Gaëlle Marteil

Jean-Philippe Gagné

Ewa Borsuk

Laurent Richard-Parpaillon

Guy G Poirier

Jacek Z Kubiak

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition

Cyclin-Dependent Kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase Promoting Factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phaseexiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: i) two forms of CDK1: Cdc2A and B, ii) a set of cyclins B with clearly diminishing number of peptides identified upon M-phase exit, iii) a number of known CDK1 substrates (e.g. peroxiredoxine)

and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 threedimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

Introduction

MPF (M-phase Promoting Factor) is the universal molecular regulator of M-phase progression [START_REF] Masui | Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes[END_REF]. Identification of the molecular nature of MPF was possible due to the appropriateness Xenopus laevis oocytes appropriateness for biochemical analysis. MPF is composed of a kinase, CDK1 (Cyclin-Dependent Kinase 1), and a regulatory subunit, Cyclin B [START_REF] Gautier | Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+[END_REF][START_REF] Lohka | Purification of maturation-promoting factor, an intracellular regulator of early mitotic events[END_REF]. CDK1/Cyclin B is associated with a third component of the complex, the p9 protein (Xe-p9 in Xenopus, Cks1 and Cks2 in mammals, orthologues of fission yeast Suc1 protein; [START_REF] Van Zon | The APC/C recruits cyclin B1-Cdk1-Cks in prometaphase before D box recognition to control mitotic exit[END_REF]. The activation of CDK1 triggers M-phase entry, whereas its inactivation is linked to M-phase exit [START_REF] Labbe | MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B[END_REF][START_REF] Riabowol | The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells[END_REF]. In Xenopus, CDK1 is activated during oocyte maturation and inactivated upon fertilization triggering embryo development. The sperm entry or a parthenogenetic treatment trigger an increase in intracellular free Ca 2+ concentration promoting CDK1 inactivation that allows MII exit and the beginning of embryo development.

This inactivation is due to the dissociation of CDK1/Cyclin B complex, Cyclin B degradation and CDK1 Thr-161 dephosphorylation [START_REF] Nishiyama | A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase[END_REF][START_REF] Chesnel | Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos[END_REF][START_REF] Chesnel | Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract[END_REF]. More precisely, the APC/C (Anaphase-Promoting Complex/Cyclosome)-dependent polyubiquitination of cyclin B targets the fully active CDK1/Cyclin B complex to the 26S proteasome. The 19S regulatory particle of the proteasome unfolds and dissociates Cyclin B from CDK1 [START_REF] Nishiyama | A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase[END_REF][START_REF] Chesnel | Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos[END_REF]. Cyclin B dissociation is the earliest and indispensable step of CDK1 inactivation. Once separated from CDK1, Cyclin B is degraded, whereas the kinase is dephosphorylated on Thr-161 by type 2C protein phosphatase [START_REF] Chesnel | Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract[END_REF]. Both CDK1 Thr-161 dephosphorylation and Cyclin B degradation ensure the irreversibility of CDK1 inactivation. CDK1 inactivation requires its Tloop refolding, which closes the access of the kinase enzymatic active site. This closure may be mediated by chaperones, assuring the rapidity of CDK1 inactivation (reviewed in [START_REF] Kubiak | Canonical and alternative pathways in Cyclin-Dependent Kinase 1/cyclin B inactivation upon M-phase exit in Xenopus laevis cell-free extracts[END_REF]. Thus CDK1 inactivation may require modifications in association with different partners.

The increasing knowledge on CDK1 makes the hypothesis of a role of CDK1-specific inhibitors and/or chaperones in CDK1 inactivation very attractive. In mammals, two types of CDK inhibitors (CKIs) have been described (for review see De [START_REF] De Clercq | Cyclin-dependent kinase inhibitors in yeast, animals, and plants: a functional comparison[END_REF]. The Ink4 family inhibits CDK4 and CDK6 by binding to CDK instead of Cyclin [START_REF] Mcconnell | Induced expression of p16(INK4a) inhibits both CDK4-and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes[END_REF][START_REF] Parry | Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors[END_REF], while Cip/Kip family members bind to and inhibit CDK/Cyclin complexes [START_REF] Chen | Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA[END_REF]. In Saccharomyces cerevisiae, Cdc6 in cooperation with Sic1 and Hct1 were postulated to participate in timely Cdc28 (CDK1-homologue) inhibition [START_REF] Calzada | Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases[END_REF]. This function has been recently suggested by Yim and Erikson (2011) in HeLa cells, but has never been shown in developmentally-regulated processes or in Xenopus. However, the CDK1 inhibitory role of Cdc6 remains controversial even in yeast [START_REF] Archambault | Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit[END_REF]. No other CKI has ever been shown to regulate mitotic CDK1 activity, and we wondered whether such regulators could exist during M-phase and participate in the regulation of CDK1 regulation in Xenopus.

Our goal in the present study was to identify CDK1 interactors during M-phase-arrest and upon oocyte activation leading to the entry into the first embryonic interphase. For several reasons, this transition in Xenopus laevis oocytes provides a unique opportunity for the biochemical study of putative changes in the composition of the CDK1 complex. First, the transition from active to inactive CDK1 is easily inducible and highly synchronous. Second, Xenopus laevis oocyte cytoplasm is abundant and rich in proteins [START_REF] Nishiyama | A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase[END_REF][START_REF] Chesnel | Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos[END_REF][START_REF] Chesnel | Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract[END_REF]. The feasibility of proteomic analysis of such oocytes and embryos was already demonstrated by our previous MS study of ubiquitinated X. laevis proteins [START_REF] Bazile | Differential Proteomic Screen To Evidence Proteins Ubiquitinated upon Mitotic Exit in Cell-Free Extract of Xenopus laevis Embryos[END_REF]. Our results show that the composition of the CDK1 complex is indeed modified during CDK1 inactivation and consequently these data open new avenues for studying the function of so far unknown protein associations with this major Mphase regulator.

Materials and Methods

Egg collection and activation

Xenopus laevis females were purchased from NASCO (Fort Atkinson, WI). Females were subcutaneously injected with human chorionic gonadotropin (hCG, 500 IU/female).

Unfertilized eggs (UFEs) were collected and washed with F1 buffer (31.25 mM NaCl, 1.75 mM KCl, 60 µM MgCl 2 , 2 mM NaHCO 3 , 10mM Hepes, 0.25 mM CaCl 2 , pH 7.6). UFEs were dejellied with 2% L-cysteine in F1 buffer, pH 7.8. Aliquots of 200 UFEs for both CDK1 IP and p9 precipitation and of 20 UFEs for Western blotting analysis were made and frozen in liquid nitrogen. Eggs were activated using 0.5µg/mL calcium ionophore A23187 for 90 sec.

Aliquots of 200 activated UFEs were taken out about 7 min after ionophore treatment for both CDK1 IP and p9 precipitation, and aliquots of 20 eggs were taken out 3, 7, 9, 15 and 30 min after treatment for Western blotting analysis.

Samples preparation for Western blotting

Eggs were homogenized in MPF-stabilizing buffer (80mM β-glycerophosphate, 50 mM NaF, 20 mM EGTA, 20 mM Hepes, 15mM MgCl 2 , 1mM DTT, pH 7.5) with mixture of protease inhibitors (1mM AEBSF and 10µg/mL of aprotinin, leupeptin, pepstatin) and 10µg/mL of sodium orthovanadate and centrifuged (10000 g, 15 min, 4°C). The egg extract was mixed with Laemmli buffer [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF], heated at 85°C and stored at -20°C.

CDK1 immunoprecipitation

To IP CDK1 during M-phase and M-phase exit, we used protein extracts of unfertilized (200 UFE) and activated eggs (200 eggs at 7 min post activation). Affi-Prep protein A beads were washed three times with TBS-Triton X100 (50mM Tris-HCl, 150 mM NaCl, 0.01% Triton X100, pH 7.5). The beads were then pre-equilibrated with or without (for IP negative control and for pre-clearing step) rabbit polyclonal antibody raised against XlCDK1 C-terminal peptide (a gift from T. Lorca, CRBM, Montpellier, France), overnight at 4°C in TBS-Triton X100 (50mM Tris-HCl, 150 mM NaCl, 0.01% Triton X100, pH 7.5), supplemented with proteases inhibitors and sodium orthovanadate (as above). We selected this antibody because it was previously successfully used to specifically immunoprecipitate CDK1 from X. laevis oocytes [START_REF] Krasinska | Cdk1 and Cdk2 activity levels determine the efficiency of replication origin firing in Xenopus[END_REF]. Fifteen hours later, aliquots of 200 eggs were homogenized in 1mL of immunoprecipitation buffer (20mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 2mM EDTA, 50 mM NaF, 50mM β-glycerophosphate, 0.2% NP-40) and centrifuged at 10 000g for 15 min at 4°C. Protein extracts were centrifuged again at 10 000g for 15 min at 4°C. Volumes of protein extracts were adjusted to 2 mL with IP buffer containing protease inhibitors, and a 15µl aliquot was collected for Western blotting analysis ("total fraction").

After three brief washes with IP buffer, naked beads (200µL per protein extract) were incubated with protein extracts and the mixture was agitated for 2 hours at 4°C to pre-clear the extract. The mixture of naked beads and egg extracts was then centrifuged at 4000 rpm, 3 min, 4°C. The supernatants were mixed with 100µL of naked beads (IP negative control) or of Affi-Prep protein A beads cross linked to CDK1 antibodies (dimethyl pimelimidate dihydrochloride was used as cross linking agent) previously washed twice with homogenizing buffer and agitated for 2.5 hours at 4°C. After centrifugation (1300g, 3 min, 4°C), the supernatants were kept for a second round of IP, but an aliquot of 15µL for each supernatant was conserved for Western blotting analysis ("unbound fraction"). The pellets were washed three times with 1.5 mL of TBS-Triton X100 with protease inhibitors. Proteins were eluted from beads using 100µL of 100mM glycine, pH 2.0. The eluted proteins were frozen at -20°C and the beads were eluted a second time with glycine and washed three times with TBS-Triton X100 and two times with IP buffer. The additional round of IP was performed as previously described by incubating the supernatant of the first round with the beads. The two aliquots of eluted proteins were pooled and neutralized with 1M Tris-HCl, pH 9.2. Laemmli buffer was added and the samples were heated at 85°C for 5min. A small aliquot was retained for Western blot analysis (bound fraction) and the remaining was analyzed by mass spectrometry.

CDK1 precipitation with p9 beads

To precipitate CDK1 with p9 beads during M-phase and M-phase exit, we used protein extracts made from unfertilized (200 UFE) and activated eggs (200 eggs taken out 7 min. after activation). p9-Sepharose beads were a kind gift from L. Meijer and O. Lozach (Marine Station, Roscoff, France;[START_REF] Vogel | Molecular cloning and characterisation of p15(CDK-BP), a novel CDK-binding protein[END_REF]. p9-bound beads and naked Sepharose beads for pre-clearing were pre-equilibrated overnight at 4°C in homogenizing buffer (25mM MOPS, pH 7.2, 60mM β-glycerophosphate, 15mM EGTA, 15mM MgCl 2 , 2mM DTT, 1mM NaF, 1mM sodium orthovanadate) with protease inhibitors and 1% BSA. Aliquots of 200 eggs were homogenized in 0.8 mL of homogenizing buffer and centrifuged at 10 000g for 15 min at 4°C. Protein extracts were centrifuged at 10 000g for 15 min at 4°C. Volumes of protein extracts were adjusted to 1.5 mL with homogenizing buffer including protease inhibitors and a 20µl aliquot was collected for Western blotting ("total fraction"). Naked Sepharose beads (200µL per protein extract) were incubated with protein extracts and agitated for 2 hours at 4°C. After centrifugation (1300g, 3 min, 4°C), supernatants were mixed to 200µL of p9 beads or naked beads (negative control) and agitated for 2.5 hours at 4°C. After centrifugation (1300g, 3min, 4°C), the supernatants (30µL) were collected for Western blotting ("unbound fraction") and the pellets of p9 beads were washed four times with 5 mL of washing buffer (Tris-HCl pH 7.4, 5mM NaF, 250 mM NaCl, 5mM EDTA, 5mM EGTA, 0.1% Nonidet P-40) with protease inhibitors. The proteins were eluted with 150 µL of Laemmli buffer and heated at 85°C for 5min.

Immunoblotting

Proteins were separated by 12% SDS-PAGE and transferred to Hybond C membranes (GE Healthcare). Membranes were probed with antibodies to: CDK1 (mouse monoclonal, MS-110-PO, Interchim), Cyclin B2 (gift from T. Lorca, CRBM, Montpellier, France; rabbit polyclonal) and MCM4 (both gifts from M. Méchali, IGH, Montpellier, France, rabbit polyclonal).

Sample preparation for mass spectrometry analysis

Eluted proteins were resolved on a 4-12% Criterion™ XT Bis-Tris gradient gel (Bio-Rad), and stained with SYPRO Ruby (Bio-Rad). Images were acquired on a Geliance CCD-based bioimaging system (PerkinElmer).

LC-MS/MS analysis

The entire protein profile of each lane on SDS-PAGE was sliced from the gel into 16 bands using a gel excision Lanepicker™ (The Gel Company). Gel slices were deposited into 96-well plates. In-gel protein digestion was performed by a MassPrep™ liquid handling station (Micromass), using sequencing-grade modified trypsin (Promega). Peptide extracts were dried using a SpeedVac™. Peptide extracts were separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by ES MS/MS. Analyses were performed on a Thermo Surveyor MS pump connected to an LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, CA). Peptide separation took place within a PicoFrit column BioBasic C18, 10 cm x 0.075 mm internal diameter (New Objective, Woburn, MA) with a linear gradient from 2 to 50% solvent B (acetonitrile, 0.1% formic acid) in 30 min, at 200 nL/min. Mass spectra were acquired using data dependent acquisition mode (Xcalibur software, version 2.0). Each full-scan mass spectrum (400 to 2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion function was enabled (30 s exclusion), and the relative collisional fragmentation energy was set to 35%.

Interpretation of Tandem-MS Spectra

The RAW files generated from MS/MS spectra were uploaded to the MASCOT search engine (Matrix Science, London, UK; version 2.2.0) for protein identifications. The parameters for database searching were as follows: (I) Protein database: Uniref_100, (II) Taxonomy:

Xenopus laevis [TaxID: 8355, 15 599 entries], (III) Trypsin digestion with up to two missed cleavage sites, (IV) Fragment and parent ion mass tolerance were 0.5 Da and 2.0 Da respectively, (V) Iodoacetamide derivative of cysteine as a fixed modification and oxidation of methionine as a variable modification.

Criteria for protein identification

Scaffold (version 03_00_01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications, and to provide confidence level (% probability) of the identification. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the PeptideProphet algorithm [START_REF] Keller | Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search[END_REF]. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 distinct peptides. Protein probabilities were assigned by the ProteinProphet algorithm [START_REF] Nesvizhskii | A statistical model for identifying proteins by tandem mass spectrometry[END_REF]. Proteins containing similar peptides that could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. A Scaffold 3 report is provided as Supporting Information. This file can be accessed with a free viewer available from Proteome Software, Inc. website (http://www.proteomesoftware.com). This file contains all the spectral information, including the accession number for each protein sequence, Mascot scores, protein sequence coverage and amino acid residues modifications, statistical probability modeling, and spectral counting.

Results

We have applied two different affinity-based approaches to identify proteins bound to CDK1.

The first approach is a classical CDK1 pull-down using an anti-CDK1 antibody [START_REF] Krasinska | Cdk1 and Cdk2 activity levels determine the efficiency of replication origin firing in Xenopus[END_REF], whereas the second is based on CDK1 precipitation via p9 beads [START_REF] Patra | Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control[END_REF]. MS analysis was used to identify CDK1-associated proteins in M-phase, when the kinase is fully active, and during its inactivation upon M-phase exit. Experiments were repeated several times to establish the method. Below, we present data from single experiments obtained using IP and p9 purifications according to the final satisfactory procedure described in Materials & Methods section. Scaffold software was used to merge the identifications, validate the peptides and proteins hits and discriminate homologous proteins (150 unique proteins, 8866 spectra, 0.1% protein false discovery rate (FDR), see Supplementary file Protein IDs. SF3)

CDK1 immunoprecipitation

As shown in Fig. 1a, the protein markers (Mcm4 and cyclin B2) confirm MII exit. CDK1 immunoprecipitations were performed on extracts from MII-arrested eggs (T0) and activated eggs (7 minutes after activation, T7). A negative IP control with naked beads instead of anti-CDK1-coupled beads was performed. To assess the yield during the stages of purification, aliquots of total egg extract (total), unbound fraction (unbound) and proteins bound to the beads (bound) were taken for Western blot analysis. CDK1 was almost completely depleted from extracts (compare unbound and bound fractions from IP T0 and T7 in Fig. 1B). To verify if this approach also enables CDK1-interacting proteins to be precipitated, we assessed the presence of Cyclin B2. As expected, Cyclin B2 was also depleted and found in beadbound fractions. We observed a clear decrease in cyclin B2 abundance between T0 and T7 extracts, confirming activation of eggs. No signal for CDK1 and Cyclin B2 was found in negative-control bound fractions, indicating that protein interactions were specific. These results showed that both CDK1 and CDK1-associated proteins were efficiently depleted. SYPRO Ruby-stained gel containing all four bound fractions (M-phase, M-phase exit and their respective negative controls) showed quantitative and qualitative differences (Fig. 1C). Control samples contained much less protein than immunoprecipitation samples, indicating that proteins interacting with CDK1 are abundant and that only few proteins interact nonspecifically with naked beads. Differences between bound fractions of M-phasearrested and activated extracts were also visible, suggesting that partners of CDK1 change upon M-phase exit. To identify CDK1 partners, protein bands corresponding to the entire gel were excised and analyzed by LC-MS/MS. We identified 30 proteins during M-phase (Table 1) and 42 during M-phase exit (Table 2). A total of 23 proteins were common to both samples. As expected, the major biological process that stands out for CDK1-associated proteins is cell cycle progression (Fig. 2). A few other purified proteins are involved in DNA/RNA metabolism, cytoskeleton/vesicle transport, fertilization for the M-phase sample.

Most importantly for the validity of the analysis, the two isoforms of CDK1, Cdc2-A and Cdc2-B, were identified in both samples. Moreover, among the 21 proteins common to both samples, four are involved in cell division, and two, Cyclins B2 and B4, are known regulatory subunits of CDK1. The two other cell cycle proteins common for M-phase and M-phase exit samples, are ERK2 (mitogen activated protein kinase 1) and calcineurin A (a serine/threonine protein phosphatase). Another category of proteins shared between M-phase and M-phase exit are chaperones belonging to T-complex protein1 (Tcp1) family and heat-shock proteins (A8 and D1).

Among proteins specifically interacting with M-phase CDK1, three are egg envelope components (XlZPA, egg envelope glycoprotein and component ZPAX). M-phase exitspecific CDK1-interactors are mainly involved in metabolism (7 out of 19) and this group apparently increases upon CDK1 inactivation. Among them, two are involved in proteolysis (arginyl aminopeptidase and LOC431925 protein). Interestingly, Septin 9 found in this group is potentially involved in late mitosis and cytokinesis [START_REF] Bi | Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis[END_REF][START_REF] Cao | The evolution, complex structures and function of septin proteins[END_REF][START_REF] Nguyen | The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis[END_REF][START_REF] Spiliotis | A mitotic septin scaffold required for Mammalian chromosome congression and segregation[END_REF]. The other proteins found exclusively during Mphase exit are involved in protein folding (a member of Tcp1 family), cytoskeleton/vesicle transport (2 proteins), DNA/RNA metabolism (2 proteins) and other functions (5 proteins). As expected, fertilization-specific proteins were missing in CDK1 complexes following oocyte activation.

Affinity purification with p9-beads

As shown in Fig. 3A, protein markers confirmed egg activation. As in CDK1 IP, p9 precipitation was performed on extracts from MII (T0) eggs, and eggs 7 min after activation (T7). CDK1 was almost completely depleted from egg extracts (compare unbound and bound fractions from p9 T0 and T7 in Fig. 3B). We also searched for Cyclin B2 in the different fractions to test whether this method allows co-precipitation of CDK1 partners. As expected, Cyclin B2 was depleted from the extract, and was found in the bound fraction of the T0 extract. We did not detect Cyclin B2 in the bound fraction of the T7 extract, due to its almost complete degradation at this time point. No signals for CDK1 and Cyclin B2 were detected in the negative control bound fractions, indicating that protein interactions were specific. These results showed that p9 precipitation depleted both CDK1 and its associated proteins.

The staining of the gel containing all four bound fractions showed fewer differences between samples than in IP experiments (Figs. 1C and3C). Nineteen proteins were identified during M-phase (Table 3) and 37 during M-phase exit (Table 4). Sixteen proteins were common for the two samples. As in IP, p9 precipitation allowed us to identify more proteins in activated oocytes compared to MII-arrested ones (Fig. 3C 3 and4) in both samples showing that other identified proteins may be partners of either Cdc2-A or Cdc2-B subunit, and/or of CDK2. Identification of Cyclins B2 and B4 confirmed the presence of CDK1-associated proteins in p9 samples. Concerning specific M-phase proteins in p9precipitates, two are B-type Cyclins (B1 and B5) and one is involved in cell adhesion (Junction plakoglobin). Proteins specific for M-phase exit (Table 4) are mainly involved in metabolism (6 out of 21), DNA/RNA metabolism (6 proteins), protein folding (3), and other functions (6).

Comparative analysis of the two approaches

Among all proteins co-purified with CDK1 and identified by these two approaches, six were common for all samples: Cdc2A, Cdc2B, Cyclin B2, Cyclin B4, Peroxiredoxin-2 and Heat shock protein A8 (Fig. 5). The presence of two CDK1 isoforms and two B-type Cyclins (B2 and B4) in all samples confirms that CDK1 partners could indeed be present among other identified proteins. None of the proteins was exclusively present in M-phase samples, whereas two proteins involved in metabolism were specific for M-phase exit samples: pyruvate kinase and the α subunit of ATP synthase (Fig. 5). The presence of pyruvate kinase and the α subunit of ATP synthase in M-phase exit samples obtained by two methods argue for their real association with CDK1.

Discussion

Validation of the proteomic screen

Our mass spectrometry analysis of CDK1 co-IPed proteins was supplemented by analysis of p9-precipitates. Large-scale proteomic screens often give rise to false-positive interactions (e.g. [START_REF] Trinkle-Mulcahy | Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes[END_REF]). The analysis of p9-precipitated proteins focused on verification of major CDK1 partners found by IP, as p9 beads also precipitate p9-specific partners, which may not be associated with either CDK1 or CDK2. Despite this shortcoming that makes the p9 precipitation less stringent for CDK1 partners than CDK1 IP, p9-beads are widely used to precipitate CDK1 in Xenopus oocytes, for instance to measure CDK1 activity [START_REF] Patra | Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control[END_REF]. In both cases two CDK1 isoforms, Cdc2-A and Cdc2-B, and CDK1 partners, cyclins B2 and B4, were identified. CDK1 was used as a positive control in a proteomic screen using HeLa cells and cyclins B were also identified as CDK1 interactors as expected (Fig. S5(B) in Hutchins at al. 2010; www.mitocheck.org ). In addition, in our screen the number of identified peptides of two cyclins B diminished in M-phase exit samples concomitant with their advanced degradation. The successful identification of remaining Cyclins B2 and B4 in activated oocytes (both with IP and p9 precipitation) demonstrates the relative sensitivity of the method used here to purify CDK1 protein complexes. These data auto-validated our screen and confirmed that we efficiently co-IPed partners of CDK1 (Cdc2-A or Cdc2-B) complexes. The absence of CDK2, the closest CDK to CDK1 in oocytes, in the IP further confirmed the specificity of this method. Thus the combination of methods used was appropriate for the purification of complexes containing CDK1 in both states: fully active and being inactivated CDK1 complex purification. Among other identified proteins only two were common for all samples (Peroxiredoxin-2 and Heat shock protein A8) and two (pyruvate kinase and the α subunit of ATP synthase) specific for M-phase exit samples and detected by both approaches. All these proteins were found often as contaminants in other proteomic analyses [START_REF] Trinkle-Mulcahy | Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes[END_REF]). However, the absence of these proteins in our control argues for their true association with CDK1 (see Tables 1234). As p9 precipitation is a method that enables the purification of CDK1, CDK2 and p9 partners, specific interactors of CDK1 are diluted among all co-precipitated proteins. As a consequence, their identification by mass spectrometry analysis could be impaired explaining why the number of overlapping proteins between the two methods is low. Another explanation could rely on differences in experimental conditions, more stringent for p9 precipitations than for IP, decreasing the number of proteins purified on p9 beads. Nevertheless, we managed to identify molecules engaged in CDK1 complexes at two studied states of activity and the major components common for the two methods.

2. CDK1 partners specifically present during M-phase exit

Our proteomic analysis revealed the presence of two proteins specifically detected during Mphase exit: pyruvate kinase and the α subunit of ATP synthase, both involved in cellular energy metabolism. Pyruvate kinase PKM2 was also identified as a CDK1 partner in human cells, but without indication of a potential cell cycle-dependent association [START_REF] Hutchins | Systematic analysis of human protein complexes identifies chromosome segregation proteins[END_REF]www.mitocheck.org). An interaction between CDK1 and the α subunit of ATP synthase has never been demonstrated and its significance remains unclear considering their functions and subcellular localization. Pyruvate kinase and ATP synthase could associate with inactive CDK1 or could be attached to a common support fixing CDK1 during its inactivation for example on cytoskeletal elements, as CDK1 associates with the spindle and its inactivation requires a functional spindle [START_REF] Kubiak | The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF[END_REF][START_REF] Thibier | In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes[END_REF]). The association between CDK1 and metabolic enzymes involved in cell cycle progression is an attractive hypothesis. For instance, 6-phosphofructo-2-kinase (PFKFB3) and fructose 2,6bisphosphate activate CDK1 to phosphorylate p27 in HeLa lysates, indicating that these proteins may have direct allosteric effects activating CDK1 [START_REF] Yalcin | Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases[END_REF]). The link between ATP cycle and CDK1 inactivation is also obvious in the light of the requirement of ATP for cyclin B polyubiquitination and M-phase exit [START_REF] Miniowitz-Shemtov | ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint[END_REF].

Interestingly, Hutchins and collaborators (2010) also found, besides pyruvate kinase, a number of metabolic enzymes associated with CDK1 in HeLa cells (e.g. carbonic anhydrase CA2, carbamoyl-phosphate synthase 1 CPS1, tRNA (cytosine-5-)-methyltransferase NSUN2, phosphoglycerate kinase 1 PGK1, 3-phosphoglycerate dehydrogenase PHGDH, thymidine phosphorylase TYMP; www.mitocheck.org).

Among potential CDK1 partners specifically present during M-phase exit following CDK1 IP, two are known to be involved in cytoskeleton functions: dynein, the microtubule motor protein [START_REF] Burakov | Dynein and dynactin as organizers of the system of cell microtubules[END_REF] and septin 9, a GTP binding protein involved in microtubule and actin function [START_REF] Cao | The evolution, complex structures and function of septin proteins[END_REF]. This is also in agreement with results obtained by Hutchins and colleagues (2010), who identified a number of cytoskeletal proteins associated with human CDK1 (e.g. ezrin EZR, actin-related protein 3 homolog ACTR3, actin binding protein coronin CORO1C, dynactin 1 DCTN1, Cytoplasmic dynein 1 heavy chain 1 DYNC1H1, non-erythrocytic spectrin SPTBN2; www.mitocheck.org). CDK1/cyclin B is localized on the spindle in mitosis and meiosis, where it regulates spindle formation and maintenance [START_REF] Brunet | Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space[END_REF][START_REF] Huo | Cell-cycledependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse[END_REF][START_REF] Nakamura | The cytoskeleton-dependent localization of cdc2/cyclin B in blastomere cortex during Xenopus embryonic cell cycle[END_REF]. Moreover, an intact spindle is necessary for cyclin B degradation and CDK1 inactivation [START_REF] Kubiak | The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF[END_REF][START_REF] Thibier | In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes[END_REF]. Thus, interactions between CDK1 and microtubule-interacting proteins seem of great importance for spindle maintenance and for correct localization of CDK1 own residual activity. The presence of actin-related proteins both in our screen and by [START_REF] Hutchins | Systematic analysis of human protein complexes identifies chromosome segregation proteins[END_REF] argues the need for a serious analysis of CDK1-actin cytoskeleton interactions.

CDK1 partners common for M phase and M-phase exit

Among common putative partners of CDK1 identified by our two approaches, the molecular chaperone HspA8, a member of Hsp70 family, seems to be particularly interesting. Hsp70 colocalizes with CDK1 on the meiotic spindle of sea urchin oocytes [START_REF] Geraci | Localization of HSP70, Cdc2, and cyclin B in sea urchin oocytes in non-stressed conditions[END_REF]. A member of the Hsp70 family (Hsp70-2) is a molecular chaperone of CDK1 involved in CDK1/cyclin B1 complex formation, and essential for the cell cycle in spermatogenesis [START_REF] Dix | Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility[END_REF][START_REF] Zhu | HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes[END_REF]. Interestingly, we did not find any members of the Hsp90 family, known as Mos-specific chaperone in Xenopus laevis oocytes [START_REF] Fisher | Hsp90 is required for c-Mos activation and biphasic MAP kinase activation in Xenopus oocytes[END_REF].

Taken together, these results show a clear molecular link between the Hsp70 family and CDK1, confirmed by our proteomic screen.

The other common protein for all samples is Peroxiredoxin-2, a protein involved in cell redox homeostasis [START_REF] Hofmann | Peroxiredoxins[END_REF]. Two members of Peroxiredoxin family (Prx family) including Prx-2 are phosphorylated in vitro by CDK1 [START_REF] Chang | Regulation of peroxiredoxin I activity by Cdc2-mediated phosphorylation[END_REF]. In vivo phosphorylation of Prx-1 on Thr90 occurs only during mitosis when CDK1 is active. A proteomic screen performed in bovine oocytes also revealed that Prx-2 is phosphorylated during oocyte maturation via CDK1 or MAPK [START_REF] Bhojwani | Molecular analysis of maturation processes by protein and phosphoprotein profiling during in vitro maturation of bovine oocytes: a proteomic approach[END_REF]. CDK1-mediated phosphorylation at Thr90 of Prx-1 leads to decrease of its activity [START_REF] Chang | Regulation of peroxiredoxin I activity by Cdc2-mediated phosphorylation[END_REF]. The significance of Prx-1 inactivation and consequently of H 2 O 2 increase on the M-phase progression remains unclear. However, one putative target of H 2 O 2 could be the Cdc25C phosphatase, a major CDK1 activator [START_REF] Morgan | Principles of CDK regulation[END_REF]Perdiguero and Nebreda, 2004;[START_REF] Wang | Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition[END_REF]. Cdc25C activity requires reducing agents, and oxidative stress induces its degradation [START_REF] Savitsky | Redox regulation of Cdc25C[END_REF]. The CDK1-Prx-2 interaction suggested by our screen could lead to CDK1-dependent inhibitory phosphorylation of Prx-2, and consequently an increase in H 2 O 2 concentration. This oxidative stress could participate in Cdc25C inactivation, and in turn CDK1 inactivation. As peroxiredoxins often appear often as contaminants in proteomic screens [START_REF] Trinkle-Mulcahy | Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes[END_REF], the potential association between Prx-2 and CDK1 will have to be carefully examined.

Among common potential CDK1 partners during M-phase and M-phase exit in both methods used in our study, several were involved in cytoskeletal functions. In the IP experiments kinectin, a membrane anchor for kinesin [START_REF] Kumar | Kinectin, an essential anchor for kinesin-driven vesicle motility[END_REF], was especially abundant. Copin, a calcium-dependent membrane binding protein involved in membrane trafficking [START_REF] Creutz | The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans[END_REF] was also identified during M-phase and M-phase exit by IP.

On the other hand, cofilin 1-A [START_REF] Bernstein | ADF/cofilin: a functional node in cell biology[END_REF] was identified in both p9 samples (Tables 3 and4). A member of cofilin family was already reported as a substrate of CDK1 and its phosphorylation is involved in the functionality of inositol (1,4,5)-trisphosphate receptors in starfish oocytes [START_REF] Santella | Activated M-phasepromoting factor (MPF) is exported from the nucleus of starfish oocytes to increase the sensitivity of the Ins(1,4,5)P3 receptors[END_REF]. Copin A knockout in Dictyostelium leads to the formation of multinucleated cells [START_REF] Damer | Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium[END_REF] and cofilin localizes at the contractile ring, suggesting their involvement in cytokinesis [START_REF] Nagaoka | Concentration of cofilin, a small actinbinding protein, at the cleavage furrow during cytokinesis[END_REF]. Together with the identification of septin 9 during M-phase exit by IP, these results suggest interplay between CDK1 and cytokinesis. Other proteins linking CDK1 and cytoskeleton are members of TCP1 complex family, identified in two IP samples. They are involved in tubulin folding [START_REF] Brown | Molecular chaperones and the centrosome. A role for TCP-1 in microtubule nucleation[END_REF] and colocalizes with CDK1 at the mitotic spindle in sea urchin embryos [START_REF] Agueli | A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATPdependent manner: a new chaperone role is proposed[END_REF]. All these results suggest that CDK1 is cytoskeleton-anchored.

Among common putative CDK1 partners during M-phase and M-phase exit present only in IP samples, two are involved in cell cycle regulation: calcineurin A and ERK2.

Calcineurin is a highly conserved phosphatase composed of a catalytic subunit, calcineurin A and a regulatory one, calcineurin B [START_REF] Rusnak | Calcineurin: form and function[END_REF]. Calcineurin is required for CSF release during MII exit in Xenopus oocytes via dephosphorylation of Apc3 and Cdc20 contributing to APC/C activation and cyclin B2 degradation [START_REF] Chung | Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint[END_REF][START_REF] Mochida | Calcineurin is required to release Xenopus egg extracts from meiotic M phase[END_REF][START_REF] Nishiyama | Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis[END_REF][START_REF] Yudkovsky | Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint[END_REF]. Calcineurin is also required for completion of meiosis in Drosophila oocytes [START_REF] Takeo | Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila[END_REF]. The association between calcineurin A and CDK1 that we found may suggest that these two proteins regulate each other, or are linked by a common support (e.g. the cytoskeleton).

ERK2 MAP kinase is involved in CDK1 activation during oocyte maturation [START_REF] Castro | c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation[END_REF]. The ERK2 pathway catalyzes the inhibitory phosphorylation of Myt1 kinase, responsible for the inhibitory phosphorylation of CDK1 on Thr14 and Tyr 15 [START_REF] Mueller | Myt1: a membraneassociated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15[END_REF], and is required for CDK1 activation. ERK2 is also involved in activation of Cdc25C at the G2/M transition [START_REF] Wang | Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition[END_REF] and Cdc25 is essential for CSF arrest [START_REF] Lorca | Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle[END_REF]. A clear feedback links ERK2 and CDK1 in meiosis [START_REF] Abrieu | The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes[END_REF], which changes during embryonic mitosis [START_REF] Bazile | Absence of reciprocal feedback between MPF and ERK2 MAP kinase in mitotic Xenopus laevis embryo cell-free extract[END_REF]). Thus, CDK1 and ERK2 are functionally related especially during meiosis. However, until now no physical association between these proteins has been shown.

Different CDK1 complexes with potentially different functions

Each of two CDK1 isoforms identified in this study (Cdc2-A and B) may be associated with different types of partners. The nature of the cyclin associated with CDK1 is essential for the substrate specificity of CDK1. Upon M-phase exit the timing of cyclin degradation is sequential [START_REF] Hochegger | New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation[END_REF]. Thus, different CDK1 complexes may exert various functions according to the type of cyclin they harbour [START_REF] Gong | The roles of cyclin A2, b1, and b2 in early and late mitotic events[END_REF][START_REF] Kõivomägi | Dynamics of Cdk1 substrate specificity during the cell cycle[END_REF]. The spectrum of different CDK1 complexes can be enlarged by association with other partners identified in the current study.

Conclusions

In this paper we have shown that CDK1 interacts with different proteins depending on the cell cycle stage (namely upon MII-arrest and oocytes activation). Our results show the necessity to study individually CDK1-other proteins associations to fully understand the regulation of this major cell cycle kinase. It also completes information on potential CDK1 associated proteins previously obtained in much larger screen of protein complexes involved in mitotic regulation by [START_REF] Hutchins | Systematic analysis of human protein complexes identifies chromosome segregation proteins[END_REF] in human cells. Our dynamic approach points the necessity to analyse the evolution of protein complexes to better understand cell cycle regulation and especially the mitotic progression.

Figure legends

  ). A functional classification indicates that the proteins identified are mainly involved in cell cycle, folding (heat shock proteins 70 kDa), metabolism (L-isoaspartate O-methyltransferase and XlGst) and DNA/RNA metabolism (Nif3-like protein 1, Ribosomal protein S30, Double-stranded RNA-binding protein A). Remaining proteins are involved in cytoskeleton/vesicle transport (Cofilin-1-A) and other functions (Traf2 and Nck-interacting protein kinase, Peroxiredoxin-2, Galectin-VIIa). As expected, p9 beads precipitated two isoforms of CDK1, Cdc2-A and Cdc2-B, next to CDK2 (represented by a single peptide, indicating a low amount; see Tables

Fig. 1 .

 1 Fig. 1. Co-immunoprecipitation of CDK1 and its partners during M-phase and M-phase exit. (A) Egg activation was checked by following Mcm4 phosphorylation status and Cyclin B2 (CycB2) abundance by immunoblotting of extracts of eggs taken 0, 3, 7, 9, 15, 30 min after calcium ionophore addition. (B) Co-immunoprecipitation of CDK1 and its partner (CycB2)during M-phase (T0) and M-phase exit (T7) was checked by following its presence either in protein fractions bound to beads (Bound) or in the non-retained fractions (Unbound). The presence of CDK1 and CycB2 was also checked in negative controls (C-, naked beads) in order to control the specificity of the interactions. (C) Comparative protein profiles of bound fraction of each samples: M-phase CDK1 IP (T0), its negative control (c-T0), M-phase exit CDK1 IP (T7) and its negative control (c-T7). Eluted proteins were resolved on SDS-PAGE and revealed with SYPRO Ruby staining.

Fig. 2 .

 2 Fig. 2. Distribution of protein functions based on Gene Ontology (GO) classification and bibliography of putative MPF interactors during M-phase (IP T0) and M-phase exit (IP T7) identified by CDK1 immunoprecipitation followed by MS analysis.

Fig. 3 .

 3 Fig. 3. Co-precipitation of CDK1 and its partners using p9 beads during M-phase and Mphase exit. (A) Egg activation was checked by following Mcm4 phosphorylation status andCycB2 abundance by immunoblotting extracts of eggs taken 0, 3, 7, 9, 15, 30 min after calcium ionophore addition. (B) Co-precipitation of CDK1 and its partner (CycB2) during Mphase (T0) and M-phase exit (T7) was checked by following its presence either in protein fractions bound to the beads (Bound) or in the non-retained fractions (Unbound). The presence of CDK1 and CycB2 was also checked in negative controls (C-, naked beads) in order to control the specificity of the interactions. (C) Comparative protein profiles of bound fraction of each samples: M-phase CDK1 p9 precipitation (T0), its negative control (c-T0), M-phase exit CDK1 p9 precipitation (T7) and its negative control (c-T7). Eluted proteins were resolved on SDS-PAGE and revealed with SYPRO Ruby staining.

Fig. 4 .

 4 Fig. 4. Distribution of protein functions based on Gene Ontology (GO) classification and bibliography of putative CDK1 interactors during M-phase (P9 T0) and M-phase exit (P9 T7)identified by CDK1 precipitation via p9 beads followed by MS analysis.

Fig. 5 .

 5 Fig. 5. Common putative CDK1 interactors between IP and p9 precipitation during M-phase and M-phase exit. (A) Six proteins are common between IP and p9 precipitation M-phase samples (Cdc2A, Cdc2B, CycB2, CycB4, HspA8 and peroxiredoxine-2). (B) Eight proteins are common between IP and p9 precipitation M-phase exit samples (Cdc2A, Cdc2B, CycB2, CycB4, HspA8, Peroxiredoxine-2, Pyruvate kinase and the alfa subunit of ATPase). (C) Six proteins are common between IP and p9 precipitation M-phase and M-phase exit samples (Cdc2A, Cdc2B, CycB2, CycB4, HspA8 and Peroxiredoxine-2).

Table 1 .

 1 Potential interactors of CDK1 identified by mass spectrometry from CDK1 immunoprecipitation in metaphase II arrest X. laevis UFE

	Protein name	Accession Number	Molecular (kDa) Weight	Unique T0 peptide c-	Unique IP T0 peptide	Biological process
	1 kinectin 1	Q08B03	97	0	12	Microtubule-based
						movement
	2 Cell division control protein 2-A	P35567	35	0	11	Cell division
	3 Heat Shock Protein A8	Q7ZTK6	71	1	9	ATP binding
	4 Cyclin B2	Q6PA39	44	0	5	Cell division
	5 Mitogen-activated protein kinase 1	P26696	41	0	4	Cell cycle
	6 Cyclin B4	Q98TI3	44	0	4	Cell division
	7 Egg cortical granule lectin	Q91719	34	0	4	Signal transduction
	8 Transitional endoplasmic reticulum	P23787	89	0	3	Transport
	ATPase
	9 Histone deacetylase 10	Q569T0	77	0	3	hydrolase activity
	10 Cell division control protein 2-B	P24033	35	0	3	Cell division
	11 T-complex protein 1 gamma subunit	P50143	61	0	3	Protein folding
	12 Sorcin	A2VDA2	22	0	2	Calcium ion binding
	13 RuvB like protein	Q0IH85	51	0	2	ATP binding
	14 Heat shock protein d1	Q6IP60	62	0	2	Protein refolding
	15 T-complex protein 1 subunit 5	A0AUT4	59	0	2	Protein folding
	16 XlZPA protein	A1L3D9	78	0	2	Single fertilization
	17 Peroxiredoxin-2	Q6ING3	22	0	1	cell redox
						homeostasis
	18 Nucleoporin	Q6PAY1	55	0	1	transmembrane
						transport
	19 Chaperonin containing TCP1, subunit	Q5XGK8	52	0	1	Protein folding
	7
	20 Calcineurin A	O57438	58	0	1	Cell division
	21 Copin	Q6P7H2	59	0	1	vesicle-mediated
						transpor
	22 Egg envelope glycoprotein	Q4VGP0	108	0	1	Unknown
	23 Nupl1 protein	A4FU91	60	0	1	transmembrane
						transport
	24 Hadh2-prov protein	Q6DCM8	27	0	1	oxidation reduction
	25 Acad9-prov protein	Q6DDF2	68	0	1	oxidation reduction
	26 Aldehyde dehydrogenase family 18,	Q6GMF0	89	0	1	proline biosynthetic
	member A1					process
	27 Egg envelope component ZPAX	B4F6R0	102	0	1	Unknown
	28 LOC100049148 protein	A4QN33	95	0	1	Unknown
	29 Thyroid hormone receptor-associated	Q5BJ39	108	0	1	regulation of
	protein 3 (TRAP150)					transcription
	30 MGC68559 protein	Q6PAA1	24	0	1	Calcium ion binding

Table 2 :

 2 Potential interactors of CDK1 identified by mass spectrometry from CDK1 immunoprecipitation in activated X. laevis UFE (proteins highlighted in grey are common between metaphase II arrested and activated UFE)

	Protein name	Accession Number	Molecular Weight (kDa)	Unique peptide c-T7	Unique peptide IP T7	Biological process (GO annotation)
	1 Kinectin 1	Q08B03	97	0	9	Microtubule-based
						movement
	2 Cell division control protein 2-A	P35567	35	2	8	Cell division
	3 Mitogen-activated protein kinase 1	P26696	41	0	4	Cell cycle
	4 T-complex protein 1 gamma subunit	P50143	61	0	4	Protein folding
	5 Heat Shock Protein A8	Q7ZTK6	71	1	4	ATP binding
	6 Transitional endoplasmic reticulum	P23787	89	0	3	Transport
	ATPase
	7 Histone deacetylase 10	Q569T0	77	0	3	hydrolase activity
	8 Nucleoporin	Q6PAY1	55	0	3	transmembrane
						transport
	9 Calcineurin A	O57438	58	0	3	Cell division
	10 Copin	Q6P7H2	59	0	3	vesicle-mediated
						transport
	11 Member of T-complex protein 1	Q6GMA6	58	0	3	Protein folding
	chaperonin family
	12 ATP synthase beta subunit	Q7ZWR6	56	0	3	ATP biosynthetic
						process
	13 Hadh2-prov protein	Q6DCM8	27	0	3	oxidation reduction
	14 Arginyl aminopeptidase	Q641C7	70	0	3	Proteolysis
	15 Cyclin B2	Q6PA39	44	0	2	Cell division
	16 Cell division control protein 2-B	P24033	35	0	2	Cell division
	17 Chaperonin containing TCP1, subunit 7	Q5XGK8	52	0	2	Protein folding
	18 RuvB like protein	Q0IH85	51	0	2	ATP binding
	19 Nupl1 protein	A4FU91	60	0	2	transmembrane
						transport
	20 Dynein, cytoplasmic 1, intermediate	Q5U238	72	0	2	Transport
	chain 2
	21 Aldehyde dehydrogenase family 18,	Q6GMF0	89	0	2	proline biosynthetic
	member A1					process
	22 ATPase family AAA domain-containing	Q58E76	67	0	2	ATP binding
	protein 3-A
	23 ATP synthase subunit alpha	Q68EY5	60	0	2	ATP biosynthetic
						process
	24 Von Willebrand factor A domain	Q6IND5	94	0	2	Unknown
	containing 5A, gene 2
	25 LOC733268 protein	Q0IH65	27	0	2	Unknown
	26 Acyltransferase	Q642P5	54	0	2	fatty-acyl-CoA
						biosynthetic process
	27 LOC431925 protein	Q6NR96	210	0	1	Proteolysis
	28 Cyclin B4	Q98TI3	44	0	1	Cell division
	29 Peroxiredoxin-2	Q6ING3	22	0	1	cell redox homeostasis
	30 Sorcin	A2VDA2	22	0	1	Calcium ion binding
	31 Heat shock protein d1	Q6IP60	62	0	1	Protein refolding
	32 T-complex protein 1 subunit 5	A0AUT4	59	0	1	Protein folding
	33 Acad9-prov protein	Q6DDF2	68	0	1	oxidation reduction
	34 Nuclear pore glycoprotein	A2BDA4	50	0	1	transport
	35 Ruvbl1 protein	Q6GR29	50	0	1	ATP binding

Acknowledgements

We are grateful to Laurent Meijer and Olivier Lozach (Roscoff, France) for p9 beads, Thierry Lorca (Montpellier, France) for the gift of a large quantity of precipitating XlCDK1 antibody , Marcel Méchali (Montpellier, France) for MCM4 antibody and James R. Hutchins (Montpellier, France) for valuable discussions and critical reading of the manuscript. The authors were supported by grants from Ligue Contre le Cancer and Association pour la Recherche contre le Cancer to JZK. GM was a recipient of a fellowship from the French Ministère de la Recherche et de l'Enseignement supérieur and MED from the Lebanese Government.


Tadeusz M Krygowski

Wojciech P Oziminski

Christopher A Ramsden

Sigma-and pi-electron structure of aza-azoles

Keywords:

The reasons behind changes of aromaticity in 10 unsubstituted aza-azoles was analysed by employing the Natural Bond Orbital (NBO) approach at the MP2/6-311+G(d,p) level of theory. Sum of occupations of p z orbitals at atoms in the ring correlates well with the magnetism based aromaticity index NICS as well as with the number of nitrogen atoms in the ring. Changes of NICS depend strongly in a linear way on the number of NN bonds.

Classification of azoles based on the number of pyridine-type nitrogen atoms vicinal to NH is supported by plotting the relative occupation of  orbitals ( occ ) against the relative occupation of  orbitals ( occ ) for all individual atoms in rings.

Introduction

Azoles belong to an interesting and important class of -electron system [START_REF] Katritzky | Handbook of Heterocyclic Chemistry[END_REF][START_REF] Katritzky | Comprehensive Heterocyclic Chemistry -III[END_REF], which fulfill the Hückel 4n+2 rule but have different degrees of aromaticity [START_REF] Cyrański | [END_REF]. In aza-azoles the aromatic stabilization energy (ASE) depends on the number and location of the pyridine-type nitrogen atoms in the ring, especially how many pyridine-type nitrogen atoms are next to the pyrroletype nitrogen atom [4]. The pyrrole-type nitrogen possesses an electron lone pair perpendicular to the molecular plane and nominally contributes two electrons to the aromatic sextet. There can also be up to four pyridine-type nitrogen atoms, each of them contributes a single electron to the electronic sextet from a 2p z orbital and their lone pairs lie in the ring plane. These two types of nitrogen atoms, and also the carbon atoms present in the ring, have their own -electron structure, which may in some way interfere with the -electron structure, and contribute to the picture of -electron delocalization. There is a long-standing dispute about how far -electron delocalization or -electron structure is responsible for aromatic equalization of bonds length and other aromatic properties [5][6][7][8][9][10]. A number of approaches have shown either significant colinearities between some aromaticity indices or lack of correlation [11][12][13]. These contradictions may be due to different physical/chemical bases for the indices themselves or to specific relationships between and electron contributions to the properties taken to measure "aromatic character". Hence, in this paper we investigate the interrelationship between the and -electron structures in aza-azoles.

Methods

Geometry optimizations and all other calculations were performed at the MP2/6-311+G(d,p) level of theory using the Gaussian 03 suite of programs [14]. All optimizations were followed by frequency calculations to ensure that the calculated stationary points are true minima on the potential energy surface. Natural Bond Orbital (NBO) [15] analysis was performed by NBO 5.G program interfaced to Gaussian03 using MP2 density matrix with the keyword density=current. The NICS [16] aromaticity index was calculated as a shielding constant (taken with minus sign) of a ghost atom located at the geometric center of the ring and NICS(1) was calculated 1Å above the ring.

Results and discussion

To analyze separately and -electron distributions we employed an occupation partitioning scheme based on Natural Population Analysis (NPA). This partitioning scheme is based on NBO analysis, which is well known for producing reliable results for organic compounds, comparing to other charge partitioning methodologies like Mulliken population analysis which is strongly basis set dependent. The advantage of NPA approach lies also in the ease of separation of and electronic systems in the case of flat aromatic molecules. If the molecule is oriented in the xy plane the s, p x and p y natural atomic orbitals (NAOs) occupations can be directly assigned to electronic system and the p z NAOs occupations to electronic system. We used similar approach in our previous study about substituted benzene molecules [17].

In our approach the -electron population of the ring is calculated as a sum of the populations of the s, p x and p y natural atomic orbitals of the ring atoms. Subtracting the "nominal" number of  electrons expected for the studied system, expressed by variable "C" (see Eq. 1), gives the extent to which the studied system deviates from the "nominal" distribution: this value we call sEDA [sigma Electron Donor-Acceptor]. The parameter "C" changes in the series of azoles because a pyrrole-type nitrogen atom contributes only three electrons to the -system (like carbon) but each pyridine-type nitrogen atom contributes four electrons to the -system. Thus, the value of "C" is equal to 15 in the case of pyrrole 1 but is incremented as the number of pyridine-type atoms increases, reaching the maximum value of 19 for pentazole 10.

Similarly, the pEDA [pi Electron Donor-Acceptor] parameter is obtained by summing up the populations of the p z natural atomic orbitals (perpendicular to the molecule plane) and subtracting the "nominal" value of 6 (two electrons from pyrrole-type nitrogen atoms and one electron from each other atom). Thus, pEDA [Eq. 2] expresses the deviation from the "nominal" or "ideal" value of the electronic sextet.

C sEDA j j     5 1  (1) 6 5 1     j j pEDA  (2)
An analysis of pEDA shows how the distribution of  electrons depends on the number of nitrogen atoms in the ring. Fig. 1 presents this relationship for which the correlation coefficient (CC) is 0.993. This means that an increase of the number of electronegative nitrogen atoms, which replace less electronegative CH units, increases the population of electrons in the ring. This necessarily means that the electron structure of the ring is less populated.

<Figure 1>

Azoles may be classified [4] according to the number (N 2,5 ) of pyridine-type nitrogen atoms neighboring the NH group in the ring. Thus, the "N 2,5 =0" group are pyrrole 1, 1H-imidazole 3 and 4H-1,2,4-triazole 7, the "N 2,5 =1" group are 1H-pyrazole 2, 1H-1,2,3-triazole 4, 1H-1,2,4triazole 6 and 1H-tetrazole 8, and the "N 2,5 =2" group are 2H-1,2,3-triazole 5, 2H-1,2,3tetrazole 9 and pentazole 10. When pEDA values are plotted against sEDA values for all ten azoles (Scheme 1), the observed correlation is expected since the -structure may only take electrons from the -structure. Fig. 2 presents this dependence (CC = -0.987).

<Scheme 1> <Figure 2>

It should be noted that changes in pEDA are dramatically smaller than those for sEDA. When the CH units are replaced by pyridine nitrogen atoms the total -population of the ring increases and the -population decreases. The decrease of -population is partially because hydrogen atoms are source of electron density for -system and the number of hydrogen atoms becomes smaller and smaller when consecutive CH groups are replaced by N atoms.

Deeper analysis of the above relationship (Fig. 2) is achieved by plotting the relative occupation of  orbitals ( occ ) against the relative occupation of  orbitals ( occ ) for individual atoms. In this way are obtained three scatter plots: two for the two types of nitrogen atom (Figs. 3 and4) and one for the carbon atoms (Fig. 5). <Figure 3> <Figure 4> <Figure 5> When all the points are taken into account, lack of a good correlation in the scatter plots in Figs. 3, 4 and 5 is not unexpected. Atoms participating in the ring differ in their electronegativities (pyrrole-type, pyridine-type and carbon) and also in their close environment. Nevertheless, a general trend is common to all three relationships: the slopes are positive indicating the necessity that if the  structure becomes richer in electrons then the  structure must lose them. However in all three cases (Fig. 345) three well-separated clusters (or subsets) can be observed for which the slopes are negative. The plots show that the changes in / relationships depend on the electronegativity of atoms involved in the whole structure of azoles, as well as their neighborhood. Moreover, the clusters are grouped following the rule dependent on the number of 2,5 pyridine-type nitrogen atoms neighboring the relevant atom.

For the dependence of orbital occupations of pyrrole-type nitrogen N1  occ on N1 occ , (Fig. 3), the type "N 2,5 =2" systems lose more  electrons than those of type "N 2,5 =1" and these still less than for type "N 2,5 =0". This rule is nicely illustrated by showing the occupation on these atoms in the four simplest cases: for pyrrole 1, imidazole 3, pyrazole 2 and 2H-1,2,3-triazole 5, as shown in Scheme 2.

<Scheme 2>

The highest occupation on the pyrrole-type nitrogen occurs in pyrrole 1, where there are no interactions with any nitrogen atoms, then for imidazole 3, where the pyridine-type nitrogen atom is distant. For the examples of neighboring one "N 2,5 =1" and two "N 2,5 =2" pyridine-type nitrogen atoms (3 and 5), the occupation on the pyrrole-type nitrogen atom decreases significantly. Thus, it may be concluded that direct interactions between pyridine-type nitrogen and pyrrole-type nitrogen atoms is the most effective factor for delocalization of p z electrons from pyrrole nitrogens towards the vicinal nitrogen atoms and then further in the ring. This observation can be rationalized as follows: electronegative nitrogen atoms replacing CH cause a decrease of  electron occupation on the p z orbital of the pyrrole-type nitrogen atom relative to the effect due to less electronegative carbon atoms. The electron pair of this nitrogen atom is drawn out to the ring as the number of electronegative pyridine nitrogen atoms increase, replacing less electronegative carbon atoms. The  system of the ring may withdraw electrons also from C-H bonds by means of an inductive effect. As the consecutive pyridine-like nitrogen atoms are introduced into the ring the number of C-H bonds is decreasing so the possibility to draw  electrons into the ring is also decreased.

For the dependence of pyridine-type nitrogen N  occ on N  occ , (Fig. 4) a similar trend is observed. Again, three classes of pyridine nitrogen form clusters, according to the number of nitrogen atoms directly bonded to the relevant nitrogen atom, and the slopes are again negative. In each cluster, the more nitrogen atoms, then the lower is  occ . Similarly, for the dependence of carbon atoms C  occ on C  occ , (Fig. 5), the slopes in clusters are also negative and the occupation of the -orbital on a carbon atom increases with a decrease of the number of nitrogen atoms.

We conclude that two factors, which are not always independent, play an important role in deciding the relationship between and -electron structure in azoles. The first factor is the number of pyridine-type nitrogen atoms (N 2,5 = 0, 1 or 2) neighboring the pyrrole nitrogen (NH), and the second is the number of CH units replaced by pyridine-type nitrogen atoms.

One more interesting symptom of the dependence of -electron structure on the relative locations of pyridine-and pyrrole-type nitrogen atoms is that mean HOMA values for the subgroups [4] "N 2,5 =0", "N 2,5 =1" and "N 2,5 =2" increase as N increases, as shown in Schemes 3. Similarly, mean ASE values (taken from [START_REF] Cyrański | [END_REF]) distinguish between these three subgroups.

<Scheme 3>

The population of electrons located on orbitals in the ring (pEDA) is nicely related to the aromaticity index NICS(1) [18] indicating that pEDA may also act as a quantitative measure of azole aromaticity. Since the population of -electrons depends on their delocalization from the pyrrole-type nitrogen atom, the less -electrons are present on this atom, the more they are delocalized on the ring and the more aromatic is the ring as measured by delocalization indices such as NICS [START_REF] Katritzky | Handbook of Heterocyclic Chemistry[END_REF]. The correlations in Figs. 6 and7 support this view. The greater value of pEDA, i.e. the population of -molecular orbitals, the more negative NICS(1), i.e. the more aromatic the system in question. The same type of dependence is present for NICS(1) or NICS(1) ZZ . When NICS [START_REF] Katritzky | Handbook of Heterocyclic Chemistry[END_REF] values are plotted against the number of NN bonds, a linear relationship is significant, as shown in Figure 8. An increase of ASE on a sequence of systems belonging to subgroups N 2,5 =0, 1 and 2 is nicely associated with the change of structure, i.e. the value of angle XNH -. For subclass "N 2,5 =0" =124.9. for subclass "N 2,5 =1", where the interaction is one sided =118.6, and for symmetric class "N 2,5 =2" =121.2. A reasonable explanation is that interaction of lone pair with N-H dipole (Fig. 9B) is attractive, and thus may be stabilizing the system in question.

<Figure 9>

Conclusions

Occupation of molecular  orbitals in azoles depends linearly on the number of nitrogen atoms: the more nitrogen atoms then the higher occupation. The linear relationship between pEDA and the number of nitrogen atoms in azoles.

Regression equation: y = 0.0076x -0.1083; CC= 0.993

Fig. 2

The inter-relationship of total-ring pEDA on sEDA. Regression equation: y=-0.0329x -0.0666; CC=0.987 Click here to download high resolution image

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Figure 9a

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Figure 9b

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Figure 9c

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  Scheme 3 show one more interesting feature. The averaged ASE and HOMA values for azole subgroups N 2,5 =0, N 2,5 =1, and N 2,5 =2 increase with an increase of the number of possible interactions between the NH group and a lone pair of a pyridine-type nitrogen atom. These interactions are associated with the change of HNN angles in molecules that are unsymmetrical and the values are smaller than those observed for HNC angles. The averaged value for three cases with one-sided (lone pair)/NH interactions [1H-pyrazole 2, 1H-1,2,3triazole 4 and 1H-124-triazole 6] is 119.1 o whereas for (lone pair)/CH interactions [pyrrole 1, imidazole 3 and 4H-1,2,4-triazole 7] the averaged value is 126.2 o . Evidently, the attraction between lone pair and NH-bond decreases the angle. Moreover, as results from Scheme 3 indicate, the number of such interactions is also associated with an increase of aromaticity, i.e. the ASE and HOMA values. It is also interesting to note that natural charges on pyrrole-type nitrogen atoms are for N 2,5 =0, N 2,5 =1 and N 2,5 =2 type atoms ~0.53e, ~0.33e and ~0.13e, respectively, indicating that pyridine/pyrrole NH interactions decrease charge on the NH nitrogen atom.

  Classification of aza-azoles by counting NH/N-lone pair interactions (N 2,5 ) is documented by dependences of pEDA on sEDA estimated for pyridine-like nitrogen, pyrrole-like nitrogen and carbon atoms. The three classes of azoles (N 2,5 =0, 1 or 2) clearly differ when aromatic stabilization energies (ASE) or HOMA values are compared.

Figure captions Scheme 1 2 3

 123 Figure captions

Fig. 3 Fig. 4 Fig. 5 CC = 1 )Fig. 6 1 2 3

 34516123 Fig. 3The inter-relationship of pyrrole-type nitrogen  occ on  occ (left: y = -0.7399x -0.0988; CC = 0.995)(middle: y = -0.5743x -0.0625; CC = 0.811)(right: y = -0.1772x -0.2904; CC = 0.851)

Figure 1

 1 Figure 1Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image

Figure 4

 4 Figure 4Click here to download high resolution image

Figure 5

 5 Figure 5 Click here to download high resolution image

Figure 6

 6 Figure 6Click here to download high resolution image

Aromaticity of azoles follows an additivity rule: NICS(1) correlates linearly with the number of NN bonds as well as with the number of nitrogen atoms.Interactions between NH bond and the lone pair of the neighboring pyridine-type nitrogen atom lead to a dramatic decrease of CNH angle: 118.6 o for pyrazole compared to 124.9 o for pyrrole.

Acknowledgments

Computational Grant G31-10 from the Interdisciplinary Centre of Mathematical and Computer Modelling (ICM) at Warsaw University is gratefully acknowledged.