min. Peptides were eluted using a 0 to 50% linear gradient of solvent B in 100 min (solvent A was 0.2% formic acid in 5% acetonitrile, and solvent B was 0.2% formic acid in 90% acetonitrile). Mass spectrometric data acquisition was performed in informationdependent acquisition mode using a dynamic peak exclusion as previously described.25 Peak lists of MS/MS spectra were created using Mascot.dll script (version 1.6b21) in Analyst QS software (version 1.1, Applied Biosystems). Data analysis was performed using the Mascot search engine (version 2.1.04), searching against the Swiss-Prot database implemented with the recombinant protein sequences, MBP-CDC25B3 and HA3CDC25B3-His6. Up to two digestion missed cleavages were allowed, and the mass tolerance for peptide and MS/MS fragment ions was 0.5 Da. Cysteine carbamidomethylation and propionamide, methionine oxidation, and serine/threonine phosphorylation were set as variable modifications. Additional variable modifications, homoserine and homoserine lactone on C-terminal methionine peptides, are considered for CNBr cleavage. Site identification was confirmed by manual interpretation of corresponding MS/MS data. The sequence coverage reached at the most 75%. Three large and/or acidic fragments (93–137; 256–280, and 494–543), amounting for 21% of the sequence, and another 5% contained into short fragments, were not detected. 2.8. Phosphorylation Site Prediction and Phosphorylation Level Estimation. We used the NetPhos 2.0 Server (http:// www.cbs.dtu.dk/services/NetPhos/) to predict phosphorylation sites on the CDC25B3 protein. The phosphorylation level was estimated by computing the peak area ratio of all peptides containing a phosphorylated given phosphorylation site over the sum of all peptides containing the same phosphorylated and nonphosphorylated phosphorylation site. Peak areas were

New Insights into the Phosphorylation Pattern of Cdc25B

research articles NanoLC-MS/MS analysis of the major band identified 17 phosphorylation sites. Except for S230 (or S229), all sites identified in untreated cells were also present in ALLN-treated cells. Six additional sites were identified: S15, T69, S182, T344, T355, and S470 (Table 2). Data concerning the other sites are displayed in Supporting Information Figures S11 to S16.

Figure 1. PAGE profile of CDC25B3 purified from U2OS cells, not treated (A) or treated (B) with proteasome inhibitor ALLN.

measured from extracted ion chromatograms (XIC) of each peptide by using the , Extract Ions . tool and the , Manually integrated . script in Analyst QS 1.1 software. For each peptide, the monoisotopic experimental mass-to-charge ratio of all observed charge states and modified forms of the peptide was considered to compute the peptide peak area. This phosphorylation level estimation may not reflect the physiological ratio of phosphorylation due to potential differences in ionization efficiencies of each peptide but rather serves as a basis of comparison between several experiments.

3. Results 3.1. Identification of 18 Phosphorylation Sites of CDC25B3 in Vivo. CDC25B is expressed at very low levels in most cell lines (ca. 10-6 of total proteins, our estimate), precluding direct analysis of post-translational modifications. To increase CDC25B expression and facilitate its purification, we constructed a derivative of U2OS cells, which conditionally expresses a Histagged CDC25B3 from a tetracycline-regulated promoter. U2OS cells expressing permanently CDC25B3 from this construct continue to divide almost normally but have a reduced clonogenicity (not presented). CDC25B3 was purified from cells expressing the transgene for 24 h. The purity of CDC25B3 was estimated to 2.5 × 10-5 in the total extract, 2 × 10-2 after NiNTA chromatography, and 0.5 after release from anti-HA beads (Figure 1A). The major band at 71 kDa was excised, digested with trypsin, and analyzed by nanoLC-MS/MS. Ten phosphorylation sites were identified unambiguously: S42, S50, S151, S160, S178, S238, S249, S323, S353, and S375 (Table 2 and Figure 2). As an example, the MS/MS analysis of the phosphorylated peptide 243-MEVEELpSPLALGR-255 allowing the identification of a new phosphorylation site at S249 is presented in Figure 3. MS/MS spectra of all other phosphopeptides analyzed are shown in the Supporting Information (Figures S1 to S9, respectively). In addition, two sites were identified with ambiguity between S89 and S91 (not shown) and between S229 and S230 (Figure S10). Even though MS/ MS data do not rule out concomitant phosphorylation of S229, phosphorylation of S230 in vivo has already been demonstrated by use of a phosphospecific antibody.31 CDC25B is an unstable protein whose degradation is at least in part dependent upon the ubiquitin-proteasome pathway.21,58 To increase CDC25B3 yield for analysis, CDC25B3 was purified from cells treated for 18 h with proteasome inhibitor (ALLN, 50 µM). Preliminary analysis had indicated that this treatment led to a 5-fold increase in the yield of CDC25B3 compared to untreated cells (not shown). The purified protein appeared as multiple bands (Figure 1B), and shifted fractions corresponding to CDC25B3 conjugated to ubiquitin as peptides revealing the presence of conjugated ubiquitin were detected (not presented).

To address the effect of protein stabilization by ALLN on CDC25B phosphorylation, MS signal intensity ratios related to the phosphorylation level of each phosphorylation site were computed. These ratios were then compared for the 11 identified sites common to CDC25B between untreated and treated cells (Table 3). All sites were more phosphorylated in ALLNtreated than in untreated cells. This indicates that ALLN treatment, in addition to stabilizing CDC25B, leads to more extensive phosphorylation. 3.2. Multiple Phosphorylation of CDC25B3 in Vivo. Although 12 sites were detected in CDC25B3 under normal growth conditions, and up to 17 after treatment with a proteasome inhibitor, the number of phosphate moieties carried by individual phosphatase molecules remains unknown. To evaluate the phosphate distribution, CDC25B3 expressed from stably transformed U2OS cells, partially purified by NiNTA chromatography, was separated by 2-D gel electrophoresis. Spots corresponding to CDC25B3 were identified either by mass spectrometry or by blotting and revelation with an antiHA antibody (not shown). Up to 14 spots containing CDC25B spanning over 0.6 pH units were clearly detected (Figure 4). This pI interval compares well with a predicted pI difference of 0.60 for 13 negative charges added to the unmodified sequence. Since phosphorylations were the only acidifying modifications detected in our analysis, we conclude that up to 13 phosphate groups may be present in CDC25B molecules and that a significant part of CDC25B molecules present in U2OS cells upon expression of the tetracycline-regulated transgene is hyperphosphorylated. 3.3. At Least 12 sites Are Phosphorylated on CDC25B3 by Chk1 in Vitro. Chk1, a major kinase activated in response to genotoxic agents, modulates the activity and stability of CDC25 phosphatases through phosphorylation of specific sites. It is now evident that Chk1 is involved in preventing premature activation of CDC25B phosphatase in unperturbed proliferation conditions.31 Thus, one might expect a substantial part of the sites detected in vivo to be substrates for Chk1. To identify the sites phosphorylated in CDC25B3 by Chk1, MBP-CDC25B3 was phosphorylated by purified Chk1 and subjected to nanoLCMS/MS analysis. Twelve sites were characterized (Table 2 and Figure 2). Eight sites (S89 or S91, S151, S178, S182, S229 or S230, S323, S353, S375) were undistinguishable from those identified in vivo, the only difference being that S182 was found only in a doubly phosphorylated peptide with S178. Here again, even though MS/MS data cannot distinguish between pS229 and pS230, a phosphospecific antibody against pS230 reacts to CDC25B3 phosphorylated in vitro by Chk1,31 suggesting that the MS/MS spectrum is indicative of S230. Spectra indicating additional phosphorylations of T167, S465, T561, and S563 are shown as Supporting Information Figures S17 to S20, respectively. T561 was found only together with S563. An examination of sequences surrounding the phosphorylation sites indicated a broad consensus (R/K)-X(2-3)-(S/T)notP. Only two sites (S89/91 and S182) did not fit this consensus, while three sites corresponding to this consensus Journal of Proteome Research • Vol. 7, No. 3, 2008 1267

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Table 2. CDC25B3 Phosphorylated Peptides Identified by NanoLC-MS/MS Analyses Mascot ion scoresb site

S15 S42 S50 T69 S89 or 91 S151

exptl calcd monoisotopic elution time Cdk1/ U2OS - U2OS + figure z mass (Da) mass (Da) (min)a Chk1 cycB ALLN ALLN no.c

peptide sequence

m/z

SSMoxEVPQPEPAPGSALpSPAGVCGGAQR PGHLPGLLLGSHGLLGpSPVR GLLGpSPVR AAASpSPVTTLTQTMH AAASpSPVTTLTQTMoxHDLAGLGSETPK AAAp(SS)PVTTLTQTMoxHDLAGLGSEpTPK LTHLp(SLS)R

911.69 686.32 439.70 798.33 894.04 920.70 503.72

3 3 2 2 3 3 2

2732.07 2055.98 877.41 1594.65 2679.12 2759.09 1005.45

2732.22 2056.10 877.45 1594.71 2679.25 2759.22 1005.51

44.5 60.0 42.1 51.8 61.1 63.2 34.4

550.72 480.69 357.84 626.76 626.77 880.70 785.95 907.36

2 2 3 2 2 3 3 3

1099.44 959.39 1070.52 1251.51 1251.54 2639.15 2354.86 2719.08

1099.50 959.40 1070.57 1251.53 1251.53 2639.15 2354.96 2719.12

32.4 20.6 38.4 18.9 20.2 33.9 42.7 36.6

785.95 3 781.98 3

2354.86 2342.93

2354.96 2343.01

42.7 47.6

781.98 813.30 762.31 564.60 564.61 615.22 832.88 560.90 592.59 593.22 723.94

3 2 2 3 3 2 2 3 3 3 3

2342.94 1624.57 1522.63 1690.79 1690.82 1228.43 1663.76 1679.82 1774.76 1776.65 2168.83

2343.01 1624.66 1522.72 1690.77 1690.77 1228.50 1663.83 1679.82 1774.84 1776.75 2168.92

45.7 51.9 60.0 NA NA 32.9 51.6 48.3 17.8 29.9 24.5

593.26 678.25 586.29 501.72 690.97 682.29 521.71

3 3 2 2 3 2 2

1776.76 2031.73 1170.57 1001.44 2069.91 1362.59 1041.42

1776.75 2031.84 1170.58 1001.45 2069.98 1362.59 1041.45

30.9 54.2 53.8 57.3 58.6 26.5 22.9

RFQpSMPVR FQpSMoxPVR S160 LLGHpSPVLR T167 NIpTNSQAPDGR S169 NITNpSQAPDGR S178 RKpSEAGSGAASSSGEDKENDGFVFK pSEAGSGAASSSGEDKENDGFVFK S178 + RKpSEAGpSGAASSSGEDKENDGFVFK S182 S182 SEAGpSGAASSSGEDKENDGFVFK S229 or EAFAQRPp(SS)APDLMoxCLSPDR 230 S238 EAFAQRPSSAPDLMoxCLpSPDR PSSAPDLMCLpSPDR S249 MEVEELpSPLALGR S321 YSKCQRLFRpSPShsl S323 YSKCQRLFRSPpShsl SPpSMoxPCSVIR SPpSMPCSVIRPILK S327 SPSMoxPCpSVIRPILK T344 LERPQDRDpTPVQNK S353 pSVTPPEEQQEAEEPK S353 + RRpSVpTPPPEEQQEAEEPK T355 T355 SVpTPPEEQQEAEEPK S375 pSLCHDEIENLLDSDHR T404 AFLLQpTVDGK S465 DAEpSFLLK S470 DAESFLLKpSPIAPCSLDK T561 LKpTRpSWAGER S563 TRpSWAGER

32 17 15 27 21 12

S11

14

31 22 17 59 56 34 9

9

11

S3

30

17

60

78 65

S4 S17 S21 S5 S13

25

39

S1 S2 S12

20 52 36 40 26 65 29

40 24 45 4

40 41

S13 S10 S6

36 33

11

3 6a 6b

26 29

28 25 23

31

36

8 37 18

41

44

S7 S22 S14 S8 S15

23 28 27 16 44 27 11

17

S9 S23 S18 S16 S19 S20

a Elution times are indicated for the U2OS + ALLN experiment. When a peptide was not present in this experiment, the measured elution times in the other experiments were corrected to match elution times of the U2OS + ALLN experiment by computing a best fit correlation function based on at least 10 different peptides spanning the whole range of elution times. For all peptides present in several experiments, their elution times after alignment were found to be within expected experimental variations. NA: not applicable. b Mascot ion scores were obtained as described in the Experimental Section except for the following search parameters: 3 missed cleavages were allowed, and cysteine carbamidomethylation was considered as a fixed modification. All MS/MS spectra were manually interpreted. c Figures displaying MS/MS spectra of phosphorylated peptides are provided either in the main text of the manuscript or in Supporting Information (figure numbers beginning with S).

(S49, S86, and S373) did not appear to be phosphorylated (Supporting Information Table 1). 3.4. At Least 14 Sites Are Phosphorylated in Vitro by Cdk1/Cyclin B. Dephosphorylation of Cdk1/cyclin B by CDC25 phosphatases is a critical event for Cdk1 activation and entry into mitosis. Conversely, active Cdk1/cyclin B phosphorylates CDC25 A, B, and C at various positions, with effects on protein stability, nuclear location, and ultimately on ensuring a rapid and stable switch into mitosis. We have investigated the identity of sites phosphorylated by Cdk1/cyclin B in vitro, using active renatured CDC25B3 as substrate. Samples containing an average of 3.8 phosphates per CDC25 molecule were subjected to nanoLC-MS/MS analysis. Fourteen sites were identified: S15, S42, S50, T69, S160, S169, S238, S249, S321, S327, T344, T355, T404, and S470 (Table 2 and Figure 2). Four sites, S169, S321, S327, and T404, were not identified from our analysis of CDC25B3 phosphorylation in vivo. The spectra for S169, S327, and T404 are shown in Supporting Information Figures S21 to S23, respectively. The case of S321 was more uncertain. 1268

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Phosphorylation of S323 in vivo (Figure S7) and by Chk1 in vitro was well established from MS/MS spectra of tryptic digests, but the exact location of the phosphate on tryptic peptide 321-SPSMPCSVIRPILK-334 phosphorylated by Cdk1/ cyclin B, other than S327, could not be ascertained. We took advantage of the presence of methionine residues to generate peptide 312-YSKCQRLFRSPSMhsl-324 upon cyanogen bromide (CNBr) treatment, where the methionine residue is modified to a homoserine lactone. MS/MS spectra clearly showed that S323 was phosphorylated by Chk1 (phosphorylated y3 ion, m/z ) 366.1) (Figure 5A), while S321 was phosphorylated by Cdk1/ cyclin B (unphosphorylated y3 ion, m/z ) 286.1) (Figure 5B). Sites phosphorylated on CDC25B3 have been compared to the well-defined consensus sequence (S/T)P-X-(R/K) deduced from high affinity sites and peptide library studies.59,60 Only two sites (S42, S238) contain a basic +2 residue. On the other hand, CDC25B3 contains 15 (S/T)P sites. Twelve were covered in this analysis and were phosphorylated to various extents except S416. Since these 11 sites were also phosphorylated in

New Insights into the Phosphorylation Pattern of Cdc25B

research articles to target a specific region of the CDC25B sequence and permitted us to distinguish and thus to unambiguously characterize S321 and S323 phosphorylation sites. CDC25B3 from ALLN-treated cells had a higher phosphorylation level than in untreated cells, possibly as a result of protein stabilization or because proteasome inhibitors are known to block cells at the G2/M transition where phosphorylation of CDC25B is highest.31,61 Neither S186/S187, phosphorylated in vitro by CK2,29 nor S169, phosphorylated by kinase Eg3,26 was detected in this analysis (Figure 2).

Figure 2. Location of CDC25B3 phosphorylation sites. Gray background indicates the catalytic domain. Dotted lines indicate undetected sequence regions. Black rectangles: (S/T) residues phosphorylated in vivo (this study). Newly identified sites are marked with arrows. White rectangles: phosphorylation in vivo established by specific antibodies. (X): (S/T) residues phosphorylated by Chk1 in vitro. (+): (S/T) residues phosphorylated by Cdk1/cyclin B in vitro. Hatched rectangles: residues phosphorylated by other kinases. The main functional features are indicated, including the nuclear export signal (NES), 14-3-3 protein binding sites, the KEN box for Cdh1-APC degradation, the β-TRCP destruction motif, and the nuclear localization signal (NLS).

vivo, (S/T)P behaves as a sufficient target for phosphorylation of CDC25B by Cdk1/cyclin B. The three additional sites phosphorylated in vitro, S169, S327, and T404, do not conform to the minimal consensus sequence (S/T)P. Since none of these amino acids were detectably phosphorylated in vivo, they may represent artifactual targets found only under in vitro conditions.

4. Discussion The aim of this study was to identify CDC25B sites phosphorylated in vivo. We report the identification of phosphorylation sites in the CDC25B3 variant overexpressed in U2OS cells. With 75% sequence coverage including 61% of all 44 possible CDC25B3 phosphorylation sites predicted according to NetPhos 2.0 and 80% coverage of the regulatory region where most modifications are present, we have identified 12 phosphorylated sites in vivo under unperturbed growth conditions and up to 17 sites in cells treated with proteasome inhibitor ALLN. Even though the CDC25B sequence coverage obtained for this 65 kDa protein was reasonably high, it could possibly be further improved by the use of endopeptidases with other cleavage specificities.17 In our hands, however, the use of endopeptidase Glu-C did not improve the sequence coverage previously obtained with trypsin and did not identify additional phosphorylation sites. CDC25B was also submitted to a chemical treatment using CNBr, which resulted in cleavages after methionine residues. This time the experiment was conducted

Out of the 18 sites disclosed by these combined analyses, four have been previously shown to be phosphorylated in vivo. These are S160, S230, S323, and S353 (Table 1 and Figure 2). S160 (S146 in CDC25B1), phosphorylated by Cdk1/cyclin B, plays a role in nuclear location and mitotic entry.23 Phosphorylation of S116, related to S160, stabilizes CDC25A,14 while in human CDC25C, phosphorylation of S160-related T130 provides a docking site for Plk1, although in an STP context not present in CDC25 A and B.24 Serine 230 is homologous to S190 of Xenopus Cdc25A, whose phosphorylation by Chk1 contributes to degradation in Xenopus egg extracts.22 In addition, it is homologous to human CDC25A S178, phosphorylated by Chk1 and Chk2.26 This site contributes slightly19,26 or not at all30 to CDC25A degradation. Its phosphorylation promotes 14–3–3 binding, and it has been suggested that 14–3–3 binding at CDC25A S178, together with binding at T507, works as a gatekeeper to prevent access to CDK substrates.30 Whether any of these functions concern CDC25B S230 is presently unknown, but it is certain that S230 is phosphorylated early in the S phase by Chk1 in the absence of DNA damage and that this phosphorylation contributes to preventing unscheduled mitosis, possibly by preventing CDC25B activity at the centrosome.31 Numerous studies (Table 1) have indicated that phosphorylation of S323 and/or its CDC25C counterparts by stress kinases CHK1,33,35–37 CHK2,38–40 C-Tak1,41 MAPKAP kinase-2,25,43 PKA,44 Eg3,45 and P-ERK1/246 leads to 14–3–3 protein binding, cytoplasmic retention,50,62–65 inhibition of catalytic activity,39,50 and degradation.46 Serine 353, phosphorylated by Aurora A,49 Akt,48 Chk1 (this study), and Chk2 on its CDC25A S293 counterpart,26 plays a role in localizing CDC25B to the cytoplasm48 and appears to play an important role for entry into mitosis after phosphorylation by Aurora A at the centrosome.49 The 14 sites not directly characterized previously are listed in Table 4. Three of these sites, S50, T69, and S151, are homologous to sites phosphorylated in other CDC25 species. Serine 50 and threonine 69 are, respectively, homologous to T48 and T67, two of the Cdk-dependent phosphorylation sites required for activation of mitotic entry by Xenopus Cdc25C.15 ERK-MAP kinases participate directly to CDC25C activation for mitosis and meiosis by phosphorylation of T48 and other sites.16 Our results indicate phosphorylation of S50 and T69, both in vivo and by Cdk1/cyclin B in vitro. Serine 151 is located within a proven 14–3–3 binding site for which serine phosphorylation by checkpoint kinases is a prerequisite for binding and biological function. Therefore phosphorylation in vivo and in vitro by Chk1 could be expected.50,66 Little is known about the 11 remaining sites identified in our analysis. Only three of them, T344, T355, and S375 are conserved in other CDC25 species (Table 4). Serine 375, conserved in CDC25B and C, is phosphorylated by kinase MAPKAP kinase-2 in response to UV irradiation25 and by Chk1 (this work). Threonine 344 and 355, also phosphorylated by Journal of Proteome Research • Vol. 7, No. 3, 2008 1269

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Figure 3. Example of a new in vivo CDC25B3 phosphorylation site identified by nanoLC-MS/MS from untreated U2OS cells. The MS/ MS spectrum of peptide 243-MEVEELpSPLALGR-255 (doubly charged precursor ion, MH22+, at m/z 762.30) displays series of b- and y-ions (according to Biemann’s nomenclature) indicating that serine residue 249 is phosphorylated. : loss of H3PO4 from sequence ions. Table 3. Relative Intensities of CDC25B Phosphorylated Peptides in Proteasome-Inhibited and Untreated Cells position

S42

S50

S89/91

S151

S160

S178

S238

S249

S323

S353

S375

U2OS - ALLN U2OS + ALLN Ratio +/- ALLN

0.40 0.60 1.5

0.02 0.68 35

0.11 0.24 2.2

0.012 0.043 3.6

0.145 0.31 2.1

0.02 0.14 6.6

0.17 0.64 3.8

0.08 0.33 4.1

0.25 0.54 2.2

0.25 0.54 2.2

0.37 0.43 1.2

Cdk1/cycB in vitro, are located within or in the vicinity of the NLS and may play a role in nucleo-cytoplasmic shuttling. Three other sites, S15, S182, and S470, were detected only in ALLN-treated cells, with a low ratio of phosphorylated to nonphosphorylated peptides. However, CDC25B S470A was recently reported to lose its ability to bind Cdk1 or Cdk2 substrates, as well as its antiproliferative properties when overexpressed, suggesting a major role of S470 phosphorylation by CDKs in phosphatase activity.67 Finally, five sites, S42, S89 (or S91), S178, S238, and S249, are previously undescribed sites with high intensity ratios between phosphorylated and nonphosphorylated peptides. S249 was recently reported to be phosphorylated in vitro by p38 SAPK, a kinase of the signaling pathway to UV-induced DNA damage.25 Out of the 18 sites characterized by MS/MS analyses, 10 fit within the minimal (S/T)P consensus for CDKs (no definitive conclusion could be reached about S321 in our analysis). In vitro, all these sites, as well as S321, were phosphorylated by Cdk1/Cyclin B. Indeed, out of the 12 (S/T)P sites covered by

Figure 4. Close-up view of the densitometric scanning of a 2-D stained gel showing CDC25B3 as multiple spots along the pI gradient axis. Two additional lawns of retarded CDC25B can also be observed. 1270

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our analysis, only one, S416, was not found phosphorylated either in vivo or in vitro. The exact identity of the CDK/cyclin complexes responsible for CDC25B bulk phosphorylation in asynchronous cells cannot be ascertained. In vitro, Cdk2-cyclin A, not Cdk1-cyclin B, appears to be required for phosphorylation-dependent degradation of CDC25B.68 Bulavin et al. proposed that Cdk1/cyclin B is specifically involved in phosphorylation of CDC25C S214 by Cdk1/cyclin B at the time of mitosis, preventing subsequent inhibitory phosphorylation of S216.33 In addition, Margolis et al. showed that this phosphorylation by Cdk1/cyclin B contributes directly to the activation of CDC25C by facilitating PP1catalyzed dephosphorylation of S216.34 CDC25C S214 and S216 are related to CDC25B3 S321 and S323, where a similar exclusion mechanism has been proposed.33 Yet a study involving RNAi and dominant-negative forms of Cdks and cyclins concluded that Cdk2, but not Cdk1, and cyclin A regulate the activation of CDC25C and CDC25B in HeLa cells.69 Whether Cdk2/cyclin A, Cdk1/cyclin B, or other CDK/cyclin combinations contribute to bulk phosphorylation in vivo, the question raised is that of the low specificity of phosphorylation by CDK/cyclin complexes. CDK/cyclin complexes phosphorylate synthetic peptides with a consensus sequence (S/T)PX(R/ K) where +2 (R/K) is essential for efficient phosphorylation.59,60 Efficient phosphorylation of natural substrates with a minimal (S/T)P motif is made possible by the binding contribution of a cyclin hydrophobic patch to an RXL (or Cy) motif present on the substrate molecule, as demonstrated for cyclins A and E and for the interaction of Myt1 with cyclin B.70–72 Other sequences may be involved in the case of cyclin B since mutation of an RRL motif of CDC25A abolishes interaction with

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New Insights into the Phosphorylation Pattern of Cdc25B

Table 4. New Cdc25B Phosphorylation Sites Identified in Vivo by NanoLC-MS/MS Analysisa CDC25B3 H. sapiens

CDC25A H. sapiens

CDC25A X. laevis

CDC25C H. sapiens

CDC25C X. laevis

S15 S42 S50 T69 S89/91 S151 S178 S182 S238 S249 T344 T355 S375 S470

— — S39 — — S107 — — — — S283 S295 S317 —

— — S36 T57 — S120 — — — — T290 S301 S321 —

— — T48 T67 — — — — — — — — S263 —

— — T48 T67 — — — — — — T308 — S343 —

a Identified phosphorylation sites (in boldface) and homologous putative phosphorylation sites (not bold) are indicated. The absence of a homologous S/T-containing motif is indicated by dashes.

Figure 6. Disorder propensity of CDC25B3, as predicted by IUPred software.75 Intrinsic disorder is predicted for regions with index values greater than 0.5.

Figure 5. (A) NanoLC-MS/MS identification for phosphorylation of S323 by Chk1 upon CNBr cleavage. The MS/MS spectrum of the phosphorylated peptide, 312-YSKCamQRLFRSPpSMhsl324 (triply charged precursor ion, MH33+, at m/z 564.60) displays fragment ions indicating the presence of a single phosphorylation site. (B) NanoLC-MS/MS identification for phosphorylation of S321 by Cdk1/cyclin B upon CNBr cleavage. The MS/MS spectrum of the phosphorylated peptide, 312YSKCamQRLFRpSPSMhsl-324 (triply charged precursor ion, MH33+, at m/z 564.62) displays fragment ions indicating the presence of a single phosphorylation site. : loss of H3PO4 from sequence ions. Cam: carbamidomethylated cysteine residue. Mhsl: induced CNBr cleavage homoserine lactone modification on the methionine residue.

cyclin A and E but not with cyclin B and D1.73 Out of the 11 p(S/T)P sites of CDC25B covered by our analysis, only two (S42 and S238) have a basic residue at position +3; therefore, nine sites would require an accessory motif for efficient kinase encounter. Four RXL motifs are present in human CDC25B3 at positions 155, 369, 391, and 571 and are strongly conserved in bird and mammal sequences. Their role in substrate recognition has not been evaluated. However, even if fully functional, the ability to phosphorylate many minimal (S/T)P sites would still be a problem. Cheng et al., discussing their and previous studies, concluded that only a minimal distance between the (S/T)P and the RXL sites is required and that

stimulation of (S/T)P phosphorylation by RXL binding can operate provided structural flexibility of the target molecule allows both sites to interact.74 An examination of the predicted disorder propensity of the CDC25B sequence (Figure 6) indicates that the N-terminal 370 amino acids, which correspond to the regulatory region and contain almost all (S/T)P sites, are predicted to be essentially unstructured (see Dyson and Wright for a recent review).76 Regions of high disorder allow protein pliability. Therefore, we hypothesize that in CDC25B structural disorder allows a small number, possibly only one, of accessory sites to help recruit a large number of minimal (S/T)P sites for phosphorylation. In addition to sites fitting the minimal (S/T)P consensus, eight sites were phosphorylated in vivo. Phosphorylation of S563 has also been demonstrated by the use of a specific antibody.31 All these sites (S89 or 91, S151, S178, S182, S229 or 230, S323, S353, S375, and S563) were also phosphorylated in vitro by stress kinase Chk1. Sites S230 and S563 are phosphorylated during the normal cell cycle by Chk1 in the absence of DNA damage.31 Other kinases such as MAPKAP kinase-2, pEg3, Akt, or Aurora A may contribute to the phosphorylation of some of these sites under the conditions of unperturbed growth used for our samples.25,26,45,48,49 Yet our results confirm that Chk1 phosphorylates CDC25B at sites S151, S230, and S323, which cooperate for 14–3–3 binding, nuclear exclusion, and maintenance of the phosphatase in an inactive state,50,77 and at site S563, involved in binding to CDK/cyclin substrates.22 Other previously unreported Chk1 sites, S89 (or S91), S178, S182, S353, Journal of Proteome Research • Vol. 7, No. 3, 2008 1271

research articles S465, and T561, may also contribute to the overall regulation and maintenance of CDC25B in an inactive state during interphase.

Acknowledgment. This work was supported by the CNRS and the University of Toulouse. It was supported in part by the Ligue Nationale Contre le Cancer, Equipe Labelisée 2005 (UMR5088 team), the Génopole Toulouse Midi-Pyrénées, and the Région Midi-Pyrénées (UMR5089 team). Supporting Information Available: MS/MS spectra indicating phosphorylation of CDC25B on S42, S50, S151, S160, S178, S238, S323, S353, S375, S229/230, S15, T69, S182, T344, T355, S470, T167, S465, T561, S563, S169, S327, and T404 (Figures S1 to S23, respectively). Comparison between CDC25B3 sites phosphorylated by Chk1 in vitro and sites respecting consensus (R/T)-X(2–3)-(S/T)-not P (A) or (R/T)-X(2–3)-(S/T)-P (B) (Supporting Information Table 1). This material is available free of charge via the Internet at http://pubs.acs.org.

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