JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066

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QUICK identification and SPR validation of signal transducers and activators of transcription 3 (Stat3) interacting

Peng Zhenga, 1, Qiu Zhongb, 1, Qian Xionga, Mingkun Yanga, Jia Zhanga, c, Chongyang Lia, Li-Jun Bic,⁎, Feng Gea,⁎⁎ aInstitute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China bIBP-ARI Joint Center for Research on Tuberculosis, Antituberculosis Research Institute of Guangdong Province, Guangzhou 510630, China cNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

ARTICLE INFO ABSTRACT

Article history: Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved Received 18 July 2011 in the pathogenesis of various human diseases and is constitutively active in human Accepted 23 October 2011 multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these Available online 30 October 2011 processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in Keywords: U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown Quantitative immunoprecipitation (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), combined with knockdown (QUICK) immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry Stable isotope labeling with amino analysis allowed us to distinguish specific Stat3 interacting proteins from background acids in cell culture (SILAC) proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting Stat3 proteins — 14-3-3ζ,PRKCBandHsp90— were further confirmed by reciprocal co- 14-3-3ζ immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results Multiple myeloma therefore not only uncover a number of Stat3 interacting proteins that possess a variety Surface plasmon resonance (SPR) of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells. © 2011 Elsevier B.V. All rights reserved.

1. Introduction regulating processes such as inflammation, survival, prolifera- tion, metastasis, angiogenesis, and chemoresistance of tumor Multiple myeloma (MM) is a B-cell malignancy characterized cells [6]. One of these members, namely Stat3, is ubiquitously by the accumulation of clonal plasma cells within the bone expressed and is functionally involved in regulating cell marrow [1]. Although the pathogenesis of the disease still proliferation, differentiation and cell survival [7]. In many remains unclear, it is well established that interleukin-6 cancer cells, Stat3 signaling has been recognized as a pivotal (IL-6) plays an essential role in the malignant progression pathway supporting survival and growth [8–10]. Stat3 is often of MM [2–3]. Numerous reports suggest that IL-6 promotes constitutively active in many human cancer cells including survival and proliferation of MM cells through the phosphory- MM, leukemia, lymphoma, and solid tumors [8, 11]. Stat3 can lation of a cell signaling , Stat3 [4–5]. The Stat proteins also be activated by certain interleukins (e.g., IL-6) and growth are a conserved family of transcription factors implicated in factors (e.g., epidermal growth factor). Upon activation, Stat3

⁎ Correspondence to: L.-J. Bi, Institute of Biophysics, Chinese Academy of Sciences, China. Tel./fax: +86 10 64871293. ⁎⁎ Correspondence to: F. Ge, Institute of Hydrobiology, Chinese Academy of Sciences, China. Tel./fax: +86 27 68780500. E-mail addresses: [email protected] (L.-J. Bi), [email protected] (F. Ge). 1 These authors contributed equally to this work.

1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.10.020 1056 JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 undergoes phosphorylation at serine 727 and at tyrosine 705, from pSiStrike/Stat3 transfected U266 cells (designated as dimerization, nuclear translocation, and DNA binding, which U266-Si) and one from pSiStrike/control transfected U266 cells in turn leads to transcription of various , including (designated as U266-Ctr). those for apoptosis inhibitors (Bcl-xL, Mcl-1, and survivin), cell cycle regulators (cyclin D1 and c-myc), and inducers of 2.2. QUICK identification of Stat3 interacting proteins in angiogenesis (VEGF) and metastasis (TWIST) [12]. U266 cells The Stat3 signaling is modulated, both positively and negatively, by its interaction with numerous other proteins, Stat3 interacting proteins were identified from U266 cells by and cross-talk occurs with various other signaling cascades, using the QUICK approach. The assay and SILAC labeling including the NF-κB, AP-1 or PI-3K pathways [13].Hence,a were done essentially as described earlier [16]. In brief, U266- key step in understanding the physiological function of Si and U266-Ctr cells were grown in SILAC RPMI 1640 Medium Stat3 and associated MM-pathology is placing Stat3 into (Pierce) containing 10% v/v dialyzed FBS, and 0.1 mg/mL heavy 13 12 biochemical pathways by the identification of its interacting [ C6] or Light [ C6] L-lysine. In the forward SILAC experiment, proteins. A systemic identification of Stat3 protein interactions the U266-Si cells were cultured in light medium, whereas the may provide new clues about the molecular mechanism of U266-Ctr cells were cultured in heavy medium. Reverse Stat3 in MM pathogenesis. Therefore, in this study, we system- SILAC experiments were also performed in which the U266- atically analyzed the Stat3 interacting proteins using the QUICK Si and U266-Ctr cells were cultured in the heavy and light (quantitative immunoprecipitation combined with knockdown) medium, respectively. To ensure full incorporation of the assay developed by Selbach and Mann [14]. This method heavy and light labeled amino acids, cells were grown for was used to identify interactions between proteins at their at least six cell doublings prior to harvest. endogenous levels and in their normal cellular environ- U266-Si or U266-Ctr cells were harvested, washed with PBS, ment by a combination of stable isotope labeling with incubated in 1.0% w/v paraformaldehyde (PFA) in PBS for amino acids (SILAC) [15], RNAi-induced knockdown, co- 10 min at 37 °C. Cross-linking reaction, antibody coupling immunoprecipitation, and quantitative MS. This highly sensi- and immunoprecipitation were performed essentially as tive and accurate approach for PPI analysis has been applied to described earlier [16]. Immunoprecipitated proteins were identify interaction partners of β-catenin and Cbl [14], 14-3-3ζ eluted and separated on a 10% SDS-PAGE gel and visualized interacting proteins [16] and Lrrk2 interaction partners [17]. with silver staining. In this study, QUICK method was undertaken to identify proteins that bind to Stat3 at endogenous level. In total, 38 2.3. Protein separation and in-gel digestion proteins were identified and 20 of them were novel Stat3 putative partners. Furthermore, we confirm the association of Stat3 with Protein bands were excised from the SDS-PAGE gel and cut 14-3-3ζ (YWHAZ), protein kinase C beta (PRKCB) and heat shock into 20 sections for in-gel tryptic digestion. In-gel tryptic protein 90 (HSP90) by reciprocal co-immunoprecipitations and digestion was performed essentially as described earlier surface plasmon resonance (SPR) analysis. Taken together, our [16]. The peptide extract and the supernatant of the gel results revealed the functional diversity of Stat3 interacting pro- slice were combined and then finally concentrated to a volume teins and provided new insight into the mechanisms that regu- of as little as 20 μL to inject into the nanoLC system. late Stat3 activity and function in MM cells. 2.4. Mass spectrometry, protein identification and quantification 2. Materials and methods Dried peptides were reconstituted in 5% acetonitrile/0.1% 2.1. Cell culture and RNA interference formic acid and analyzed essentially as described earlier [16]. All identified peptides were subjected to relative quantifi- The human myeloma cell line U266 was purchased from cation analysis using the program Census [19]. Only proteins American Type Culture Collections (Rockville, MD). All cells with a minimum of 2 quantifiable peptides were included in were routinely maintained in RPMI 1640 supplemented with our final dataset. The protein ratios were calculated from the 1% penicillin/streptomycin, 1 mmol/L L-glutamine, and 10% average of all quantified peptides. The quantification was fetal bovine serum at 37°C, 5% CO2 in air. based on four independent SILAC and LC-MS/MS experiments, Stable Stat3 knockdown was achieved by transfecting U266 which included two forward and two reverse SILAC labelings. cells with a plasmid using the siSTRIKE U6 Hairpin Cloning Grubbs test [14] was utilized to test whether the determined ra- System (Promega, Madison, WI). The siRNA which targeted tios were significantly different from the 1:1 ratios characteristic the Stat3 sequence corresponded to nucleotides 823 to 841 of of background proteins and a p-value of 0.05 was selected as human Stat3 [18]. A negative control scrambled siRNA threshold for significant enrichment of Stat3 interacting pro- (TTCTCCGAACGTGTCACGT) was used as a control. The teins. To reduce errors caused by possible interfering peaks, resulting pSiStrike/Stat3 and pSiStrike/control vectors were we manually confirmed peptide SILAC ratios for the proteins in- purified and used to transfect the U266 cells. The plasmids cluded in Table 1. Only those proteins with p<0.05 and quanti- were introduced into U266 cells using the Nucleofector X005 fied in all four sets (including two forward and two reverse) (Amaxa, Cologne, Germany), according to the Optimized of SILAC measurements were reported as Stat3-interacting Protocol for the U266B1 cell line. Transfected cells were selected proteins. The detailed description of the quantified peptides for puromicin resistance for 3 weeks. One clone was selected and proteins is available in Tables S1 and S2. JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 1057

Table 1 – Summary of Stat3 interacting proteins identified by QUICK method. Proteins are categorized according to their molecular function with names, protein names, average ratios and S.D. listed. Gene name a) Protein name b) UniPort ID c) Average ratio S.D. p-value (H/L)

Chaperone HSP90B1 Heat shock protein 90 kDa beta P14625 4.08 0.47 <0.01 YWHAQ 14-3-3 protein theta P27348 2.32 0.55 <0.01 YWHAB 14-3-3 protein beta/alpha P31946 2.58 0.34 <0.01 YWHAG 14-3-3 protein gamma P61981 2.46 0.36 <0.01 YWHAE 14-3-3 protein epsilon P62258 2.35 0.51 <0.01 YWHAZ 14-3-3 protein zeta/delta P63104 4.02 0.26 <0.01 HSPH1 Heat shock protein 105 kDa Q92598 1.79 0.15 0.002

Kinase PGK1 Phosphoglycerate kinase 1 P00558 2.4 0.2 <0.01 MAP3K4 Mitogen-activated protein kinase kinase kinase 4 Q9Y6R4 3.26 0.3 <0.01 MAPK1 Mitogen-activated protein kinase 1 P28482 6.01 0.22 <0.01 MAPK14 Mitogen-activated protein kinase 14 Q16539 4.92 0.29 <0.01 MAPK3 Mitogen-activated protein kinase 3 P27361 5.08 0.2 <0.01 PRKCB Protein kinase C beta type P05771 3.89 0.39 <0.01 PRKCA Protein kinase C alpha type P17252 3.69 0.66 <0.01 PRKDC DNA-dependent protein kinase catalytic subunit P78527 4.24 0.41 <0.01 PTK2 Focal adhesion kinase 1 Q05397 2.5 0.34 <0.01 SRPK1 Serine/threonine–protein kinase Q96SB4 2.57 0.76 <0.01

Phosphatase PPP2R1A Serine/threonine–protein phosphatase 2A 65 kDa P30153 4.11 0.68 <0.01 regulatory subunit A alpha isoform PPP2R1B Serine/threonine–protein phosphatase 2A 65 kDa P30154 4.14 0.45 <0.01 regulatory subunit A beta isoform PPP2CA Serine/threonine–protein phosphatase 2A catalytic P67775 2.9 0.89 <0.01 subunit alpha isoform PPP2R5D Serine/threonine–protein phosphatase 2A 56 kDa Q14738 3.9 0.58 <0.01 regulatory subunit delta isoform

Enzyme modulator CDC37 Hsp90 co-chaperone Cdc37 Q16543 3.19 0.26 <0.01

Isomerase FKBP4 FK506-binding protein 4 Q02790 5.05 0.4 <0.01

Nucleic acid binding HNRNPK Heterogeneous nuclear ribonucleoprotein K P61978 3.02 0.19 <0.01 HNRNPH1 Heterogeneous nuclear ribonucleoprotein H P31943 6.11 0.24 <0.01 HNRNPU Heterogeneous nuclear ribonucleoprotein U Q00839 3.25 0.44 <0.01 EIF4G1 Eukaryotic translation initiation factor 4 gamma 1 Q04637 3.76 0.63 <0.01 NCL Nucleolin P19338 4.96 0.71 <0.01 ANXA1 Annexin A1 P04083 3.19 0.19 <0.01 PTPRC Leukocyte common antigen P08575 3.19 0.48 <0.01 EEF2 Elongation factor 2 P13639 4.25 0.38 <0.01

Oxidoreductase HDAC1 Histone deacetylase 1 Q13547 3.72 0.53 <0.01 HDAC2 Histone deacetylase 2 Q92769 2.83 0.3 <0.01

Transcription factor STAT3 Signal transducer and activator of transcription 3 P40763 5.06 0.57 <0.01 STAT5A Signal transducer and activator of transcription 5A P42229 5.13 0.3 <0.01 STAT5B Signal transducer and activator of transcription 5B P51692 4.7 0.38 <0.01 CAND1 Cullin-associated NEDD8-dissociated protein 1 Q86VP6 2.15 0.25 <0.01 ILF2 Interleukin enhancer-binding factor 2 Q12905 2.3 0.32 <0.01 a) Name of the corresponding gene according to the IPI database. b) Name of identified proteins according to the IPI database. c) Accession numbers are derived from the UniProt database. 1058 JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066

2.5. Bioinformatics analysis of the Stat3 interacting proteins otherwise stated were from Sigma (St. Louis, MO, USA). Purchased proteins were: Stat3, 14-3-3γ (YWHAG), 14-3-3ζ All identified Stat3-interacting proteins were classified based (YWHAZ), protein phosphatase 2 catalytic subunit (PPP2CA), on the PANTHER (Protein ANalysis THrough Evolutionary protein kinase C beta (PRKCB), heat shock protein 90 kDa Relationships) system (http://www.pantherdb.org), which beta (HSP90B1) and enolase 2 (ENO2) (OriGene Technologies, is a unique resource that classifies genes and proteins by Rockville, MD). The surface of CM5 chip was activated following their functions [20].Someproteinswereannotatedmanually a standard 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide based on literature searches and closely related homologues. hydrochloride/N-hydroxysuccinimide amine coupling Biacore The differentially expressed protein interaction network protocol. For immobilization of proteins, the Stat3 protein was was built automatically by the STRING (Search Tool for the injected in 100 μL of 10 mM sodium acetate (flow rate: 10 μL/ Retrieval of Interacting Genes/Proteins) system with default min). After immobilization, each surface was blocked by 1 M setting except that organism, confidence (score), and interactors ethanolamine at pH 8.5 for 6 min. For interaction measure- shown were set to “human”, “0.40”,and“no more than 10 inter- ments, recombinant human proteins (YWHAZ, PPP2CA, actors”, respectively [21]. The gene name list of these proteins PRKCB, HSP90B1 and ENO2) were dissolved according to the was input to search against the database which contains instructions of the manufacturer (OriGene Technologies). known and predicted protein–protein interactions. The retrieve Various concentrations of proteins were injected over this included a detailed network which highlights several hub interaction surface for 5 min in a buffer containing 150 mM proteins. NaCl, 5 mM CaCl2, 0.005% (v/v) Tween 20, 20 mM Hepes To determine if any types of proteins are overrepresented, (pH7.4) with a flow rate of 5 μL/min. The sensor surface was enrichment analysis of (GO) terms [22] and regenerated between sample injections by washing with 6 M Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways guanidine-HCl for 0.5 min at the flow rate of 50 μL/min. An [23] was performed using the web-accessible program Database equivalent volume of each protein sample (analyte) was for Annotation, Visualization and Integrated Discovery (DAVID ) injected over a chip surface with no protein immobilized to 6.7 [24–25]. The default human proteome was used as the serve as a blank phase for the background subtraction. background list. The significance of the enrichments was statis- ENO2, which was identified as a background protein by our tically evaluated with a modified Fisher's exact test, and a QUICK method, was injected as a negative control in the p-value for each term was calculated by applying a Benjamini– same condition. All experiments were repeated at least – Hochberg false discovery rate correction [24 25]. For GO terms three times. The kinetic parameters (ka: association rate con- enrichments, the GO fat annotation available in DAVID was stant; kd: dissociation rate constant; KD = kd/ka: equilibrium used. The GO fat is a subset of the GO term set created by filtering dissociation constant) for each interaction were determined out the broadest ontology terms in order to do not overshadow by globally fitting the experimental data with BIAevaluation more specific ones. GO slims are cut-down versions of the GO software 4.1 (GE Healthcare). ontologies containing a subset of the terms in the whole GO. They give a broad overview of the ontology content without 2.8. Statistical analysis the detail of the specific fine grained terms. The enrichment of GO biological process terms was also analyzed using the Data are expressed as the mean±standard error of the mean Cytoscape and its Plugin the Biological Networks Gene Ontology from at least three separate experiments performed in triplicate, tool (BiNGO) 2.3 [26],usingthecompleteGOtermsetanda unless otherwise noted. Statistical analysis was performed hypergeometric statistical test with Benjamini–Hochberg false using a two-tailed Student's t-test. Results were considered discovery rate correction. The GO categories, the distribution of significant if p-values were less than 0.05. cellular components, molecular functions and biological pro- cesses of the Stat3 interacting proteins were analyzed. 3. Results 2.6. Co-immunoprecipitation and Western blot analysis 3.1. QUICK identification of specific Stat3 interacting proteins Co-immunoprecipitation and Western blot analysis was per- formed essentially as described earlier [16]. The antibodies To identify Stat3 interacting proteins we conducted the QUICK and sources of the antibodies used in this study were as follows: approach which is a SILAC-based quantitative strategy to cap- 14-3-3ζ, GAPDH antibodies (Santa Cruz Biotechnology, Santa ture endogenous protein–protein interactions with very high Cruz, CA), Stat1, Stat2, Stat5, Stat3, pY705-Stat3, pS727-Stat3 confidence [14]. The strategy for purification and identifica- antibodies (Cell Signaling, Danvers, MA), PRKCB antibody tion of interacting proteins associated with Stat3 by QUICK (BD Biosciences, San Jose, CA), Hsp90 antibody (Abcam, Inc., method was illustrated in Fig. 1A. A prerequisite for the Cambridge, MA). QUICK assay is the availability of RNAi cells exhibiting reduced expression of the protein of interest. Stat3 gene 2.7. Surface plasmon resonance (SPR) analysis silencing was achieved by transfecting U266 cells with the Stat3 shRNA plasmids as described earlier [27].Stat3knock- SPR experiments were carried out at 25 °C using a BIAcore down efficiency was monitored by western blot analysis using 3000 instrument (BIAcore AB, Uppsala, Sweden). SPR buffers, the Stat3-specific antibody. As shown in Fig. 1B, Stat3 knock- regeneration solutions and sensor chips were purchased down U266 cells (U266-Si) showed diminished Stat3 protein from GE Healthcare (Uppsala, Sweden). Chemicals unless levels comparing with the parental U266 and U266 transfected JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 1059

Fig. 1 – QUICK identification of STAT3 interacting proteins in U266 cells. (A) Schematic showing application of forward and reverse SILAC coupled with in vivo cross-linking and RNAi to identify specific STAT3-interacting proteins. U266-Si and U266-Ctr Cells were differentially labeled by growing them in medium containing light or heavy amino acids (SILAC). Cells were lysed, combined, immunoprecipitated and analyzed by quantitative proteomics (LC-MS/MS). (B) Expression of STAT isoforms in U266 parental (U266) and control cells (U266-Ctr) and U266/STAT3 shRNA cells (U266-Si). Compared with the parental U266 and U266-Ctr cells, U266-Si cells showed diminished STAT3 protein levels. Depletion of STAT3 showed no obvious effect on the expression of other STAT isoforms. GAPDH blotting was performed to ensure equal loading. (C) Functional distribution of identified STAT3 interacting proteins. Categorizations were based on information provided by the online resource PANTHER classification system.

with negative control shRNA (U266-Ctr). Depletion of intra- both conditions and therefore considered as background cellular Stat3 showed no obvious effect on the expression of proteins (Table S2). The detailed information of all quantified other Stat isoforms and internal control GAPDH (Fig. 1B). The proteins is shown in Tables S1 and S2. immunoprecipitated complex was eluted, separated on SDS- Comparison of our data set with 799 known and pre- PAGE and visualized with silver staining (Fig. S1). Stained gel dicted interacting proteins of Stat3 (http://www.genecards. was cut into 20 sections and subjected to in-gel trypsin digestion org/cgi-bin/carddisp.pl?gene=STAT3&search=stat3&rf=/home/ (Fig. S1). The resulting peptide mixtures were extracted from the /current/website/carddisp.pl&interactions=799#int)re- gel and analyzed by LC-MS/MS analysis. As expected, Stat3 was vealed overlap but also differences (Table S3). About 17 (46%) significantly enriched 5.06-fold in the wt compared to the of the proteins identified in this study have been reported in knockdown condition (Table 1). According to the criteria previous studies as putative Stat3 binding partners and 21 pro- described in Materials and methods, a total of 38 proteins teins were newly identified Stat3 interacting partners (Table we identified as potential Stat3 interaction partners (Table 1). S3). It is worth mentioning that six previously reported putative Moreover, we found 33 proteins being equally abundant in Stat3 binding partners, COPB1, DDB1, HSPA8, IQGAP1, PDIA3, 1060 JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 and PSMD1, had SILAC ratio close to 1, indicating that under our Among the 38 identified proteins, 34 of them can be linked experimental conditions they are likely nonspecific interacting through direct interaction into a protein–protein interaction proteins (Tables S2 and S3). The novel proteins identified in network based on the prediction results of STRING system our experiment might be a result of differences in cell lines, (Fig. 2). Notably, several 14-3-3 proteins were hubs in this net- cell culture conditions, purification strategies, and mass spec- work and have the greatest number of connections. By trometry platforms used. comparing with 427 known and predicted interacting pro- teins of 14-3-3ζ (http://www.genecards.org/cgi-bin/carddisp.pl? 3.2. Functional categories and protein association network gene=YWHAZ&rf=/home/genecards/current/website/carddisp. of identified proteins pl&interactions=446#int), 16 out of the 37 proteins are known 14-3-3ζ interactors. In our previous study, we also found that To understand the biological relevance of the Stat3 interacting Stat3 is one of the novel 14-3-3ζ interacting proteins [16]. These proteins, PANTHER classification system was used to classify results suggested that 14-3-3ζ can interact with Stat3 in vivo these interactors according to their functions. The PANTHER and may play an important role in regulating Stat3 activity in classification system revealed that the interactors can be clas- MM cells. sified into 8 groups according to their functions (Fig. 1C). The largest group of Stat3-interacting proteins is kinase (26%). Sig- 3.3. GO analysis of the Stat3 interacting proteins nificant numbers of Stat3 targets are also implicated in nucleic acid binding (22%), chaperone (16%), and phosphatase (11%), in- To gain insights into functional roles of Stat3, the over- dicating the functional diversity of Stat3 interacting proteins. representation (enrichment) of ontology terms and components

Fig. 2 – The protein–protein interaction network of the identified proteins. The network containing 37 identified proteins was mapped using the STRING system (http://string.embl.de/) based on evidence with different types. In the evidence view, the links between proteins represent possible interactions. Different line colors represent the types of evidence for the associations, which are shown in the legend. Neighborhood stands for runs of genes that occur repeatedly in close neighborhood in genomes. Co-occurrence: the presence of linked proteins across species. Co-expression: the genes that are co-expressed in the same or in other species (transferred by homology). Experiments: the protein interaction information was gathered from other protein–protein interaction databases. Databases: the protein interaction information was gathered from curated databases. Text mining: the protein interaction information was extracted from the abstracts of scientific literature. Fusion stands for the individual gene fusion events per species. JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 1061 of molecular pathways in the Stat3 interacting proteins was adenyl ribonucleotide binding and nucleotide binding. In the analyzed in comparison to their occurrence in the human pro- GO cellular component category, we found that most of the teome. First, a GO slim Generic assignment gave us an overview Stat3 interacting proteins identified were localized in the nuclear − − of the GO distribution. The GO terms can also be visualized as a lumen (p-value=1.01×10 7), cytosol (p-value=7.75×10 5)and − graph where directed links describe the hierarchy and relation- intracellular organelle lumen (p-value=1.25×10 4). ships between terms. Fig. 3 shows the graphical representation of the results. The colored nodes are those determined to be 3.4. Signaling pathway analysis overrepresented with statistical significance. We found that by looking at the closest branch points of the overrepresented GO To validate some of the proteins identified by QUICK, co- terms, protein modification process, protein binding function immunoprecipitation experiments and Western blot analysis and cytosol proteins strongly enriched in the Stat3 interacting were performed. We first examined the phosphorylation proteins (Fig. 3). Next, we performed a GO biological process status of Stat3 in U266 cells by using phospho-tyrosine analysis, and a molecular function and cellular component and phospho-serine specific Stat3 antibodies. Consistent analysis (Table 2 and Tables S4–S6). GO biological process analy- with previous report [8], Stat3 is constitutively activated in sis provides a comprehensive picture of the Stat3 interacting U266 cells (Fig. S2). Based on how well their biological functions proteins in which protein kinase cascade, regulation of apopto- and importance are known, we selected three putative Stat3 sis, regulation of programmed cell death, regulation of cell binding proteins for validation. As shown in Fig. 4A, Stat3, death and regulation of phosphate metabolic process were all 14-3-3ζ, PRKCB and Hsp90 were detected in the Stat3 immune overrepresented (Table 2). In the GO molecular functions cate- complex (Stat3) and the U266 cell lysate (Input) but not in gory, we found that the most overrepresented functions in the the non-immune IgG control (IgG). Furthermore, reverse GO molecular functions category were involved in regulating immunoprecipitation assay using specific antibodies for binding activity, such as phosphoprotein binding, ATP binding, these proteins followed by Western blotting with 14-3-3ζ

Fig. 3 – Overview of the GO slim generic distribution of the identified Stat3 interacting proteins using BINGO 2.3, a plugin of Cytoscape. The GO terms enriched in the Stat3 interacting proteins are shown as nodes connected by directed edges that indicate hierarchies and relationships between terms. Node size is proportional to the number of Stat3 interacting proteins belonging to the functional category. Node color indicates the corrected p-value for the enrichment of the term according to the legend. Information on all annotations is provided in Tables S4–S6. 1062 JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066

Table 2 – GO biological process terms enriched in the Stat3 interacting proteins. The top 5 GO biological process, molecular function and cellular component terms enriched in the Stat3 interacting proteins are listed. A complete list can be found in Tables S4–S6. Term Description Count a) % b) P-value c)

GO:0007243 Protein kinase cascade 11 29.72 1.52E−08 GO:0042981 Regulation of apoptosis 14 37.83 3.03E−08 GO:0043067 Regulation of programmed cell death 14 37.83 3.41E−08 GO:0010941 Regulation of cell death 14 37.83 3.56E−08 GO:0007242 Intracellular signaling cascade 16 43.24 1.03E−07 GO:0051219 Phosphoprotein binding 4 10.81 5.47E−05 GO:0005524 ATP binding 14 37.83 9.40E−05 GO:0032559 Adenyl ribonucleotide binding 14 37.83 1.08E−04 GO:0000166 Nucleotide binding 17 45.94 1.34E−04 GO:0004674 Protein serine/threonine kinase activity 8 21.62 1.51E−04 GO:0005829 Cytosol 16 43.24 1.01E−07 GO:0031981 Nuclear lumen 13 35.13 7.75E−05 GO:0070013 Intracellular organelle lumen 14 37.83 1.25E−04 GO:0043233 Organelle lumen 14 37.83 1.59E−04 GO:0005654 Nucleoplasm 10 27.02 1.72E−04

a) The number of Stat3 interacting proteins. b) The percentage of mapped proteins associated with each term. c) The statistical significance of the difference between the fraction of Stat3 interacting proteins assigned to this GO term and the fraction of all proteins within the human protein set assigned to this GO term.

Fig. 4 – Western blot analysis after co-immunoprecipitation. Immunoprecipitation assays of Stat3, 14-3-3ζ, PRKCB and Hsp90 proteins were carried out in U266 cells as described in the Materials and methods section. (A) Immunoblot analysis demonstrated that Stat3 protein binds with 14-3-3ζ, PRKCB and Hsp90 proteins. No band of Stat3 was observed in the negative control (IgG). Input stands for the total cell lysate extracted from U266 cells. Similarly, reverse immunoprecipitation assays confirmed the binding of (B) 14-3-3ζ, (C) PRKCB and (C) Hsp90 protein with Stat3. JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 1063

(Fig. 4B), PRKCB (Fig. 4C) and Hsp90 (Fig. 4D) confirmed their selected five putative Stat3 interacting proteins for SPR analysis. binding to Stat3. Thus, compelling evidence shows that Stat3 These included YWHAG, HSP90B1, YWHAZ, PPP2CA and PRKCB. interacts with 14-3-3ζ, PRKCB and Hsp90 proteins in U266 cells. We also included ENO2, which was identified as a background protein by QUICK method as a negative control for these SPR 3.6. SPR analysis experiments. Injection of solutions of individual proteins in the running buffer into a chip containing covalently immobilized The kinetic parameters for the binding of several Stat3 inter- Stat3 resulted in the appearance of a characteristic response acting proteins to Stat3 were analyzed using SPR method. (Fig. 5). Response unit (RU) values were proportional to sample We first test the phosphorylation status of the purified Stat3 concentrations within certain ranges (Fig. 5). This in vitro assay protein by using phospho-serine or -tyrosine specific Stat3 system clearly demonstrated that YWHAG, YWHAZ, PPP2CA, antibodies. As shown in Fig. S3, purified Stat3 protein was PRKCB and HSP90B1 have affinity for immobilized Stat3, phosphorylated at both tyrosine 705 and serine 727. Then, we whereas no interaction between ENO2 and Stat3 occurred

Fig. 5 – Characterization of the interaction of YWHAG, YWHAZ, PPP2CA, PRKCB, HSP90B1 and ENO2 with Stat3 by SPR assays. SPR analysis was carried out as described in the Materials and methods section. Sensorgrams of the interactions of (A) YWHAG (25, 50, 100, 200, 400 nM) with Stat3; (B) YWHAZ (25, 50, 100, 200, 400 nM) with Stat3; (C) HSP90B1 (5, 10, 50, 100, 150 μM) with Stat3; (D) PPP2CA (5, 10, 50, 100, 150 μM) with Stat3; (E) PRKCB (5, 10, 50, 100, 200 μM) with Stat3; (F) ENO2 (5, 10, 50, 100, 200 μM) with Stat3. 1064 JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 under the same conditions (Fig. 5). Evaluation of the sensor- monooxygenase activation protein, zeta polypeptide), has grams has shown that the equilibrium dissociation con- been shown to play a central role regulating multiple path- stants (KD) for the interaction of YWHAG, YWHAZ, PPP2CA, ways responsible for cancer initiation and progression [40]. PRKCB and HSP90B1 with immobilized Stat3 were: YWHAG, Involvement of 14-3-3ζ in multiple signaling pathways has

KD =700nM; YWHAZ, KD =56 nM; PPP2CA, KD =110nM; PRKCB, been reported and activities of various signaling mediators are ζ KD =610nM; HSP90B1, KD =390 nM, respectively (Table 3). Fur- differentially regulated by 14-3-3 via direct physical association thermore, subsequent injection of the running buffer caused [40]. However, whether 14-3-3ζ also regulates the Stat family rapid dissociation of YWHAG and YWHAZ, whereas in the was unknown. In this study, both co-immunoprecipitation case of PPP2CA, PRKCB and HSP90B1 their dissociation occurred and SPR results clearly showed that 14-3-3ζ is a Stat3 interacting much slower. This suggests different affinity of the proteins protein. Therefore, it is tempting to suggest that 14-3-3ζ may tested towards Stat3. Especially, the relatively high affinity play an important role in regulating Stat3 activity in MM cells. between YWHAZ and Stat3 raised the possibility that Experiments testing this hypothesis are ongoing in our YWHAZ may have biological importance in Stat3 pathway. laboratory. Our proteomic studies also identified several PKC isoforms and PP2A subunits as Stat3 interacting proteins (Table 1). Protein 4. Discussion kinase C (PKC) comprises a family of Ser/Thr kinases and is divided into three subfamilies termed conventional (cPKC: Stat3 activity is tightly regulated by its interacting proteins α, βI, βII, and γ), novel (nPKC: δ, ε, η and θ) and atypical and multiple signaling cascades and its prolonged activation (aPKC: ζ and ι) [41]. The PKCs play a crucial role in many is associated with various malignancies, including MM [8, 28]. intracellular signal-transducing pathways [42]. These pathways To further understand the regulation of Stat3 activity in MM are involved in various vital functions, including regulation of cells, we have performed a comprehensive analysis of the cell proliferation and differentiation, cell-to-cell interactions, interactome of Stat3 in myeloma cells using the QUICK method. secretion, gene transcription, apoptosis and drug resistance As a result, a total of 38 Stat3 interacting proteins were identi- [42]. PKC pathways have been implicated in MM cell prolifera- fied with very high confidence. Importantly, the current study tion, apoptosis and migration and tumor induced angiogenesis revealed a number of novel Stat3 binding partners. Furthermore, [43–46]. Recently, evidence indicated that PKC interacts with the interaction of Stat3 with 14-3-3ζ, PRKCB and Hsp90 was Stat3, phosphorylates Stat3 Ser727, and increases both DNA confirmed by co-immunoprecipitation/Western blot analysis binding and transcriptional activity of Stat3 [47]. and SPR study. Protein phosphatase 2A (PP2A) refers to a large family of In this study, five 14-3-3 proteins, e.g. 14-3-3θ (YWHAQ), 14-3- heterotrimeric serine–threonine phosphatases that account 3β (YWHAB), 14-3-3γ (YWHAG), 14-3-3ε (YWHAE), 14-3-3ζ for the majority of serine–threonine phosphatase activity in (YWHAZ), were identified as Stat3 interacting proteins most cells and tissues [48]. PP2A have been implicated in cell (Table 1). In particular, as suggested by bioinformational analysis cycle regulation, cell morphology and development [49] and (Fig. 2), 14-3-3 proteins may play an important role in regulating is an essential factor for survival and growth of myeloma Stat3 activity in MM cells. The 14-3-3 proteins are a family of cells [50]. PP2A also plays a prominent role in the regulation ubiquitously expressed regulatory molecules and seven of Stat3 phosphorylation, subcellular distribution, and DNA isoforms, designated δ, η, γ, ε, θ, β and ζ, have been described binding activity [51]. previously [29–30]. The 14-3-3 proteins have raised to a position Consistent with these reports, both QUICK and SPR results of integrators of diverse signaling cues that impact cell fate and clearly showed that protein phosphatase 2 catalytic subunit cancer development [31]. Through regulated interactions with (PPP2CA) and protein kinase C beta (PRKCB) are Stat3- crucial signaling mediators, such as PKC [32–33],MAPK[34–36], associated proteins. These data, along with the results on or AKT [37–39], 14-3-3 controls diverse cellular responses Stat3/14-3-3ζ interaction, support the hypothesis that multi- ranging from , cell cycle, metabolism, and ple signaling proteins, including PKC and PP2A, impinge on apoptosis [31]. Among seven 14-3-3 proteins, 14-3-3ζ,also Stat3 and that 14-3-3ζ serves as a coordinator for different termed as YWHAZ (tyrosine 3-monooxygenase/tryptophan 5- pathways to regulate Stat3 activity in MM cells. This speculative idea, however, is not yet supported by experimental data, and further experiments will be required to determine the functional implication of 14-3-3ζ/Stat3 interaction. – Table 3 Kinetic parameters of proteins binding to Stat3. In conclusion, we demonstrated QUICK as a powerful and ka: association rate, kd: dissociation rate, and KD: kd/ka, unambiguous method for quantitative analysis of protein in- dissociation constant; ND: not determined due to low teractions. By using this approach, we systematically profiled interaction signal. proteins interacting with Stat3 in MM cells. Many reliable −1 −1 −1 Interacting ka (M S )kd (S )KD (M) (kd/ka) Stat3 interacting proteins were identified and some interesting proteins clues were given. In addition to those previously described, we − − Stat3/YWHAG (3.3±0.3)×104 (2.3±0.6)×10 2 (7.0±1.4)×10 7 have identified many novel proteins that are involved in − − Stat3/YWHAZ (1.2±0.4)×106 (6.7±1.6)×10 2 (5.6±1.1)×10 8 various biological processes, many of which have not been − − Stat3/HSP90B1 (1.2±0.2)×103 (4.7±1.3)×10 4 (3.9±1.7)×10 7 − − previously described in association with Stat3. It is now im- Stat3/PPP2CA (1.5±0.7)×103 (1.6±0.1)×10 4 (1.1±0.6)×10 7 − − portant to further characterize the interactions between the Stat3/PRKCB (4.4±1.2)×102 (2.7±0.3)×10 4 (6.1±0.5)×10 7 Stat3 and individual target proteins and to define the signal Stat3/ENO2 ND ND ND transduction pathways that control binding of Stat3 to their JOURNAL OF PROTEOMICS 75 (2012) 1055– 1066 1065 multiple binding partners in response to changing physio- [15] Ong SE, Mann M. A practical recipe for stable isotope labeling logical and pathological conditions. by amino acids in cell culture (SILAC). Nat Protoc 2006;1: 2650–60. Supplementary materials related to this article can be [16] Ge F, Li WL, Bi LJ, Tao SC, Zhang ZP, Zhang XE. Identification found online at doi:10.1016/j.jprot.2011.10.020. of novel 14-3-3zeta interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK). J Proteome Res 2010;9:5848–58. 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