TPPII, MYBBP1A and CDK2 form a protein–protein interaction network

Archives of Biochemistry and Biophysics 564 (2014) 128–135

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TPPII, MYBBP1A and CDK2 form a protein–protein interaction network Jarmila Nahálková ⇑, Birgitta Tomkinson Uppsala University, Department of Medical Biochemistry and Microbiology (IMBIM), BMC, Box 582, SE-751 23 Uppsala, Sweden

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Article history: Received 22 June 2014 and in revised form 24 September 2014 Available online 7 October 2014 Keywords: TPPII MYBBP1A CDK2 Cell cycle Apoptosis

a b s t r a c t Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein–protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein–protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein–protein interaction network of these proteins. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Cytosolic proteolytic degradation is primarily performed by the ubiquitin–proteasome system (UPS1) complemented downstream by oligo- and aminopeptidases. TPPII complements UPS [12] by removing N-terminal tripeptides from longer peptides [2]. TPPII is a major degrading enzyme capable to cleave substrates with specific length of the polypeptide chain > 15 amino acid residues produced by the proteasome only in limited amounts [20,29], and this ability can represent a regulatory effect for some biologically important peptides. TPPII has also raised attention in cancer research due to its overexpression in several cancer cell lines and by its regulatory effect on apoptosis. Inhibition of TPPII activity directly induces apoptosis in Burkitt-lymphoma cells (BL), which are generally resistant to the presence of proteasome inhibitors. TPPII is required for cell growth and survival in these cells [5]. Specific inhibition of ⇑ Corresponding author. E-mail addresses: [email protected], jarmila.nahalkova@gmail. com (J. Nahálková). 1 Abbreviations used: UPS, ubiquitin–proteasome system; BL, Burkitt-lymphoma cells; IMDM, Iscove’s Modified Dulbecco’s Medium; DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; PBS, phosphate buffered saline; DPBS, Dulbecco’s modified PBS; co-IP, co-immunoprecipitation; WB, Western blot; PLA, Proximity Ligation Assay; SB, sample buffer; HRP, Horseradish peroxidase; CTCF, corrected total cell fluorescence; CDK2, cyclin-dependent kinase 2. http://dx.doi.org/10.1016/j.abb.2014.09.017 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

enzymatic activity of TPPII by butabindide and genetic knockdown of TPPII gene by siRNA suppress centriole aberrations and cause significant cell death and suppression of cell growth in c-myc overexpressing BL cells [4]. On the other hand, TPPII over-expression induces accelerated growth and resistance to apoptosis in HEK293 cells [23], thus suggesting a functional link between TPPII and cell cycle regulation. TPPII knockout mice have induced cell-type specific death programs expressed by early immunosenescence and shorter lifespan due to premature aging of animals [9]. TPPII may also participate in the apoptotic cascade upstream of caspase-1 by promoting pro-caspase-1 maturation [7]. Furthermore, TPPII contributes to cellular stress response by translocation from cytoplasm to nucleus upon ROS production and DNA damage, which may represent a functional link connecting mitochondrial respiration to DNA damage signaling in the nucleus [19]. The investigation of TPPII interactions with other proteins can promote an understanding of the regulatory effect of TPPII on cell cycle, apoptosis and senescence and it may assist in identifying the appropriate signaling pathways. The present study demonstrates that TPPII physically interacts with the tumor suppressor MYBBP1A, a protein with an activation effect on p53 and with the key cell cycle regulator protein CDK2 that is involved in the apoptotic regulation by cell cycle arrest at G1. In addition, the interaction found between CDK2 and MYBBP1A in this study, link the functional mutual interactions among TPPII, MYBBP1A and

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CDK2 with the cell cycle regulation, tumor suppression and cell death mechanisms.

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Co-immunoprecipitation assay HeLa and HEK 293 transformants over-expressing murine highwere washed once with cold DPBS (pH 7.4) and harvested by cell scraper. The cells where washed twice more by ice-cold DPBS using 6 min centrifugation at 125g. Cellular pellets were extracted by 10 min incubation with RIPA buffer (50 mM Tris– HCl, pH 7.4; 150 mM NaCl; 1% (w/v) NP-40; 0.25% (w/v) sodium deoxycholate; 15% glycerol (v/v) and 0.1% SDS) with the addition of Protease inhibitor cocktail (P8340, Sigma–Aldrich Sweden AB) by adding 50 ll of buffer/106 cells followed by 30 min centrifugation at 16,000g. For preparation of nuclear fractions, HEK293 cells overexpressing highactTPPII were fractionated according to the protocol of Suzuki et al. [28] and the resulting nuclear fractions were used for co-IP assay. The protein concentration in the supernatants was quantified by measuring the absorbance at 280 nm using NanoDrop 2000c Spectrophotometer (Thermo Scientific, Inc.). The cell lysates (500 lg total protein/250 ll) were mixed with 1 lg IgG of appropriate antibody or unimmunized IgG (IgG control) and incubated overnight at 4 °C at slow speed on a tube rocker. Negative controls were prepared by omitting primary antibodies and by adding corresponding volume of PBS into the mixture. Then, 20 ll of the Bio-Adembeads Protein A/G (Ademtech, Pessac, France) was added and incubated for 3 h at 4 °C followed by three washing steps with RIPA buffer. Samples were eluted by PAG elution buffer (Ademtech, Pessac, France) and after 2 min incubation at room temperature supernatants were neutralized using 1 M Tris–HCl buffer (pH 7.5). Beads were subsequently eluted with LDS sample buffer (SB) (Bolt™, Novex by Life Technologies™, Invitrogen, Sweden) for 5 min at 95 °C and supernatants were loaded together with fractions from first elution step on SDS–PAGE. actTPPII

Material and methods Cell lines and cell culture Chronic myelogenous leukemia K562 cells (ATCC CCL-243™) were cultivated in Iscove’s Modified Dulbecco’s Medium (IMDM, GibcoÒ by Life Technologies™, Sweden); HeLa and HEK293 (ATCC CRL 1573) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, GibcoÒ by Life Technologies™. Sweden). All cells were grown with addition of 10% (v/v) fetal bovine serum (FBS, Sigma–Aldrich AB), 100 U/ml penicillin and 100 lg/ml streptomycin. Protein–protein interactions of TPPII were performed using HEK293 transformant overexpressing murine TPPII (highactTPPII; called m(-)TPP in [24] and forming active high-molecular weight multimers, in contrast to human TPP II overexpressed in these cells [25]. Control cells are HEK293 cells transformed with empty pcDNA 3 expression vector. HEK293 cells were grown in DMEM supplemented by geneticin (100 lg/ml; G418 disulfate salt solution) and amphotericin (2.5 lg/ml). All antibiotics were purchased from Sigma–Aldrich AB. The inhibitory study using TPPII inhibitor butabindide was performed in ExCELLÒ 293 Serum-Free Medium for HEK293 cells (SAFC Biosciences, Inc., Sigma–Aldrich AB) supplemented with 6 mM L-glutamine. HEK 293 cells were grown for 24 h prior addition of butabindide at concentration 1 or 10 lM followed by incubation for 22 h and cell fixation. The effect of CDK2 inhibitor II (Calbiochem, Merck KGaA, Germany) was studied in HeLa cells starved for 48 h in DMEM without presence of FBS. The inhibitor at the concentration of 100 lM or corresponding volume of dimethylsulfoxide (DMSO; mock treatment) was added into the medium 6 h prior to cell fixation. Anoikis assay was performed in 2 ml of ExCELLÒ 293 SerumFree Medium by plating HEK 293 cells on polystyrene cell culture dishes with diameter 6 cm at density 5  106 cells/plate. The plates for cell growth at detached conditions were prepared by coating them overnight using 1% (w/v) Pluronic Poloxamer (O-BASF, Ludwigshafen, Germany) in phosphate buffered saline (PBS; pH 7.4) at 4 °C. Cells were incubated in either coated or uncoated plates for 4 h at 37 °C, collected gently by cell scraper and washed by Dulbecco’s modified PBS (DPBS, pH 7.4) prior to RNA extraction and quantitative RT-PCR. Triplicate experiments were performed with each treatment and cell type.

Antibodies For co-immunoprecipitation (co-IP), Western blot (WB) and in situ Proximity Ligation Assay (PLA) the following primary antibodies have been exploited: Chicken anti-TPPII raised by Immunsystem AB, Uppsala, Sweden and immunopurified according to Tomkinson & Zetterquist [26] (WB); TPPII (E-17) sc-15148 (WB, co-IP, PLA) and CDK2 (D-12) sc 6248 (WB, co-IP, PLA) were obtained from Santa Cruz Biotechnology, Inc, Heidelberg Germany; pAB anti-MYBBP1A (NB100-61050) (WB, co-IP, PLA) was purchased from Novus Biologicals, Cambridge, UK. Secondary antibodies for Western blot were the following: Rabbit anti-goat IgG antibody [HRP] NB710-H was purchased from Novus Biologicals, Cambridge, UK; ECL™ anti-mouse IgG, HRP-linked F(ab´)2 fragment (from sheep) Na9310V and ECL™ anti-rabbit IgG, HRP-linked F(ab´)2 fragment (from donkey) Na9340V were from GE Healthcare, Uppsala, Sweden; Donkey anti-chicken IRDyeÒ 800 CW 926-32218 was purchased from LiCOR Biosciences UK Ltd.

SDS–PAGE and immunoblotting Protein samples quantified by Microplate BCA Protein Assay Kit (Fisher Scientific AB, Göteborg, Sweden) were mixed with LDS sample buffer, reduced and separated on Novex 4–12% Bis-Tris Plus gels using MES SDS Running buffer (20) (Bolt™, Novex by Life Technologies™, Invitrogen, Sweden) and transferred to Immobilon-P Membrane (PVDF, 0.45 lm; Millipore AB, Solna, Sweden). Horseradish peroxidase (HRP)-conjugated secondary antibodies were detected after incubation with PierceÒ ECL Western Blotting Substrate (Fisher Scientific, AB, Göteborg, Sweden) by means of ChemiDoc™ MB System equipped with Image Lab™ Software, Biorad, Sundbyberg, Sweden. IRDye labeled secondary antibodies were visualized by ODYSSEY Infrared Imaging System, LI-COR Biosciences UK Ltd., Cambridge UK.

LC–MS/MS The samples obtained by co-IP were run on Novex 4–12% Bis-Tris Plus gels (Bolt™, Novex by Life Technologies™, Invitrogen, Sweden) and stained by ‘blue silver’ staining method [3]. In-gel digestion, reduction and alkylation steps were performed according to the protocol of Leibniz Institute for Age Research – Fritz Lipmann Institute, Germany (http://www.fli-leibniz.de/vbmf/SELDIMALDI/InGelDigest_Silverstain.pdf) adapted from Shevcenko et al. [22]. Sequencing Grade Modified Trypsin (Promega Biotech AB, Nacka, Sweden) was used for in-gel digestion of the samples. The samples were analyzed on a 6330 Ion Trap LC/MS system and protein identification was performed by Spectrum Mill database search (Agilent Technologies Sweden AB).

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In situ Proximity Ligation Assay (PLA) Cells were grown in Lab-Tek II 8-well chamber slides (Sigma– Aldrich Sweden AB) on media with/without presence of FBS and inhibitors according to conditions specified at Cell lines and cell culture Section. Specific growth conditions are also described in figure legends. Then cells were fixed/permeabilized using absolute methanol by 10 min incubation on ice followed by washing by PBS (pH 8.0), blocking step and incubation for 60 min with the primary antibody solution according to the Cell Signaling TechnologyÒ Immunofluorescence protocol (http://www.cellsignal.com) and further processed according to the instruction of DuolinkÒ In Situ Detection Reagents Orange (Sigma–Aldrich, St. Louis, US). Primary antibody dilutions were firstly optimized by immunofluorescence using serial dilutions of single antibodies. Control experiments were performed for each used combination of PLA probes by omitting both primary antibodies and for each protein–protein interaction by omitting one of the primary antibodies. Interaction experiments were performed at least 3-times for each primary antibody pair and inhibitory experiments were performed at least twice using the same cell type. Each chamber slide well was scanned at 3-different spots creating cca 18–25 planar sections/scan. The scanned images were visually evaluated for presence of the nuclear and cytoplasmic signal. Differences between nuclear and cytoplasmic signal are shown by presenting the image scan taken from the confocal depth approximately equal to the cell radius and its distance from cell surface section is marked on the image. In situ PLA signal of middle planar sections was quantified using ImageJ software 1.49e (National Health Institute USA), where only red channel corresponding to in situ PLA signal was used for analysis with the intention to differentiate it from fluorescence corresponding to nuclear DAPI staining. The in situ PLA nuclear signal was defined by selecting of image areas overlapping with DAPI staining. The residual intracellular signal obtained by subtraction of nuclei from images using Adobe Photoshop 7.0 was defined as in situ PLA cytoplasmic signal. Fluorescent signals from cytoplasm and nuclei of 3-times 10 cells obtained from three different images were quantified compared to control background areas picked from 5 different points of the images. The results were expressed as CTCF (corrected total cell fluorescence) = Integrated Density  (Area of selected cell  Mean fluorescence of background readings) [6]. The results were plotted as a function of CTCF depending on cell type using Microsoft Excel software. The images well representing all observations are presented for each interaction. Confocal microscopy The signal was visualized by LSM 700 Microscope System equipped with Zen 2009 software (Carl Zeiss, Jena, Germany). DuolinkÒ In Situ Detection Reagent Orange has been visualized by means of Cy3 settings using laser line/MBS 514/532 nm and emission filter >530, max at 566 nm. DAPI has been detected by 405 nm laserline equipped with emission filter >385/420 nm, max at 461 nm. RNA extraction and quantitative real-time PCR Total RNA was extracted from cells by RNEasy Mini Kit (Qiagen, GmbH, Hilden, Germany) followed by cDNA synthesis using QuantiTect Reverse Transcription Kit (Qiagen, GmbH, Hilden, Germany). Real-time PCR analysis was performed using MiniOpticon realtime PCR detection system equipped with CFX Manager™ software and using iQ™ SYBR Green Supermix (Bio-rad). Primers and PCR conditions were following:

b-Actin ActinF: 50 -AGAGCTACGAGCTGCCTGAC-30 and ActinR: 50 TGCGGATGTCCACGTCACAC-30 (350 nM each); 95 °C for 3 min, 40 cycles of 1 s at 95 °C and 20 s at 61 °C. TPPII QPCR1F: 50 -CTCGTTGGTGGGCAAGTCT-30 and QPCR1R: 50 -ATGCAGGGAGCCAGATCTTC-30 (200 nM each); 95 °C for 3 min, 40 cycles of 1 s at 95 °C and 20 s at 61 °C. MYBBP1A [27] MYBBP1AF primer: 50 -CGCACCACCTGTGCCGTGCCCGGCG-30 and MYBBP1AR primer: CTTGGTCAGGAAGGAGCTCAGTGCTG-30 (500 nM each); 95 °C for 2 min, 40 cycles of 10 s at 95 °C and 45 s at 67 °C. p21 [1] p21F primer: 50 -GGAGACTCTCAGGGTCGAAA-30 and p21R primer: 50 -TTAGGGCTTCCTCTTGGAGA-30 (200 nM each); 95 °C for 2 min, 40 cycles of 10 s at 95 °C and 15 s at 57 °C. Results The protein–protein interactions of TPPII were examined by different approaches for identification of interaction partners with a characterized functional link to known signaling pathways in order to explain the previously observed regulatory effects of TPPII on apoptosis in cancer cells. Previously, Neganova et al. [15] reported a large-scale proteomic study, which identified TPPII as one of the cyclin-dependent kinase 2 (CDK2) interacting proteins. In the present study, we have confirmed this interaction by co-IP of TPPII with anti-CDK2 antibody in cell lysates prepared from HEK293 cells overexpressing murine highactTPPII. Immunoblotting of fractions eluted from magnetic beads by both PAG and LDS-sample buffers showed immunoreactivity with anti-TPPII (Fig. 1A). Two subsequent elution steps were used for elution of antigens from immune complexes. The first elution step using low pH conditions (PAG buffer) released antigen very specifically (Fig. 1A). The second elution step using heating beads in LDS sample buffer eluted an additional portion of antigen, but also small amount of non-specifically interacting protein was detected through negative control (Fig. 1A and D). Non-immunized IgG controls, where antigen-specific antibody was replaced by IgG from the same or from unrelated species revealed weak bands, which could correspond to antigen non-specifically interacting with Protein A/G beads and to non-reduced IgG. However, the total density of bands corresponding to antigens eluted by both buffers from anti-TPPII, anti-CDK2 and antiMYBBP1A were much higher compared to controls, thus demonstrating the specificity of detected interactions (Fig. 1A, C and D). The interaction between TPPII and CDK2 was also confirmed using cell lysates prepared from HeLa cells (data not shown). The fractions eluted from anti-TPPII and anti-CDK2 immune complexes were separated by SDS–PAGE and several major protein bands were excised for identification by LC–MS/MS in order to identify additional interaction partners. MYBBP1A (AL024407; AU019902; DBP; FLJ37886; MBB1A; Myb-binding protein 1A; MYB binding protein (P160) 1a; PAP2; P160; p53-activated protein-2; p67MBP; RP23-48A2) was identified in the sample eluted from immune complex with anti-CDK2 antibody (Fig. 1B, the protein band marked with *), suggesting interaction between these two proteins. A protein band with similar relative molecular weight (Mr) was also detected in the sample using anti-TPPII (Fig. 1B) and anti-p53 co-IP-s (data not shown). It was previously reported, that MYBBP1A physically interacts with p53 [1]. The interaction of

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Fig. 1. Mutual interactions of TPPII, CDK2 and MYBBP1A detected by co-IP assay in HEK 293 cells constitutively overexpressing murine highactTPPII (A–C) and in HeLa cells (D). (A) Co-IP of TPPII with CDK2 and TPPII antibodies showing physical interaction of TPPII with CDK2. Eluted fractions were probed by immunoblotting using TPPII antibody (sc15148). (B) ‘Blue silver’ stained gel of PAG buffer eluted fractions after co-IP using anti-CDK2 and anti-TPPII antibodies. The protein band marked with asterisk was identified by LC–MS/MS as MYBBP1A. (C) MYBBP1A-CDK2 interaction detected by co-IP of MYBBP1A by CDK2 antibody in nuclear fraction prepared by subcellular fractionation. Eluted fractions were probed by immunoblotting using MYBBP1A antibody. (D) TPPII–MYBBP1A interaction detected by co-IP of MYBBP1A with anti-TPPII antibody and by probing of eluted fractions on immunoblot by anti-MYBBP1A; PAG and LDS sample buffer (SB) elutions – buffers used for elution of immune complexes from pA/G magnetic beads; Control () represents negative control with omitting of primary antibody; r (m, g) IgG – positive control containing the same quantity of rabbit (mouse, goat) IgG as the antibody used for co-IP assay.

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Fig. 2. The protein–protein interactions between TPPII and MYBBP1A detected by in situ Proximity Ligation Assay (PLA). (A) K-562 cells; (B) HeLa cells; (C) empty vector transformed (control) HEK293 cells and (D). HEK293 cells overexpressing murine highactTPPII grown for 48 h in IMDM and DMEM media both supplemented with 10% (v/v) FBS. The inhibitory effect of butabindide on TPPII–MYBBP1A interaction detected in control HEK293 cells grown in ExCELLÒ 293 Serum-Free Medium (E–L); (E and F) mock treated control HEK293 cells grown for 46 h; control cells grown for 24 h with additional 22 h incubation at the presence of (G) 1 lM butabindide and (H) 10 lM butabindide. HEK293 cells overexpressing murine highactTPPII grown in Serum-Free Medium (I–K); (I) Mock treated cells; Cells grown at the presence of (J) 1 lM butabindide and (K) 10 lM butabindide. Subcellular localization of the interaction between TPPII and MYBBP1A in control HEK293 cells (L and M); (L) control cells grown for 46 h in DMEM medium containing 10% (v/v) FBS; (M) cells grown for 24 h in ExCELLÒ 293 Serum-Free Medium with an additional 22 h incubation at the presence of 10 lM butabindide. Subcellular localization of TPPII–MYBBP1A interaction in HEK293 cells overexpressing enzymatically active murine highactTPPII (N and O) grown for 46 h in (N) DMEM medium containing 10% (v/v) FBS (O) in ExCELLÒ 293 Serum-Free Medium with an additional incubation for 22 h in the presence of 10 lM butabindide. Distance measured from the cell surface is indicated in upper right corner. Bar represents 10 lm.

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Fig. 3. The protein–protein interactions between MYBBP1A and CDK2 visualized by in situ Proximity Ligation Assay (PLA) in HeLa (A and B) and HEK293 cells (C and D). (A) HeLa cells starved for 48 h on DMEM at the absence of fetal bovine serum (FBS); (B) cells grown in DMEM at the presence of 10% (v/v) FBS; (C) vector-transformed (control) HEK293 cells and (D) HEK 293 cells overexpressing murine highactTPPII grown for 48 h in the DMEM containing 10% (v/v) FBS. Bar represents 10 lm.

Fig. 4. Quantitative analysis of in situ Proximity Ligation Assay (PLA) signal analyzed by ImageJ software. Comparison of TPPII–MYBBP1A interaction signal in (A) cytoplasm and in (B) nucleus of empty vector transformed (control) HEK293 cells and HEK293 cells overexpressing murine highactTPPII. Cells were grown in DMEM containing 10% (v/v) FBS or in ExCELLÒ 293 Serum-Free Medium for 24 h with additional incubation for 22 h at the presence of 10 lM butabindide or with addition of appropriate volume of DPBS (mock treatment). Quantitative analysis of MYBBP1A-CDK2 interactions in cytoplasm and nuclei of (C) control HEK293 cells and the cells overexpressing murine highactTPPII grown for 48 h in DMEM supplemented with 10% (v/v) FBS; (D) starving HeLa cells maintained in DMEM without addition of serum and the cells grown for 48 h in DMEM with addition of 10% (v/v) FBS. CTFC – corrected total cell fluorescence; Error bars – standard deviation of measurements of total cytoplasmic and nuclear red channel fluorescent signal produced by PLA. Measurements were performed 3-times using 10 cells obtained from three different middle planar section images.

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MYBBP1A with CDK2 was further confirmed by co-IP of MYBBP1A with anti-CDK2 antibody using nuclear extract prepared from HEK293 cells overexpressing highactTPPII (Fig. 1C). MYBBP1A coimmunoprecipitated with anti-TPPII as a protein band with the appropriate size equal to cca 175 kDa, which was eluted from magnetic beads with pA/G bound anti-TPPII and anti-MYBBP1A antibodies (Fig. 1D). Cytoplasmic interaction of TPPII with the tumor suppressor MYBBP1A is inhibited by the presence of butabindide TPPII–MYBBP1A interaction was further verified by in situ PLA technique in several mammalian cell lines including K-562 (Fig. 2A), HeLa (Fig. 2B) and HEK293 cells (Fig. 2C–O) and the fluorescent signal was quantitatively analyzed by ImageJ (Fig. 4A and B). In order to verify specificity of TPPII–MYBBP1A interaction, the assay was performed using previously characterized HEK293 transformant cells overexpressing murine highactTPPII forming high molecular weight active enzymatic complex [24]. In situ PLA signal frequency increased with overexpression of murine highactTPPII in HEK293 cells compared to the vector transformed control cells (Figs. 2C, D and 4A, B), which contain a low endogenous expression of TPP II. The requirement of enzymatically active TPPII for establishment of its interaction with MYBBP1A was further examined by the study of the inhibitory effect of the specific reversible TPPII inhibitor butabindide [21]. This was performed by adding butabindide into ExCELLÒ 293 Serum-Free Medium due to its instability in media containing serum [20]. Generally, the frequency of the interaction increased by cell growth in this medium (Fig. 2C compared to Figs. 2F and 4A, B). Butabindide suppressed cytoplasmic TPPII– MYBBP1A interactions both in HEK293 cells transformed with empty vector (Figs. 2E–H and 4A) and in the cells overexpressing murine highactTPPII (Fig. 2I–K), however, nuclear interactions in the butabindide treated cells had tendency to increase in the cells overexpressing TPPII (Fig. 4B). The presence of cytoplasmic and nuclear in situ PLA signal was demonstrated in both control cells and the cells overexpressing highactTPPII by scanning middle planar specimen sections (Fig. 2L–O). MYBBP1A interacts with the cell cycle regulator CDK2 MYBBP1A-CDK2 interaction was also confirmed by in situ PLA in both HeLa and HEK293 transformants (Figs. 3 and 4C, D). In starving HeLa cells grown in the absence of FBS, the resulting in situ PLA signal had medium frequency (Fig. 3A) which was not suppressed by the presence of 100 lM CDK2 inhibitor II (data not shown), whereas the signal frequency decreased by growing cells at the presence of FBS (Figs. 3B and 4D). Overexpression of TPPII as a third interaction partner of both CDK2 and MYBBP1A decreased the frequency of in situ PLA signal in cytoplasm of the cells overexpressing murine highactTPPII compared to the vector-transformed control HEK293 cells (Fig. 4C). TPPII reduces expression of MYBBP1A after cell detachment Since both TPPII and MYBBP1a have been previously functionally connected to apoptosis and anoikis [7,1], we have further investigated if the gene expression level of TPPII affects expression of its interaction partner during cell detachment. Overexpression of TPPII in detached cells caused downregulation of MYBBP1A mRNA compared to vector transformed control cells (Fig. 5). Further we have investigated expression level of cyclin-CDK2 inhibitor p21; however the results of the expression study did not show a clear effect in neither control cells nor in the cells overexpressing TPPII (Fig. 5).

Fig. 5. Gene expression of TPPII, MYBBP1A and p21 in vector transformed (control) HEK293 cells and cells overexpressing murine highactTPPII during anoikis assay (highactTPPII). Cells were incubated for 4 h at attached or detached conditions and gene expression was analyzed by quantitative real-time PCR. Expression data were normalized to b-actin and expression fold was related to the expression of vector transformed cells incubated at attached conditions with expression equal to 1: Bars: ±standard error of mean, n = 3.

Discussion The described mutual physical interactions functionally associate proteins with cellular functions in protein degradation, tumor suppression, regulation of the cell cycle and apoptosis. All the interacting proteins described here were previously implicated in the research for development of tumor suppressing agents. MYBBP1A has multiple functions in cancer cells via its p53 activation effect [1], by being a substrate of Aurora kinase B considered as drug target for cancer therapy [18] and by its function as a corepressor of the transcription factor NFjB [17]. TPPII inhibitors have been suggested as potential tumor-suppressing agents [4], and some inhibitors of CDK-s with effect on cell cycle arrest at G1/S checkpoint have cytostatic effects [16]. TPPII gene expression is broadly upregulated in most cancer tissues as it was observed by data mining of the Oncomine™ Research Edition database. This is in agreement with observations using a BL cellular model, where TPPII is required for cancer cell growth and survival [5]. Previous studies already suggested that TPPII might be involved in the direct or indirect regulation of cell division, since it can facilitate survival of cells even with severe mitotic aberrations [23]. An increased level of enzymatically active TPPII in HEK293 cells, similar to the conditions in cancer cells, stimulates cytoplasmic protein–protein interaction with tumor suppressor MYBBP1A. The interaction can be partially suppressed by the inhibitor butabindide, which specifically binds into the active site of TPPII. Functionally, MYBBP1A shares some similarity with TPPII concerning its regulatory effect on apoptosis and cell cycle suggesting that they might affect common signaling pathways. Depletion of MYBBP1A causes increased level of p21 leading to cellular senescence, occurring in the phenotype of TPPII knockout mice [9]. However in our experimental study gene expression of p21 was not affected by changes in TPPII and MYBBP1A gene expressions. According to Mori et al. [14], MYBBP1A down-regulation causes growth inhibition in HeLa cells, cell cycle arrest at G2/ M phase of the cell cycle with certain effects at the G1/S phase and mitotic aberrations. However, the opposite effect of MYBBP1A has been observed in the immortalized cell line NIH3T3, where cell growth stimulation was detected upon MYBBP1A depletion [14]. According to our study, upregulation of TPPII in HEK293 cells causes downregulation of MYBBP1a during cell detachment. This

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effect on expression of the tumor suppressor MYBBP1A could represent the mechanism by which enzymatically active TPPII promotes malignant growth in cancer cells. There exist several alternative pathways how MYBBP1A can further affect cell cycle and apoptosis and mediate its tumor suppressor activity. It might occur via its ability to act as a p53 transcription activator [11,1], by regulation of p21 [13], NFjB repression [17] and/or directly by its novel interaction with CDK2 and these effects might be influenced by its interaction with TPPII. Deregulation of p53 and NF-jB was indeed observed in TPPII deficient fibroblasts and T-cells [9] supporting the idea, that these effects are mediated through regulation of MYBBP1A expression. CDK2 itself mediates DNA damage responses and apoptosis by triggering G1 checkpoint through ATM-CHK2-p53-p21 pathway [15]. Interacting proteins MYBBP1A, CDK2 and TPPII can both occur within cytoplasm and nucleus. TPPII commonly occurs in cytoplasm of normal and cancer cells; however Preta et al. [19] detected nuclear localization upon gamma irradiation, which can be inhibited by butabindide treatment. Our observations also indicate that the interactions of TPPII with MYBBP1A occur in both cytoplasmic and nuclear compartments. The cytoplasmic TPPII– MYBBP1A interactions seems to be mediated through the active site of TPPII, since treatment by butabindide decreases the total frequency of the interactions; however at the same time overexpression of TPPII stimulates nuclear interactions with MYBBP1A. Subcellular localization of CDK2 is also crucial for its function, since cytoplasmic CDK2 is involved in triggering of apoptotic pathway and nuclear localization occurs in proliferating cells [8]. Cytoplasmic MYBBP1A-CDK2 interaction decreases with overexpression of TPPII in HEK293 cells, while the presence of FBS in medium also affects the interaction in HeLa cells. As it was previously described, subcellular compartmentalization regulates activity of MYBBP1A by shuttling between cytoplasm and nucleus. The protein contains C-terminal basic amino acid repeats which are responsible for its nuclear targeting, and after their cleavage the protein become localized in cytoplasm [10]. The shuttling of all three interacting proteins between nucleus and cytoplasm might be important for their interaction. In conclusion, our study demonstrates novel protein–protein interactions of TPPII with the tumor suppressor MYBBP1A and with the cell cycle regulator CDK2. It is probable that these interactions mediate the regulatory effect of TPPII on the cell cycle and apoptosis with importance in tumorigenesis, but the exact mechanism remains to be determined. The novel interaction between MYBBP1A and CDK2 described here may represent an alternative pathway to p53 activation and how MYBBP1A affects cell cycle and apoptosis. Acknowledgements We would like to acknowledge Eva Andersson and Dr. Lars Tjernberg for valuable assistance with performance of LC–MS/MS

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analysis. The experimental research was financially supported by Försäkringskassan, O. E. och Edla Johanssons vetenskapliga stiftelse and Stiftelsen Sigurd och Elsa Goljes Minne. The funding sources did not have any role in the experimental design, analysis, and data interpretation or in making publishing decisions. References [1] K. Akaogi, W. Ono, Y. Hayashi, H. Kishimoto, J. Yanagisawa, BMC Cancer 13 (2013) 65. [2] R.-M. Bålöw, U. Ragnarsson, Ö. Zetterqvist, J. Biol. Chem. 258 (19) (1983) 11622–11628. [3] G. Candiano, M. Bruschi, L. Musante, L. Santucci, G.M. Ghiggeri, et al., Electrophoresis 25 (9) (2004) 1327–1333. [4] S. Duensing, S. Darr, R. Cuevas, N. Melquiot, A.G. Brickner, et al., Genes Cancer 1 (9) (2010) 883–892. [5] R. Gavioli, T. Frisan, S. Vertuani, G.W. Bornkamm, M.G. Masucci, Nat. Cell Biol. 3 (3) (2001) 283–288. [6] O. Gavet, J. Pines, Dev. Cell 18 (4) (2010) 533–543. [7] H. Hilbi, R.J. Puro, A. Zychlinsky, Infect. Immun. 68 (10) (2000) 5502–5508. [8] K. Hiromura, J.W. Pippin, M.J. Blonski, J.M. Roberts, S.J. Shankland, Oncogene 21 (11) (2002) 1750–1758. [9] J. Huai, E. Firat, A. Nil, D. Million, S. Gaedicke, et al., Proc. Natl. Acad. Sci. U.S.A. 105 (13) (2008) 5177–5182. [10] R.A. Keough, E.M. Macmillan, J.K. Lutwyche, J.M. Gardner, F.J. Tavner, et al., Exp. Cell Res. 289 (1) (2003) 108–123. [11] T. Kuroda, A. Murayama, N. Katagiri, Y. Ohta, E. Fujita, et al., EMBO J. 30 (6) (2011) 1054–1066. [12] F. Lévy, L. Burri, S. Morel, A.L. Peitrequin, N. Lévy, et al., J. Immunol. 169 (8) (2002) 4161–4171. [13] K.F. Macleod, N. Sherry, G. Hannon, D. Beach, T. Tokino, et al., Genes Dev. 9 (8) (1995) 935–944. [14] S. Mori, R. Bernardi, A. Laurent, M. Resnati, A. Crippa, et al., PLoS One 7 (10) (2012) e39723 (7). [15] I. Neganova, F. Vilella, S.P. Atkinson, M. Lloret, J.F. Passos, et al., Stem Cells 29 (4) (2011) 651–659. [16] T. Owa, H. Yoshino, K. Yoshimatsu, T. Nagasu, Curr. Med. Chem. 8 (12) (2001) 1487–1503. [17] H.R. Owen, M. Elser, E. Cheung, M. Gersbach, L. Kraus, et al., J. Mol. Biol. 366 (3) (2007) 725–736. [18] C. Perrera, R. Colombo, B. Valsasina, P. Carpinelli, S. Troiani, et al., J. Biol. Chem. 285 (16) (2010) 11775–11785. [19] G. Preta, R. Klark, G. Glas, Biochem. Biophys. Res. Commun. 389 (4) (2009) 575–579. [20] E. Reits, J. Neijssen, C. Herberts, W. Benckhuijsen, L. Janssen, Immunity 20 (4) (2004) 495–506. [21] C. Rose, F. Vargas, P. Facchinetti, P. Bourgeat, Bambal, et al., Nature 380 (6573) (1996) 403–409. [22] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68 (5) (1996) 850– 858. [23] V. Stavroupoulou, J. Xie, M. Henriksson, B. Tomkinson, S. Imreh, et al., Cancer Res. 65 (4) (2005) 1361–1368. [24] B. Tomkinson, M. Hansen, W.-F. Cheung, FEBS Lett. 405 (1997) 277–280. [25] B. Tomkinson, B. Ní Laoi, K. Wellington, Eur. J. Biochem. 269 (2002) 1438– 1443. [26] B. Tomkinson, Ö. Zetterqvist, Biochem. J. 267 (1) (1990) 149–154. [27] G.A.A. Sanhueza, L. Faller, B. George, J. Koffler, V. Misetic, et al., BMC Cancer 12 (1) (2012) 1–10. [28] K. Suzuki, P. Bose, R.Y.Y. Leong-Quong, D.J. Fujita, K. Riabowol, BMC Res. Notes 3 (1) (2010) 294–299. [29] I.A. York, N. Bhutani, S. Zendzian, A.L. Goldberg, K.L. Rock, J. Immunol. 177 (3) (2006) 1434–1443.