Proteomic and biochemical analysis of 14-3-3-binding proteins during C2-ceramide-induced apoptosis Mercedes Pozuelo-Rubio Centro Andaluz de Biologı ´ a Molecular y Medicina Regenerativa, Consejo Superior de Investigaciones Cientı ´ ficas, Sevilla, Spain Keywords 14-3-3; apoptosis; C2-ceramide; Hela cells; proteomics Correspondence M. Pozuelo-Rubio, CABIMER (SC4), Americo Vespucio s ⁄ n, Sevilla 41092, Spain Fax: +34 954 461664 Tel: +34 600826730 E-mail: merce_pozo@yahoo.es (Received 14 March 2010, revised 28 May 2010, accepted 3 June 2010) doi:10.1111/j.1742-4658.2010.07730.x 14-3-3 is a family of proteins comprising several isoforms that, in many cases, promote cell survival by association with proapoptotic proteins. This study was designed to obtain further understanding of the 14-3-3 role in apoptosis regulation, by analyzing apoptosis-related protein–14-3-3 interactions. Western blot analysis of an eluted fraction from the 14-3-3-affinity chroma- tography column identified proapoptotic proteins as receptor-interacting protein 3 and Bcl-2-antagonist ⁄ killer as new phophorylation-dependent 14-3-3-binding proteins under physiological conditions. The apoptosis indu- cer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis showed that depletion of the 14-3-3f isoform sensitized to cell death, whereas overexpression of this isoform delayed cell death. A combination of tandem affinity purification and liquid chromatography–tandem MS techniques identified 15 proteins involved in cell survival processes whose 14-3-3-binding status changed during C2-ceramide-induced apoptosis. Under physiological conditions, desmin was clearly identified as a new 14-3-3-interactor protein, and vasodilator-stimulated phosphoprotein, nucleophosmin and calmodulin, whose 14-3-3 binding was suggested by others on the basis of MS analysis, were confirmed here as phosphorylation-dependent 14-3-3-associated pro- teins. Interestingly, proteins related to the regulation of DNA double-strand break repair in the early stages of apoptosis, such as DNA-dependent protein kinase, or the regulation of cell shrinkage during apoptosis, such as vasodila- tor-stimulated phosphoprotein and death promoters like receptor-interacting protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding status changed when apoptosis was initiated. The functional diversity of these identified proteins suggests that 14-3-3 may regulate the apoptotic pro- cess through new mechanisms, in addition to others previously characterized. Structured digital abstract l A list of the large number of protein–protein interactions described in this article is available via the MINT article ID MINT-7899808 Abbreviations CAN, acetonitrile; ASK1, apoptosis signal-regulating kinase 1; B23, nucleophosmin; BAD, Bcl-xL ⁄ Bcl-2-associated death promoter; BAK, Bcl2-antagonist ⁄ killer; BAX, Bcl2-associated X protein; BMH1 ⁄ 2, yeast 14-3-3 homolog; CaM, calmodulin; COX IV, cytochrome c oxidase subunit IV; DIG, digoxigenin; DNA-PK, DNA-dependent protein kinase; FADD, Fas-associated death domain; FOXO, forkhead box protein; G418, geneticin; GADPH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HIP-55, hematopoietic progenitor kinase 1-interacting protein of 55 kDa; JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide; LC-MS ⁄ MS, liquid chromatography-tandem MS; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor-jB; RIP1, receptor-interacting protein 1; RIP3, receptor-interacting protein 3; siRNA, small interfering RNA; Smac, second mitochondrial-derived activator of caspase; STAT3, signal transducer and activator of transcription 3; TAP, tandem affinity purification; TNF-a, tumor necrosis factor-a; TSC2, tuberous sclerosis protein 2; VASP, vasodilator-stimulated phosphoprotein. FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3321 Introduction The term 14-3-3 denotes a large family of acidic pro- teins that exist primarily as homodimers and heterodi- mers within all eukaryotic cells [1,2]. In mammals, there are seven 14-3-3 isoforms, designated by Greek letters (a ⁄ b, g, e, c, s ⁄ h, f ⁄ d, and r) and encoded by seven different genes [3,4]. 14-3-3 proteins play central regulatory roles in eukaryotic cells by binding to diverse target proteins, thereby modulating the function of the associated partners [5]. In most cases, 14-3-3 proteins regulate cellular processes by binding to spe- cific phosphoserine and phosphothreonine motifs within target proteins [6]. Two optimal 14-3-3 phos- phopeptide ligands with the consensus sequences RSX(pS ⁄ T)XP and RX(Y ⁄ F)X(pS ⁄ T)XP (where pS ⁄ T represents phosphoserine or phosphothreonine, and X is any amino acid) have been defined [7]. Alternatively, some 14-3-3 proteins bind to phosphorylated motifs that are completely different to the consensus sites described above [8], or even bind to unphosphorylated motifs [9]. 14-3-3 binding can alter the enzymatic activity, sub- cellular localization, protein–protein interactions, dephosphorylation and proteolysis of individual target proteins [10]. Many 14-3-3 target proteins have been shown to be involved in cancers, diabetes, Parkinson’s disease, and other neurological diseases [11]. More- over, 14-3-3 proteins have been shown to be key regu- lators of a large number of processes, such as control of cell proliferation, the cell cycle, regulation of human metabolism, and apoptosis in mammalian cells [12–20]. In a number of cases, interaction of 14-3-3 proteins with their target proteins promotes events that support cell survival, mediating an essential antiapoptotic signal in cells [21]. Apoptosis is an active process of cell death that plays a critical role in normal development, mainte- nance of tissue homoeostasis and elimination of dam- aged or unwanted cells through a balance of antiapoptotic and proapoptotic factors, which may be shifted by extracellular signals [22]. It has been reported that 14-3-3 binds members of the Bcl-2 fam- ily, named Bcl-xL ⁄ Bcl-2-associated death promoter (BAD) and Bcl-2-associated X protein (BAX), inhibit- ing their proapoptotic activities [23,24]. 14-3-3 inhibits cell death caused by other death promoters, such as apoptosis signal-regulating kinase 1 (ASK1) [25]. Fur- thermore, 14-3-3 protein binds to a member of the family of forkhead transcription factors named fork- head box protein (FOXO), blocking its translocation to the nucleus and later activation of death genes [26]. These functions of 14-3-3 proteins have been reported to be dependent on their dimeric structure. The dimeric status of 14-3-3 proteins is regulated by site- specific serine (Ser58) phosphorylation by sphingosine- dependent kinase 1. This serine is located within the dimer interface of 14-3-3 proteins, and its phosphoryla- tion promotes the formation of a monomeric form of 14-3-3. Thus, phosphorylation of Ser58 on 14-3-3f controls its ability to modulate target protein activity, and this may have significant implications for the regu- lation of many cellular processes, including apoptosis, by preventing dimer-dependent inactivation of proa- poptotic BAD or BAX [27]. Ceramide, a bioactive lipid mediator, was found to be an apoptosis inducer that activates sphingosine-dependent kinase 1, regu- lates Bcl-2 expression, blocks survival signals, and acti- vates phosphatases (protein phosphatase 1 and protein phosphatase 2A) [28–31]. Several studies have pro- posed that ceramide and its metabolic derivatives be therapeutically applied in cancer-suppressing strategies [32–36]. Inhibition of apoptosis by 14-3-3, through known processes such as association with BAD, FOXO, and ASK1, and other unknown processes that involve mitogen-activated protein kinase (MAPK) and phos- phoinositide 3-kinase cascades, suggests that 14-3-3 has an important antiapoptotic function. Expression of a polypeptide that prevents 14-3-3 proteins from bind- ing to targets in mammalian cells triggers apoptosis and decreases viability in prostate, lung and cervix cancer cell lines [37,38]. Furthermore, treatment with 2-methoxyestradiol resulted in decreased 14-3-3 expres- sion that, in parallel with apoptosis induction, decreased cell growth [39], and the use of 14-3-3f anti- sense in cancer cell lines increased the sensitivity of the cells to stress-induced apoptosis, such as that induced by UV light, IR light, and doxorubicin [40–42]. On the other hand, several studies found increased expression of 14-3-3f in lung, stomach and breast cancers [42–47]. These data suggest that 14-3-3 proteins have a role in regulating cancer cell proliferation and, as such, could be targeted by cancer therapies. Several proteomics studies have been performed to find new 14-3-3-interactor proteins under physiological conditions or even during mitosis [12–16,18–20]. Never- theless, the work reported here is the first study to include a comprehensive proteomics analysis of 14-3-3-binding proteins under physiological conditions as compared with apoptosis stimulation, with the aim of increasing our knowledge of the role of 14-3-3 proteins in the apoptotic pathway. Because antineoplastic thera- pies ultimately eliminate tumor cells by the induction of 14-3-3-binding status during apoptosis M. Pozuelo-Rubio 3322 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS apoptosis, a comprehensive understanding of how 14-3-3-mediated survival pathways inhibit apoptosis may allow the use of 14-3-3 antagonists to sensitize tumor cells for effective therapy. Thus, to identify novel cellular survival functions of 14-3-3 proteins, global proteomics and biochemical analyses were carried out to identify proteins that bind 14-3-3 proteins during apoptotic and survival conditions. These 14-3-3-interacting proteins were purified from extracts of both control and C2-cera- mide-stimulated HeLa cells, using tandem affinity purification (TAP) methodology. The proteins, identi- fied by liquid chromatography–tandem MS (LC- MS ⁄ MS) analysis, were involved in multiple cellular biological processes, but a pool of these proteins had important functions in apoptosis through regula- tion of intermediate filament integrity, cell blebbing, formation of apoptotic bodies, DNA repair, and regulation of oncogenic or death promoters dur- ing apoptosis. Using the small interfering RNA (siRNA) technique, the survival role of 14-3-3f during C2-ceramide-induced apoptosis was characterized. The involvement of identified C2-ceramide-regulated 14-3-3-binding proteins with several processes that control apoptosis suggests possible survival roles of 14-3-3 proteins in addition to others that have been previously characterized. Results Identification of 14-3-3-binding proteins related to apoptosis A few years ago, in a proteomics study of 14-3-3-affin- ity purification of over 200 human phosphoproteins, new links of 14-3-3 proteins with the regulation of cellular metabolism, proliferation and trafficking were shown [12]. Related to the functions of 14-3-3 proteins as regulators of cell survival with central roles in inhib- iting apoptosis, several apoptotic-related 14-3-3-bind- ing proteins were identified in our study. Thus, we found further 14-3-3-interactor proteins that are regu- lators of apoptosis, such as receptor-interacting protein kinase 1 (RIP1), programmed cell death protein 6 ⁄ ALG2 (apoptosis-linked gene 2), second mitochon- drial-derived activator of caspase (Smac), signal trans- ducer and activator of transcription 3 (STAT3), and hematopoietic progenitor kinase 1-interacting protein of 55 kDa (HIP-55). Using both MS and MALDI- TOF ⁄ TOF MS tryptic mass fingerprinting, those proteins were identified as 14-3-3-interactor proteins; however, studies of their presence in the eluted fraction from the 14-3-3-affinity chromatography column to confirm these data were not performed at the time. Here, western blotting analysis showed the presence of the corresponding protein with the appropriate molec- ular mass in the ARAApSAPA elution pool from the 14-3-3-affinity chromatography column (Fig. 1). These data show that proteins such as RIP1, Smac, STAT3 and HIP-55 were eluted from the affinity column, con- firming these proteins as 14-3-3-interactor proteins under physiological conditions. Note that none of these proteins was eluted from the column by either extensive washing under high-salt conditions or mock elution with control phosphopeptides that do not bind to 14-3-3 proteins. These results indicate that isolated proteins bind to the phosphopeptide-binding sites on the 14-3-3 proteins, either directly or as components of protein complexes. As mentioned above, 14-3-3 interacts with apopto- sis-related proteins such as BAD, FOXO or ASK1 to perform its apoptosis-suppressing role in cells. Here, Crude Flow through 1st Wash 2nd Wash 3rd Wash Control 14-3-3BP BAX BID Caspase-8 Caspase-9 FADD HIP-55 BAK BAD RIP1 Smac STAT3 RIP3 Fig. 1. 14-3-3-affinity chromatography of human HeLa cell extracts. Clarified HeLa cell extract was subjected to chromatography on 14-3-3–Sepharose, as described in Experimental procedures. Column fractions were subjected to SDS ⁄ PAGE, using 10% Tris ⁄ glycine gels, and transferred to nitrocellulose membranes. The amounts of protein subjected to SDS ⁄ PAGE were as follows: extract, flow through and beginning of salt wash (1st Wash), 40 lg of each; middle and end of salt wash (2nd Wash and 3rd Wash, respectively), protein undetectable; control (phospho)peptide pool, < 1 lg; and ARAApSAPA elution pool, 2 lg. Western blots were probed with antibodies against the indicated proteins related to apoptosis. M. Pozuelo-Rubio 14-3-3-binding status during apoptosis FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3323 14-3-3 interaction with other apoptosis-related proteins was analyzed by its presence in the 14-3-3-affinity chromatography elution pool. Thus, proapoptotic pro- teins such as receptor-interacting protein kinase 3 (RIP3) and Bcl-2-antagonist ⁄ killer (BAK) were eluted from the 14-3-3-affinity chromatography column, sug- gesting a broad role of 14-3-3 proteins in apoptosis regulation. Note that the well-known apoptosis-related 14-3-3-binding protein BAD [23] was eluted from the affinity chromatography column, giving confidence in this technique. Additionally, the proapoptotic protein BAX [24], which is known to be a 14-3-3-interactor protein, did not appear to be eluted from the column, probably because its defined interaction with 14-3-3 proteins is independent of phosphorylation (which is a requirement for elution from the column). On the other hand, members of extrinsic apoptosis pathways, such as caspase-8, Fas-associated death domain (FADD), and Bcl-2-interacting domain, did not bind to 14-3-3 proteins under the conditions tested. C2-ceramide promotes changes in 14-3-3-binding patterns in HeLa cells during C2-ceramide- induced apoptosis With the aim of further analyzing the role of 14-3-3 proteins in apoptosis, an evaluation of the ability of proteins to bind and to be regulated by 14-3-3 proteins during C2-ceramide-induced apoptotis was carried out. Previous results have established C2-ceramide as an inducer of programmed cell death [28]. Thus, C2-cera- mide-induced cell death in HeLa cells was analyzed, and the time when this death occurred was established. HeLa cells were left untreated or exposed to C2-cera- mide (50 lm) for the indicated times (Fig. 2A). Sample extracts were processed, and cell death was determined as a percentage of the sub-G 1 population. The results in Fig. 2A show that 50 lm C2-ceramide promoted cell death in HeLa cells in a time-dependent manner. In order to evaluate the 14-3-3-binding status of proteins from HeLa cell extracts during C2-ceramide- induced cell death, cells were treated in the presence or absence of C2-ceramide (50 lm), and clarified extracts were run into a gel and electrotransferred to a nitrocel- lulose membrane. Ponceau dyes showed differential protein expression, probably because ceramide is linked to nuclear factor-jB (NFjB) and SAPK ⁄ JNK cascades, which control protein expression in cells [48,49], or perhaps because death initiation requires caspase-dependent cleavage of specific targets [50–54]. Nevertheless, a digoxigenin (DIG)–14-3-3 overlay assay showed protein bands with a significantly decreased 14-3-3-binding signal during C2-ceramide-induced cell death (Fig. 2B). These data are intriguing, and may suggest deregulation of the association of 14-3-3 pro- teins with their targets during C2-ceramide treatment. To further investigate the role of 14-3-3 proteins dur- ing C2-ceramide treatment, downregulation of 14-3-3 proteins was performed and its effects on C2-ceramide cell death were analyzed in HeLa cells. First, levels of expression of seven human 14-3-3 iso- forms were analyzed in a cervical cancer cell line (HeLa) and in several breast cancer cell lines (Fig. 3). The data showed that four different 14-3-3 isoforms were expressed in HeLa cells, 14-3-3f and 14-3-3h being the best expressed. Note that similar results were A 0 10 20 30 40 50 0h 8h 24h % of apoptosis Time B 94 kDa 67 kDa 43 kDa 30 kDa DIG–14-3-3 Ponceau C C2 C C2 Fig. 2. C2-ceramide induces changes in the pattern of 14-3-3 bind- ing in HeLa cell protein extracts. (A) HeLa cells were incubated with 50 l M C2-ceramide for the indicated times. Apoptosis was measured as percentage of cells with sub-G 1 DNA content, as described in Experimental procedures. Columns represent the aver- age of three different experiments. (B) Clarified extract from control HeLa cells or HeLa cells treated with 50 l M C2-ceramide overnight were subjected to SDS ⁄ PAGE, using 10% Tris ⁄ glycine gels, and transferred to a nitrocellulose membrane. Line (C) corresponds to a nontreated control sample, and (C2) corresponds to an extract from C2-ceramide-treated cells. The membrane was stained for protein (Ponceau) and analyzed by DIG–14-3-3 overlay assay. 14-3-3-binding status during apoptosis M. Pozuelo-Rubio 3324 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS found in different breast cancer cell lines, where 14-3-3f and 14-3-3h were expressed well and uni- formly. Meanwhile, other human 14-3-3 isoforms showed low expression levels in HeLa cells, and were also differently expressed in several types of breast cancer cell line. Previous reports suggested that 14-3-3f overexpression occurs in a high percentage of breast tumors in the early stage of the disease, contributing to the transformation of cells and also to the further progression of breast cancer [42]. On the other hand, downregulation of 14-3-3f reduced anchorage-indepen- dent growth and sensitized cells to stress-induced apoptosis [42]. These data suggest an important role of 14-3-3f overexpression in cancer; it is considered to be a molecular marker for disease recurrence in breast cancer patients, and may serve as an effective thera- peutic target in patients whose tumors overexpress 14-3-3f. On the other hand, many reports suggest important regulatory functions of this isoform in the apoptotic pathway, through interactions with specific components of the apoptotic process [55,56]. Downregulation of 14-3-3f with siRNA oligonucleotide enhances C2-ceramide-induced apoptosis in HeLa cells To investigate the role of 14-3-3f downregulation during C2-ceramide-induced apoptosis, sensitization effects on cell death were analyzed in Hela cells in which 14-3-3 binding was blocked by decreasing the levels of 14-3-3f expression, using 14-3-3f siRNA. Clarified extracts from HeLa cells, transfected with 14-3-3f siRNA or scrambled siRNA, were immunob- lotted with antibodies against all human 14-3-3 iso- forms (note that all mammal isoforms were tested, but only four of them were visible in HeLa cells). Fig- ure 4A shows specific downregulation of 14-3-3f iso- forms by 14-3-3f siRNA oligonucleotide, but no difference was observed in other human isoforms. Cell death was determined as the percentage of the sub-G 1 population, in order to evaluate the effects of 14-3-3f downregulation on C2-ceramide induced apoptosis in transfected HeLa cells with 14-3-3f or scrambled siRNA. The results in Fig. 4B show that 14-3-3f siRNA did not promote cell death on its own after 48 h of transfection (or after an additional 24 h; data not shown). Otherwise, downregulation of endogenous 14-3-3f sensitized HeLa cells to cell death promoted by C2-ceramide at 50 lm. Previously, it was reported that downregulation of 14-3-3 proteins sensitized cells to stress-induced apoptosis, such as that induced by UV light and doxorubicin [41,42]. To my knowledge, this is the first study to analyze in detail the effects of 14-3-3 downregulation on C2-ceramide-induced apoptosis. These results suggest an important role of 14-3-3f in C2-ceramide-induced cell death, probably by binding to and regulation of specific targets that play important roles in C2-cera- mide-induced cell death. Knockdown of 14-3-3f promotes C2-ceramide- induced activation of caspase-8 and regulation of the mitochondrial apoptotic pathway Mitochondrial dysfunction appears to be important in C2-ceramide signaling of apoptosis. In vitro studies have shown that C2-ceramide itself is not an efficient inducer of nuclear apoptosis, unless mitochondria are present [57]. It is still a matter of debate whether C2-ceramide acts directly or indirectly on mitochon- dria, but some data suggest that C2-ceramide could signal mitochondrial apoptosis by inhibiting the pro- tein kinase Akt, which is responsible for BAD phos- phorylation, hence leading to inhibition of the antiapoptotic protein Bcl-2 by BAD [58–60]. More- over, C2-ceramide induces cytochrome c release from mitochondria in a caspase-independent fashion, leading to the activation of executioner caspases and also activation of the initiator caspase-8 [61], effects that are completely abolished by Bcl-2 and Bcl-xL [62,63]. MDA-MB- 435 MCF-7/C4 MDA-MB- 231 MCF-7/E6 EvsaT BT 474 HeLa SKBR3 14-3-3γ 14-3-3ζ 14-3-3ε 14-3-3σ 14-3-3θ Tubulin Fig. 3. Analysis of expression levels of several 14-3-3 isoforms in cervical and breast cancer cell lines. Extracts from cervical cancer cells (HeLa) and several breast cancer cell lines (EvsaT, MDA-MB- 435, MDA-MB-231, MCF-7 ⁄ E6, MCF-7 ⁄ C4, BT-474, and SKBR3) (30 lg), grown under physiological conditions, were subjected to SDS ⁄ PAGE, using 10% Tris ⁄ glycine gels, and transferred to a nitro- cellulose membrane. Western blots were probed with antibodies against several isoforms of 14-3-3 proteins. M. Pozuelo-Rubio 14-3-3-binding status during apoptosis FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3325 As downregulation of 14-3-3f has been seen to enhance C2-ceramide-induced cell death, the aim was to obtain further insights into the mechanism of sensi- tization to C2-ceramide with 14-3-3f siRNA by investi- gating the C2-ceramide-induced mitochondrial apoptotic pathway. Therefore, western blot analysis was performed to examine the presence of cyto- chrome c in cytosolic and membrane fractions from extracts of HeLa cells transfected with 14-3-3f siRNA and treated with C2-ceramide. The results in Fig. 4C show lowered cytochrome c levels in the mitochondria- containing membrane fraction and the release of cyto- chrome c to the cytosolic fraction on C2-ceramide treatment when 14-3-3f was downregulated. To confirm that the apoptosis cascade was fully active in 14-3-3f siRNA-transfected HeLa cells treated with C2-ceramide, the proteolytic degradation of the nuclear protein poly(ADP-ribose) polymerase (PARP), a substrate of effector caspases, and of the effector cas- pase-8 were analyzed. As shown in Fig. 4D, PARP cleavage was clearly induced in C2-ceramide-treated HeLa cells previously transfected with 14-3-3f siRNA, but no PARP cleavage was observed in untreated HeLa cells. Cell extracts of indicated samples were analyzed by western blot to determine caspase-8 acti- vation. Procaspase-8 is first cleaved to the p43 ⁄ p41 intermediate fragments, releasing the small subunit p12, and then subsequently processed to generate the large, catalytically active p18 subunit [64]. On the other hand, procaspase-8 has been reported to be cleaved in the presence of C2-ceramide, both native and exogenous, releasing active caspase-8, showing that caspase-8 plays a role downstream of C2-ceramide in the cell death process [65,66]. As shown in Fig. 4D, neither the downregulation of 14-3-3f nor C2-ceramide treatment alone promoted caspase-8 activation at the indicated times, but a combination of both led to the processing of procaspase-8 to its 43 and 41 kDa Fig. 4. Downregulation of endogenous 14-3-3f sensitizes cells to C2-ceramide-dependent apoptosis. (A) HeLa cells were transfected either with siRNA oligonucleotide targeting 14-3-3f or with a scram- bled RNA oligonucleotide, as described in Experimental procedures. After 48 h, extracts from untransfected cells (C) or cells transfected either with siRNA 14-3-3f (14-3-3) or with scrambled siRNA (SC) were harvested for immunoblot analysis to verify knockdown of endogenous 14-3-3f but not other isoforms (14-3-3r, 14-3-3e, and 14-3-3h). Tubulin was used as a protein loading control. (B) HeLa cells transfected either with 14-3-3f or scrambled siRNA oligonu- cleotide, or without siRNA (control), were treated with 50 l M C2-ceramide for the indicated times. Apoptosis was measured as percentage of cells with sub-G 1 DNA content, as described in Experimental procedures. Columns represent the average of three different experiments. (C) HeLa cells were transfected as in (A) and treated with 50 l M C2-ceramide for an additional 4 or 8 h. Follow- ing treatment, cells were lysed, and cytosolic proteins were sepa- rated from mitochondria as described in Experimental procedures. Levels of cytochrome c in cytosolic and membrane fractions were determined by western blot. COX IV was used as a mitochondrial loading control, and tubulin was used as a cytosolic protein loading control. (D) HeLa cells untransfected (C) or transfected either with siRNA oligonucleotide targeting 14-3-3f (14-3-3) or with a scram- bled RNA oligonucleotide (SC) were treated in the presence or absence of 50 l M C2-ceramide for an additional 4 h. HeLa cells were harvested for immunoblotting to analyze caspase-8 process- ing with mouse monoclonal antibody against human caspase-8. Both the 55 ⁄ 53 kDa native forms and the 43 ⁄ 41 kDa intermediate cleavage products are indicated by arrows. PARP cleavage was detected by immunoblotting with antibody against PARP; intermedi- ate cleavage products are indicated by arrows. 14-3-3f antibodies were used to verify knockdown of this isoform, and tubulin was used as a protein loading control. 14-3-3-binding status during apoptosis M. Pozuelo-Rubio 3326 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS intermediate fragments. In conclusion, downregulation of endogenous 14-3-3f sensitizes HeLa cells to the C2-ceramide-induced mitochondrial apoptotic pathway and activation of caspase-8 and PARP cleavage. These data suggest an extensive and important role of 14-3-3 proteins in C2-ceramide-induced apoptosis, probably through regulation of already known apoptosis-related 14-3-3-binding proteins, some of them most likely still to be identified. Purification of 14-3-3-binding proteins from HeLa cells stably expressing green fluorescent protein (GFP)–TAP–14-3-3f by TAP method The data shown above suggest an interesting role of 14-3-3 proteins in C2-ceramide-induced apoptosis, taking into consideration that 14-3-3 downregulation sensitizes cells to C2-ceramide-induced apoptosis. Thus, it was considered that 14-3-3 proteins modulated C2-ceramide-induced apoptosis by binding to well- known apoptosis-related proteins, but possibly also by association with other targets with central roles in the apoptotic process that remain to be identified. There- fore, the aim was to identify new targets of 14-3-3 proteins involved in C2-ceramide-induced apoptosis. To identify proteins associated with 14-3-3 in vivo, a TAP tag approach was used, which allows the isola- tion of native protein complexes from cells ectopically expressing the tagged protein of interest [67]. The TAP tag was fused to 14-3-3f as previously described [68]. This construct, generously provided by D. Alessi (MRC, Dundee, UK), was successfully used to analyze LKB1 phosphorylation-dependent 14-3-3 binding of protein kinases closely related to AMP-activated pro- tein kinase, such as QSK and SIK, in 293 cells [68]. Here, HeLa cells stably expressing GFP–TAP–14-3- 3f were generated and analyzed to determine the size, level of expression and distribution of stably transfect- ed fusion protein (Fig. 5A,B). Western blot analysis with polyclonal antibody against 14-3-3f showed GFP– TAP–14-3-3f of the expected size with a similar level of expression to that of endogenous protein (Fig. 5A). Moreover, the fusion protein showed a cytoplasmic localization identical to the previously described locali- zation for endogenous 14-3-3f [4,69] (Fig. 5B). With regard to the goal of purifying and identifying new 14-3-3-binding proteins involved in C2-ceramide- induced apoptosis, HeLa cells stably expressing GFP–TAP–14-3-3f were used for subsequent protein purification and identification by the TAP method. Thus, stably transfected HeLa cells were either exponen- tially proliferating (untreated) or treated with C2-cera- mide to induce apoptosis (see Experimental procedures). Eluted pools from control and C2-ceramide-treated GFP–TAP–14-3-3f-expressing HeLa cells, purified by TAP, were further analyzed by LC-MS ⁄ MS. Identification of 14-3-3-affinity purified proteins by LC-MS ⁄ MS analysis Analysis by LC-MS ⁄ MS of purified 14-3-3-binding proteins from cells undergoing control and C2-cera- mide-induced apoptosis showed different potential ligands of 14-3-3f in both conditions. The 14-3-3 inter- actors were grouped according to the processes in which they had previously been involved (Tables 1 and S1). The identified 14-3-3-binding proteins included proteins involved in cell signaling, metabolic pathways, A 115 kDa 82 kDa 49 kDa 64 kDa 37 kDa 26 kDa GFP-TAP-14-3-3ζ Endogenous 14-3-3ζ GAPDH HeLa HeLa 14-3-3ζ Wb: Anti-GFP Wb: Anti-14-3-3 19 kDa HeLa HeLa 14-3-3ζ GAPDH B GFP-TAP-14-3-3 ζ Fig. 5. Stable expression of GFP–TAP–14-3-3f in HeLa cells. (A) Cell extracts from HeLa cells stably expressing GFP–TAP–14-3-3f or control HeLa cells were harvested for immunoblotting to verify expression of 14-3-3f fusion or endogenous protein with GFP (left panel) and 14-3-3f (right panel) antibodies from Santa Cruz. Molecu- lar masses of the transfected (GFP–TAP–14-3-3f ) and endogenous protein indicated by western blotting were in agreement with expected masses. GAPDH was used as a protein loading control. (B) HeLa cells stably expressing GFP–TAP–14-3-3f were fixed in 3% (v ⁄ v) paraformaldehyde, and GFP localization was visualized directly by observing GFP fluorescence. The cells were viewed with a Leica CTR 6000 confocal microscope. A full color version of this figure can be found in FEBS Journal online edition. M. Pozuelo-Rubio 14-3-3-binding status during apoptosis FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3327 cytoskeletal dynamics, RNA binding, DNA binding and chromatin structure, cellular trafficking, and pro- tein folding. Some of them were previously shown to be associated with 14-3-3 isoforms (indicated in Table S1). Detection of those 14-3-3 ligands already Table 1. Comparative analysis of 14-3-3-binding proteins identified by TAP–MS from control or C2-ceramide-treated GFP–TAP–14-3-3f- expressing HeLa cells. This is an abbreviated version of Table S1; proteins identified by TAP and LC-MS ⁄ MS analysis were grouped into functional classes, and data were searched against the Euro- pean Bioinformatics Institute ⁄ International Protein Index human database, using the MASCOT search algorithm (see Experimental procedures). The data were obtained by LC-MS ⁄ MS analysis of tandem affinity-purified 14-3-3f-associated proteins from GFP–TAP– 14-3-3f-expressing HeLa cells left untreated (control) or stimulated with C2-ceramide to induce apoptosis. Each protein identification was manually confirmed to ensure that no other human proteins matched the peptide sequences obtained. Interactions validated by biochemical methods are indicated in bold. Control Ceramide Chromatin structure, DNA binding Histone H1.0 Histone H1.0 Histone H1.3 Histone H1t Histone H2A.x Histone H2A type 1 Histone H2B type 1 Histone H2B type 1 Histone H4 Histone H4 B23 Ttransforming growth factor-b -induced transcription factor 2-like protein RNA binding Heterogeneous nuclear ribonucleoproteins C1 ⁄ C2 Heterogeneous nuclear ribonucleoproteins A2 ⁄ B1 RNA-binding protein Raly Translation 40S Ribosomal protein S3 Elongation factor 1 a1 Protein folding and processing E3 ubiquitin-protein ligase CBL Cellular trafficking Voltage-dependent L-type calcium channel subunit a1S Metabolism ATP synthase subunit a, mitochondrial precursor ATP synthase subunit a, mitochondrial precursor Ubiquitin C-terminal hydrolase 42 ATP synthase subunit b Carbamoyl-phosphate synthase, mitochondrial precursor Hydroxymethylglutaryl- CoA synthase U6 snRNA-specific terminal uridylyltransferase 1 Cellular signaling Histone H1.2 Myosin regulatory light chain 2 Myosin light chain kinase 2 Titin Table 1. (Continued). Control Ceramide CaM Centrosomal Nek2-associated protein 1 TSC2 Myosin light chain kinase 2 14-3-3f ⁄ d 14-3-3f ⁄ d 14-3-3e 14-3-3e 14-3-3c 14-3-3h 14-3-3h 14-3-3g B-cell scaffold protein with ankyrin repeats 14-3-3r 14-3-3b ⁄ a DNA-PK catalytic subunit Serine ⁄ threonine protein kinase WNK4 Cellular organization Vimentin Lamin-A ⁄ C a-Actinin-2 a-Actinin-3 Desmin VASP Myosin-2 Myosin-3 Myosin-7 (myosin heavy chain 7) Myosin-7 Myosin-9 Myosin-9 Myosin-11 Myosin-13 Myosin-13 Myosin light polypeptide 3 Myosin light polypeptide 3 a-Actin-2 Actin, cytoplasmic 1 c-Actin Actin, cytoplasmic 1 Tubulin b Tubulin a Ankyrin repeat domain-containing protein 18A Ankyrin repeat domain-containing protein 18A Heat-shock protein b1 a-Actin-2 Unclassified Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 8 Keratin, type I cytoskeletal 17 Keratin, type I cytoskeletal 17 Keratin, type I cytoskeletal 18 Tropomyosin-1 a chain 14-3-3-binding status during apoptosis M. Pozuelo-Rubio 3328 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS known implies that the conditions used here for the TAP tag purification allowed the identification of genuine 14-3-3 ligands. Detailed analysis of the 14-3-3-asociated proteins found showed that 46 of them were exclusively present in one of the conditions ana- lyzed and 15 of them were involved, to a greater or les- ser extent, in the apoptotic process, according to previous reports (detailed in Table 2). It is interesting to note that 14-3-3f copurified with other 14-3-3 isoforms, which is in accordance with previous reports showing heterodimerization among different 14-3-3 isoforms [1]. Detection of 14-3-3-binding motifs on purified and identified 14-3-3-binding proteins The TAP tag approach allows the isolation of native protein complexes from cells ectopically expressing the tagged protein of interest, so proteins associated with 14-3-3 proteins were purified and identified in this study (Tables 1 and 2). Frequently, 14-3-3 proteins regulate cellular processes by binding to phosphory- lated motifs (phosphoserine and phosphothreonine) within target proteins [6], but, because of the methodo- logical characteristics of the TAP tag approach, this phosphorylation-dependent binding of identified proteins is not evident. Two optimal 14-3-3 phosphopeptide ligands with the consensus sequences [RSX(pS ⁄ T)XP and RX(Y ⁄ F)X (pS ⁄ T)XP] have been defined [7], although some 14-3-3 proteins bind to phosphorylated motifs that are com- pletely different to the consensus sites, or even bind to unphosphorylated motifs [9]. To investigate the phos- phorylation-dependent binding to 14-3-3 proteins of the identified proteins, the presence of putative 14-3-3 consensus binding sites was determined for identified 14-3-3f-associated proteins, using the software scan- site [70] (Table 2) (detailed in Table S3). Low-strin- gency settings of the scansite algorithm were applied to analyze 14-3-3-binding consensus motif mode I [RSX(pS ⁄ T)XP] on identified proteins. Note that most proteins studied were identified in normal cell growth conditions, and lost association with 14-3-3 in the treat- ments with ceramide. To determine whether this associ- ation was phosphorylation-dependent, extracts from GFP–TAP–14-3-3f HeLa cells were loaded onto an IgG–agarose chromatography column. Phosphoryla- tion-dependent 14-3-3-binding proteins were eluted using a phosphopeptide (ARAApSAPA) that competes with proteins for 14-3-3 binding in a phosphorylation- dependent manner. The data in Fig. 6A show desmin to be a protein eluted from the affinity column. To my knowledge, desmin, a protein that has been shown to actively participate in the execution of apoptosis [51], was clearly identified here for the first time as a phosphorylation-dependent 14-3-3-associated protein under normal growth conditions, using LC-MS ⁄ MS (Table 2) and biochemical validation (Fig. 6A). Fur- thermore, the data shown here confirm vasodilator- stimulated phosphoprotein (VASP), nucleophosmin (B23) and calmodulin (CaM), whose 14-3-3 binding was suggested in previous studies, as phosphorylation- dependent 14-3-3-associated proteins (Fig. 6A). The data in Fig. 6A show vimentin to be a phos- phorylation-dependent 14-3-3-binding protein in con- trol conditions. Analysis using the highest-stringency settings in the scansite algorithm showed Ser39 in vimentin to be the most probable 14-3-3-binding site (Table S3). These data support previous findings sug- gesting that 14-3-3 binding of vimentin is a phosphory- lation-dependent mechanism [71]. Tuberin [tuberous sclerosis protein 2 (TSC2)], a tumor suppressor protein that antagonizes the mTOR signaling pathway, was also found to be a phosphorylation-dependent 14-3-3-binding protein. These data support previous results showing that Akt phosphorylation of Ser939 in TSC2 is required for its association with 14-3-3 [72]. Both results gave confidence in this technique. On the other hand, the TAP tag approach and phos- phopeptide-specific elution from IgG–agarose chroma- tography columns allows the isolation of native proteins from cells either directly or as components of protein complexes. To determine whether isolated proteins undergo direct interactions with 14-3-3, immunoprecipi- tation assays for several isolated apoptotis-related 14-3-3-binding proteins were performed. Figure 6B shows VASP and B23 to be phosphorylation-dependent 14-3-3-associated proteins that undergo direct interac- tions with 14-3-3 proteins. TSC2 also showed a direct interaction with 14-3-3 proteins, supporting previous results [72], and giving confidence in this technique. Biochemical validation of identified 14-3-3-associated proteins related to apoptosis The combination of TAP and LC-MS ⁄ MS allowed iden- tification of 14-3-3-binding proteins from both control cells and those subjected to C2-ceramide treatments. These data showed a pool of 14-3-3-interactor proteins involved in apoptosis, the 14-3-3-binding pattern being regulated during C2-ceramide-induced apoptosis (Table 2). Silver staining of a gel loaded with the eluted fractions from TAP purification showed different bands of 14-3-3-binding proteins between control and C2-cera- mide-induced apoptosis conditions (Fig. 7). These data also support the idea that C2-ceramide-induced apop- tosis promoted changes in the 14-3-3-binding pattern M. Pozuelo-Rubio 14-3-3-binding status during apoptosis FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3329 Table 2. Selected apoptosis-related 14-3-3-associated proteins identified by TAP–MS and immunoblotting analysis. Apoptosis-related 14-3-3- binding proteins identified in this study by LC-MS ⁄ MS and ⁄ or western blot analysis are listed and grouped by their functions. The role of every protein in the apoptotic process is reported. References are cited for consensus binding sites (CBSs) for every protein; for the details of every site found, see Table S3. Underlining indicates proteins that undergo 14-3-3-binding under control conditions but lose this associa- tion after C2-ceramide treatment. Nonunderlined proteins bind to 14-3-3 proteins under conditions of C2-ceramide-induced apoptosis. Function GI accession no. Name CBS Notes Chromatin structure, DNA binding 121992 Histone H2A.x a [2] Histone H2AX induction occurs only in apoptotic nuclei in cells, and is implicated in the restoration of genomic integrity in response to DNA double-strand breaks [80] 114762 B23 b,c [4] B23 negatively regulates p53 and antagonizes stress-induced apoptosis in human normal and malignant hematopoietic cells [76] RNA binding 108935845 Heterogeneous nuclear ribonucleoproteins C1 ⁄ C2 a [4] Upregulation of hnRNP C1 ⁄ C2 during ischemia or staurosporine-induced apoptosis in mice may foster the synthesis of XIAP as a protective pathway against apoptotic effects [95] Protein folding and processing 115855 E3 ubiquitin-protein ligase CBL a [1] Caspase-3 negatively regulates Bim expression by stimulating its degradation through E3-ubiquitin ligases Cbl, thus creating a negative feedback loop in the Bim caspase axis [88] Metabolism 4033707 Carbamoyl-phosphate synthase, mitochondrial precursor a [2] Carbamoyl-phosphate synthase (CPS) is part of a multienzymatic protein (CAD) required for the de novo synthesis of pyrimidine nucleotides and cell growth. CAD is a target for caspase-dependent regulation during apoptosis, in this case a fast inactivation of CPS occurs [89] Cellular signaling 417101 Histone H1.2 a [1] Histone H1.2 is translocated to mitochondria and associates with BAK in cells undergoing bleomycin-induced apoptosis. Upon DNA damage, histone H1.2 acts as a positive regulator of apoptosome formation, triggering activation of caspase-3 and caspase-7 via APAF-1 and caspase-9 [96–98] 127169 Myosin regulatory light chain 2 a [1] Myosin regulatory light chain phosphorylation is critical for apoptotic membrane blebbing and the active morphological changes during apoptosis [90] 108861911 Titin a [9] Titin expression is induced by cyclosporin A via activation of MAPK pathways, and this may promote proliferation, promote invasion and inhibit apoptosis of human first trimester trophoblasts [91] 49037474 CaM a,b [0] CaM has been shown to regulate apoptosis in tumor models. CaM-specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation [92] 1717799 TSC2 b,c [17] TSC2 is a tumor suppressor that antagonizes the mTOR signaling pathway, thus regulating cell growth and proliferation. TSC2 activates BAD to promote apoptosis and negatively regulate Bcl-2’s antiapoptotic effects on low serum deprivation-induced apoptosis [99–101] 74751216 B-cell scaffold protein with ankyrin repeats (BANK1) a [2] It has been reported that the BANK1 gene is one of the most downregulated genes in colorectal cancer patients, and this suggests that it can be used as a novel blood marker for colorectal cancer [93] 4506539 RIP1 b [4] RIP1 is a specific mediator of the p38 MAPK response to TNF-a [94] 205371831 RIP3 b [4] Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, being capable of selectively binding to large prodomain initiator caspases and attenuating both RIP1 and TNF-a receptor-1-induced NF-jB activation [102–104] 38258929 DNA-PK catalytic subunit b,c [6] The crucial survival role of DNA-PK in the repair of DNA double strand breaks is highlighted by the hypersensitivity of DNA-PK(– ⁄ –) mice to IR light. DNA-PK is robustly activated in apoptotic cells during C2-ceramide treatment [82] 14-3-3-binding status during apoptosis M. Pozuelo-Rubio 3330 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS [...]... combination of TAP purification and LC-MS ⁄ MS identified 15 proteins involved in cell survival processes, their 14-3-3-binding status being changed when apoptosis was promoted; and (f) immunoblot analysis showed that the 14-3-3-binding status of VASP, RIP1 and RIP3 decayed during induced apoptosis, whereas the association of DNA-PK with 14-3-3 increased during cell death Several regulators of apoptosis, ... proteolysis Here, analysis of Ponceau staining of proteins in cell extracts suggests that some proteins could be cleaved during apoptosis initiation; this fact suggests an interesting hypothesis concerning the role of 14-3-3-binding in protecting 14-3-3 targets from proteolysis in the early stages of apoptosis Here, LC-MS ⁄ MS analysis and biochemical validation suggest that apoptotic-related proteins such... many of the proteins identified here are likely to form complexes with 14-3-3, either directly or indirectly Analysis in detail of identified 14-3-3-binding proteins showed that more than half of the proteins lost their ability to bind in C2-ceramide-induced HeLa cells, and just 8% of the total identified proteins increased their binding after C2-ceramide treatment A detailed study of identified proteins. .. 14-3-3binding proteins whose association was lost during C2-ceramide-induced apoptosis Meanwhile, the catalytic unit of DNA-dependent protein kinase (DNAPK), a protein with an essential role in DNA doublestrand break repair in the early stages of apoptosis, raised their 14-3-3-binding status after treatment with C2-ceramide (Fig 7) Thus, LC-MS ⁄ MS and biochemical validation analysis confirms a pool of apoptosisrelated... Pozuelo-Rubio during early stages of apoptotic Finally, this biochemical and functional analysis proposes 14-3-3 as a survival factor during C2-ceramide-induced apoptosis, and identifies novel 14-3-3 interactor proteins under survival or death conditions in HeLa cells Future research can now focus on dissecting the details of the signaling pathways involved in phosphorylation and 14-3-3-binding of identified... others, using MS ⁄ MS analysis, such as RIP1, Smac, STAT3, B23, and CaM, were confirmed here by immunoblot analysis to be phosphorylationdependent 14-3-3-associated proteins; (c) C2-ceramideinduced apoptosis promoted decay of the 14-3-3-binding signal of proteins in cell extracts; (d) depletion of 14-3-3f sensitized cells to C2-ceramide-induced cell death, whereas overexpression of this isoform delayed cell... apoptosisrelated proteins whose 14-3-3-binding status changes during apoptosis, suggesting an extensive role for 14-3-3 proteins during apoptosis initiation Stable expression of GFP–TAP–14-3-3f in HeLa cells delays C2-ceramide-induced cell death Previous studies have clearly shown that 14-3-3 proteins are survival proteins with antiapoptotic effects in cells, by binding to well-known antiapoptotic proteins and. .. [75] All of these previous proteomics studies on 14-3-3-binding proteins using TAP purification increased the possibility of finding genuine 14-3-3-associated proteins In fact, half of the 14-3-3-binding proteins found here were previously described as 14-3-3 targets, such as vimentin and TSC2 However, several of the identified proteins have not previously been shown to bind 14-3-3, such as desmin and RIP3... collected, and the column was washed with 600 column volumes of 50 mm Tris ⁄ HCl (pH 7.5), 500 mm NaCl, and 1 mm dithiothreitol (buffer A), and then with 12 mL of a control synthetic phosphopeptide that does not bind 14-3-3 proteins (1 mm RSRTRTDpSYSAGQSV in buffer A) Proteins that bind to the phosphopeptide-binding site of 14-3-3 proteins were 14-3-3-binding status during apoptosis eluted with 12 mL of 1... immunoblot analysis was not performed at the time [12] Here, a combination of LC-MS ⁄ MS and western blot analysis showed VASP to be a 14-33-binding protein that loses its association with 14-3-3 during C2-ceramide-induced apoptosis It has been reported that VASP binds to aII-spectrin and this complex has a role in the regulation of cell shrinkage, membrane blebbing and the formation of apoptotic bodies during . Proteomic and biochemical analysis of 14-3-3-binding proteins during C2-ceramide-induced apoptosis Mercedes Pozuelo-Rubio Centro Andaluz de. functions of 14-3-3 proteins, global proteomics and biochemical analyses were carried out to identify proteins that bind 14-3-3 proteins during apoptotic and