Mercedes Pozuelo Rubio

Regulation of autophagic activity by 14-3-3ζ proteins associated with class III phosphatidylinositol-3-kinase

Keywords: 14-3-3, Autophagy, Starvation, C2-ceramide, hVps34

14-3-3s are binding proteins with survival functions in cells by interaction with proteins involved in the regulation of cell fate. The role of 14-3-3 during autophagy was investigated, thus, a forced expression of 14-3-3ζ reduces C2-ceramide-induced autophagy, while depletion of 14-3-3ζ promotes autophagy. The 14-3-3 role in autophagyc-related proteins was also investigated. The human vacuolar protein sorting 34 (hVps34), the class III phosphatidylinositol-3-kinase (PI3KC3) mediates multiple vesicle-trafficking processes such as endocytosis and autophagy, its activation being a requirement for autophagy initiation. Using chromatography techniques, hVps34 were eluted from a 14-3-3 affinity column, showing also a direct interaction with 14-3-3 proteins under physiological condition. Further analysis suggests that hVps34/14-3-3 association is a PMAdependent phosphorylated mechanism promoting a strong inhibition of the hVps34 lipid kinase activity, Proteins kinase C being the likely kinase involved in phosphorylation and 14-3-3-binding of hVps34 under physiological conditions. Meanwhile, stimulation of autophagy leads to the dissociation of the 14-3-3/hVps34 complex enhancing hVps34 lipid kinase activity. Forced expression of 14-3-3ζ reduces hVps34 kinase activity and depletion of 14-3-3ζ promotes upregulation of this activity. Here, 14-3-3ζ proteins are shown as a negative regulator of autophagy through regulation of a key component of early stages of the autophagy pathway, such as hVps34.

INTRODUCTION

Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved and regulated pathway characterized by an increase in the number of autophagosomes, vacuoles surrounded by a double membrane that sequesters cytoplasm or organelles. Subsequently, autophagosomes fuse with lysosomes to form autolysosomes, where sequestered material is digested by lysosomal hydrolases. [START_REF] Kroemer | Lysosomes and autophagy in cell death control[END_REF] The autophagy can be enhanced by several cancer-related stimuli, for example, radiation [START_REF] Paglin | A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles[END_REF] , starvation [START_REF] Inbal | DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death[END_REF] , as well as cytokines such as TNF [START_REF] Jia | Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells[END_REF] and ceramide. [START_REF] Lavieu | Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?[END_REF] Focusing on an autophagy pathway component, one of the earliest characterized mammalian autophagy proteins is Beclin-1 (encoded by Becn1, the orthologue of yeast Atg6/Vps30). [START_REF] Liang | Induction of autophagy and inhibition of tumorigenesis by beclin 1[END_REF] This protein is essential for the initiation of autophagy, perhaps via its interaction with the phosphatidylinositol-3-kinase class III (PI3KC3)/hVps34 (mammalian homologue of yeast Vps34) [START_REF] Zeng | Functional specificity of the mammalian Beclin-Vps34 PI 3kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking[END_REF] forming a complex that participates in autophagosome formation. [START_REF] Kihara | Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network[END_REF] hVps34 specifically produces the lipid PtdIns3P [START_REF] Volinia | A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system[END_REF] , this activity being essential for autophagy. [START_REF] Juhasz | The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila[END_REF] An inhibitor of hVps34, 3-methyladenine, is a known inhibitor of autophagy, shown to inhibit deprivation-induced lysosomal proteins degradation in human IMR-90 fibroblasts. [START_REF] Hendil | Both endocytic and endogenous protein degradation in fibroblasts is stimulated by serum/amino acid deprivation and inhibited by 3-methyladenine[END_REF] Yeast has at least two Atg6/Vps34 protein complexes: one containing Atg14 (Autohagy-regulating gene 14) and participating in autophagy, and the other containing Vps38 and functioning in nonautophagic pathways. [START_REF] Kihara | Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae[END_REF] Recently, such regulatory mechanisms directing Atg6/hVps34 specificity have been identified in mammals, thus newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin-1-interacting protein) were identified in a complex with Beclin-1/hVps34. Characterization of the new proteins revealed that Atg14L enhances hVps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces hVps34 activity and downregulates autophagy. [START_REF] Zhong | Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex[END_REF] According to these results it seems clear that autophagy is strictly controlled maybe because of the multiplicity of functions that this process can promote in vivo. In this sense, it is still unclear if autophagy is a survival or death mechanism, or both. Some evidence has come to light during the last decade to help understand this complexity.

First, autophagy seems to have a prosurvival function at the cellular level. Thus, in the case of starvation or growth factor deprivation, autophagy genes are essential to maintain cellular ATP energy production, macromolecular synthesis, and cell survival. [START_REF] Lum | Autophagy in metazoans: cell survival in the land of plenty[END_REF] In addition, the absence of autophagy genes promotes apoptosis of starved HeLa cells. [START_REF] Boya | Inhibition of macroautophagy triggers apoptosis[END_REF] Several other connections have been found between autophagy and cell death and in this regard it is worth mentioning that C2-ceramidepromoted autophagy may also represent a form of caspases-independent cell death. [START_REF] Daido | Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells[END_REF] Antiapoptotic protein Bcl-2 binds to Beclin-1 which disrupts autophagy and autophagy gene-dependent cell death. [START_REF] Pattingre | Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy[END_REF] Recently, the FLICE-like inhibitor protein (cFLIP) that protect cells from apoptosis mediated by a death receptor, has been shown to regulate autophagosome biogenesis and to suppress caspases-independent cell death with autophagy. [START_REF] Lee | FLIP-mediated autophagy regulation in cell death control[END_REF] In this study, the aim was to obtain further understanding of the autophagy mechanism in context of survival or cell death by analyzing the role of 14-3-3 proteins on autophagy regulation. 14-3-3 is a binding protein with survival functions in cells by interaction with large numbers of proteins involved in the regulation of apoptosis, thereby modulating the function of the associated partners. [START_REF] Porter | Dynamic 14-3-3/client protein interactions integrate survival and apoptotic pathways[END_REF] In most cases 14-3-3-mediated sequestration of pro-apoptotic client proteins mediates suppression of apoptosis.

Here, mammalian cells were used to assess whether or not 14-3-3 proteins regulate autophagy.

It was shown that overexpression of 14-3-3ζ protein blocks autophagy, while depletion of 14-3-3ζ promotes autophagy. Furthermore, 14-3-3 directly interacts with hVps34, allowing a negative regulation of hVps34 kinase activity under physiological conditions, Proteins kinase C being the likely kinase involved in phosphorylation and 14-3-3-binding of hVps34. Meanwhile, starvation or C2-ceramide-induced autophagy promotes dissociation of the 14-3-3/hVps34 complex leading to hVps34 activation, which is required during autophagy. These data provide, to my understanding, the first evidence that 14-3-3ζ functions as a negative regulator of autophagy through inhibition of hVps34 kinase activity, which is essential for the autophagosome membrane assembly in early stages of the autophagic process.

RESULTS

Over expression of 14-3-3ζ proteins inhibit C2-ceramide-induced autophagy

Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Previous reports clearly show that C2-ceramide triggers autophagy by several mechanisms such as inhibition of the class I PI3K/AKT pathway and activation of protein phosphatases (PP2A and PP1) which promote inactivation of a number of pro-growth cellular regulators (revised in [START_REF] Zheng | Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy[END_REF] ). According to these data, Figure 1 clearly shows that 10 μM of C2-ceramide in a range of time triggers autophagy. Thus, C2-ceramide treatment induces the appearance of the structural component of autophagosomes LC3-II (also known as Atg8), decreases the levels of a general autophagy marker such as p62/SQSTM1 and inhibits mTOR activity (by analysis of P-p70S6K and P-S6) (Fig. 1A). C2-ceramide also increases the number of cells with autophagosome accumulation and the number of autophagosomes per cell seen by using microscopy analysis of autophagosome-associated protein LC3 (fused to green fluorescent protein (GFP-LC3)) distribution (Fig. 1B). Autophagosome formation was significant at 16-24 h after the addition of 10 μM of C2-ceramide. Significantly, C2-ceramide within this concentration and time failed to induce death in HeLa cells (See Supplementary Fig. S1). These data support C2-ceramide-induced autophagosome accumulation being dependent on the classic autophagy pathway through mTOR inactivation, reflecting an increase in functional autophagy as was previously reported. [START_REF] Scarlatti | Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1[END_REF] Ceramide clearly induces autophagy but whether ceramide-induced autophagy is a survival or cell death mechanism is under discussion and requires in-depth scrutiny. Thus, taking into consideration this controversy it becomes a matter of interest to study the function of survival 14-3-3 proteins during C2-ceramide-induced autophagy. Several reports suggest that 14-3-3ζ isoform has an important role in cell survival regulation, in fact overexpression of this isoform leads the resistance to cell death (see Supplementary Fig. S2). Thus, C2-ceramide-induced autophagy was evaluated in HEK293T cells transfected or not with GFP-14-3-3ζ. As was shown for HeLa cells, untransfected HEK293T cells responded to C2-ceramide by increasing autophagy (Fig. 1C,1D). It is noteworthy that overexpression of GFP-14-3-3ζ blocks autophagy induction (see ratio LC3-II/Tubulin ratio in Fig. 1C) even in the presence of low levels of mTOR activity checked by phosphorylation of its p70S6K substrate. This fact raises the possibility that the autophagy pathway could be directly regulated downstream of mTOR by 14-3-3 proteins. Interestingly, overexpression of HA-14-3-3ζ in C2-ceramide treated HEK293T cells was able to considerably reduce the presence of the typical punctae structures of GFP-LC3 in autophagosomes formation during C2-ceramide treatment.

Downregulation of endogenous 14-3-3ζ sensitizes cells to autophagy.

To further investigate the role of 14-3-3 in autophagy, 14-3-3ζ expression was knocked down in serum-grown cells by RNA interference (siRNA). Autophagosome formation was analysed using fluorescence microscopy by checking increased punctae of GFP-LC3 in the transiently transfected HeLa cell. In control or scrambled siRNA-treated cells, few GFP-LC3 puncta were observed. In contrast, 14-3-3ζ siRNA transfection resulted in accumulation of a large GFP-LC3 puncta (Fig. 2A).

Additionally, Hela cells, expressing GFP-LC3, 14-3-3ζ siRNA resulted in an increased LC3-II/Tubulin ratio in comparison with control or scramble siRNA (Fig. 2B). Furthermore, 14-3-3ζ siRNA resulted in the processing of GFP-LC3 into GFP caused by the autophagic-dependent degradation of LC3. Similar results were found using GFP-LC3 stable transfected MCF-7 breast carcinoma cell line (Supplementay Figure S3A andS3B). It is worth mentioning that downregulation of endogenous 14-3-3ζ does not affect levels of expression of autophagy-related proteins under siRNA transfection conditions used in this study (Supplementary Figure 4). On the other hand, it has been well establish in the literature that 14-3-3 isoforms have the tendency to work redundantly in cell survival mechanisms, moreover, the 14-3-3σ isoform has an additional function of controlling cell cycle progression. The first analysis suggests that 14-3-3θ and 14-3-3ζ are best expressed in different cell types; 14-3-3σ was also identified in the MCF-7 cell line (Supplementary Figure S5A).

Aimed at analysing the impact of 14-3-3θ and 14-3-3σ isoforms on autophagy initiation, their expressions were knocked down in serum-grown cells by siRNA. While this work was in revision, Wang et al (2010) [START_REF] Wang | 14-3-3Tau regulates Beclin 1 and is required for autophagy[END_REF] using U2OS cell lines stably expressing inducible or transient siRNA against 14-3-3τ (θ), using in house 19-nt target sequences for si14-3-3τ under the control of tetracycline operon in pSUPERIOR.puro vector, a significant decreases of the Beclin-1 protein expression was observed upon depletion of 14-3-3τ, becoming a positive regulator of autophagy. Nevertheless, siRNA against either 14-3-3ζ, 14-3-3σ or 14-3-3θ from Santa Cruz Biotechnology clearly depletes each 14-3-3 isoform expression specifically and induce autophagy, while it does not affect levels of expression of autophagyc-related proteins, included Beclin-1 (Supplementary Fig. S4 and Supplementary Fig. S5).

All these data suggest that knocked down 14-3-3ζ protein clearly induces autophagy in cervix and breast cancer cell lines, while participation of the rest of the isoforms cannot be ruled out because at least another two additional isoforms (σ and θ) seem to work redundantly in autophagy regulation probably because of its tendency to form heterodimers. Phenomenon observed also during other processes that control cell survival, such as apoptosis. The regulation of autophagy by 14-3-3 proteins raises the possibility that the autophagy pathway could be directly regulated by 14-3-3 proteins through binding to specific autophagy-related proteins.

14-3-3-binding of proteins involved in autophagic pathway

To approach this possibility, human proteins were isolated that bind to 14-3-3-Sepharose and were eluted specifically with a competing 14-3-3-binding phosphopeptide, ARAApSAPA. [START_REF] Rubio | 14-3-3s regulate fructose-2,6bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase[END_REF] The ARAApSAPA elution pool is highly enriched in many human proteins that can bind directly to 14-3-3s in a phosphorylation dependent manner. Using this method, among 200 14-3-3-affinity-purified proteins were identified in the past, a big proportion of them being studied in detail by their interaction to 14-3-3 proteins. [START_REF] Rubio | 14-3-3affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF] This fact enhances the confidence in the sensitivity and selectivity of the 14-3-3 affinity chromatography procedure. A crude extract of human HeLa cells continuously growing in serum were passed through a 14-3-3-Sepharose column. After extensive washes and 'mock' elution with control peptides that do not bind to 14-3-3, proteins were eluted with a 14-3-3binding phosphopeptide (ARAApSAPA, where pS is phosphorylated serine), with the aim of selectively purifying those proteins that were bound to the phosphopeptide-binding site on the immobilized 14-3-3s. The 14-3-3 overlay shows that the ARAApSAPA elution pool was highly enriched in 14-3-3-binding proteins compared with the crude extract, and that 14-3-3-binding proteins were not eluted with the control non-binding (phospho)peptide (Fig. 3). Inmunoblotting analysis confirmed the presence of hVps34 and Beclin-1, with its appropriate molecular mass, in the ARAApSAPA elution pool. None of these proteins were eluted from the column by either extensive washing under high-salt conditions or mock elution. These results indicate that isolated proteins bind to 14-3-3 proteins, either directly or as components of protein complexes. Interestingly, both proteins involved in autophagosome formation during early and regulatory steps of autophagy have been reported to form a complex. [START_REF] Kihara | Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network[END_REF] These data suggest the possibility that 14-3-3 proteins can regulate the autophagy initiation process through association with essential proteins of this pathway. To test this hypothesis, 14-3-3association with autophagy-related proteins was studied in detail.

hVps34 dissociates from 14-3-3 and is activated during C2-ceramide-induced autophagy Autophagy-related proteins with essential regulatory roles in autophagy progression are Beclin-1 and PI3-kinase class III (PI3KC3/hVps34). Using anti-Beclin-1 and anti-hVps34 antibodies in the experiment of endogenous proteins co-immunoprecipitation, the amount of hVps34 associated with Beclin-1 complex was found to be similar under normal growth conditions or in C2-ceramideinduced autophagy in HeLa (Fig. 4) and HEK293T cells (Supplementary Fig. S6). Analysing DIG-14-3-3 overlay shows no sign of Beclin-1 size but a band that matched hVps34 size was found during serum-growth conditions which become lost during C2-ceramide-induced autophagy in both cell lines (Fig. 4, Supplementary Fig. S6). An unknown 14-3-3-binding protein band (*) was observed in Beclin-1 immunoprecipitates whose identity is now the focus of study. Considering that hVps34/14-3-3 association is a phosphorylated-dependent mechanism (see Fig. 3) and that C2ceramide may activate protein phosphatases (PP2A and PP1) 20 , 14-3-3/hVps34 dissociation may cause by protein phosphatase activation during C2-ceramide treatments. It is noteworthy also that hVps34/14-3-3 association/dissociation does not affect hVps34/Beclin-1 complex. On the other hand, 14-3-3 endogenous proteins co-immunoprecipitated with endogenous hVps34 under physiological conditions, this interaction being lost during autophagy (Fig. 4B). An interesting observation is the fact that hVps34 lipid kinase activity, in both hVps34 and Beclin-1 immunoprecipitates, increases when it loses its association with 14-3-3 proteins during autophagy in HeLa and HEK293T cells. This raises the possibility that hVps34 kinase activity can be regulated by association with 14-3-3 proteins. To examine this hypothesis, the hVps34 kinase activity in mammalian cells overexpressing and lacking 14-3-3ζ proteins was measured.

Increased expression of 14-3-3ζ decreases hVps34 activity meanwhile blocking C2ceramide-dependent autophagy

In order to examine the role of 14-3-3ζ proteins on hVps34 kinase activity and autophagy initiation, HeLa and HEK293T cells were transfected with GFP-14-3-3ζ. Those transfected with GFP-14-3-3ζ exhibit a significant decrease of hVps34 activity in hVps34-immunoprecipitates from cells grown under physiological conditions (Fig. 5A, Supplementary Fig. S7A). These data suggest that 14-3-3 proteins may directly inhibit hVps34 activity. Additionally, data obtained with transfected HeLa and HEK293T cells suggest that GFP-14-3-3ζ overexpression blocks C2ceramide-dependent autophagy (Fig. 5B, Fig. 1C andD, Supplementary Fig. S7B). According to these data, overexpression of 14-3-3ζ in C2-ceramide-induced autophagy in both cell lines significantly decreases hVps34 kinase activity associated with Beclin-1 complex, in comparison with those levels found in untransfected cells treated with C2-ceramide (Fig. 5C and Supplementary Fig. S7C). The hVps34 function on autophagy progression being essential, the role of 14-3-3 proteins decreasing hVps34 kinase activity may contribute to 14-3-3-dependent inhibition of autophagy.

Downregulation of endogenous 14-3-3ζ increases hVps34 activity

Several assays were performed to determine the role of 14-3-3 in autophagy. Previously, it was shown that 14-3-3ζ overexpression promotes decrease of hVps34 activity and inhibition of autophagy; secondly, 14-3-3ζ expression was knocked down in serum-grown cells by RNA interference (siRNA) and autophagy was induced. When, hVps34 kinase activity was analysed, a significant increase of activity was observed during the 14-3-3ζ knocked down in HeLa and MEF-7 in comparison with control cells (Fig. 6 and Supplementary Fig. 8). These data suggest that knocked down of 14-3-3ζ proteins clearly induces autophagy in cervix and breast cancer cell lines and the autophagy induction is coordinated with an increase in hVps34 lipid kinase activity.

hVps34 dissociates from 14-3-3 and is activated during Starvation-induced autophagy

Class III PI3K (phosphoinositide 3-kinase), hVps34 is required for the induction of autophagy during nutrient deprivation. [START_REF] Backer | The regulation and function of Class III PI3Ks: novel roles for Vps34[END_REF] To further analyse the regulation of 14-3-3 protein on hVps34 activity during the initial step of autophagy, HeLa cells and MCF-7 were left untreated or starved for several hours to induce autophagy. Starvation clearly induces autophagy in Hela and MCF-7 cells, expressing GFP-LC3, when GFP-LC3 punctae per cell was analysed by microscopy, while starvation promotes an increased ratio of LC3-II/Tubulin by Western-blot analysis (Fig 7A, Supplementary Fig. 9A). It was observed that hVps34 lipid kinase activity increases when it loses its association with 14-3-3 proteins during starvation-induced autophagy in HeLa and MCF-7 cells (Fig 7B, Supplementary Fig. 9B). On the other hand, Rapamycin and Etoposide promote hVps34/14-3-3 dissociation as well while inducing autophagy, 3-MA blocks Rapamycin-and Etoposide-induced autophagy while hVps34 remains associated with 14-3-3 (Supplementary Fig. S10). Taking into consideration that hVps34/14-3-3 binding seems to be a phosphorylated-dependent mechanism (see Fig. 3), several reports suggesting that Rapamycin and Etoposide promote phosphatase activities (PP2A, Calcineurin phosphatases, etc; [START_REF] Meske | Coupling of mammalian target of rapamycin with phosphoinositide 3kinase signaling pathway regulates protein phosphatase 2A-and glycogen synthase kinase-3dependent phosphorylation of Tau[END_REF][START_REF] Chen | Lithium inhibits ceramide-and etoposide-induced protein phosphatase 2A methylation, Bcl-2 dephosphorylation, caspase-2 activation, and apoptosis[END_REF] ) may explain the dephosphorylation of hVps34 leading its dissociation with 14-3-3 proteins. These data suggest that hVps34 binds 14-3-3 proteins under physiological conditions, this association being lost during autophagy initiation by starvation, C2-ceramide, Rapamycin or Etoposide.

PMA-dependent 14-3-3-binding of hVps34 promotes a decreased hVps34 lipid kinase activity.

Aimed at further analysing 14-3-3/hVps34 binding, pull down of proteins using precipitation with 14-3-3-Sepharose was used. Thus, hVps34 was found to be present in 14-3-3-Sepharose precipitates from HeLa cell and lung mouse tissue extracts continuously serum-growing in the presence of growth factors (Fig. 8A, Supplementary Fig. 11).

In most cases, it was essential for the 14-3-3-binding that 14-3-3-interactor proteins were previously phosphorylated in vivo, thus, 14-3-3 proteins usually bind to phosphorylated serine or threonine residues in target proteins. [START_REF] Tzivion | 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation[END_REF] Data shown in Figure 3 suggests that hVps34 was eluted specifically from the 14-3-3-Sepharose column using a competing 14-3-3-binding phosphopeptide, ARAApSAPA [START_REF] Rubio | 14-3-3s regulate fructose-2,6bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase[END_REF] , suggesting that hVps34 binds to 14-3-3s in a phosphorylation dependent manner.

Aimed at analysing, hVps34/14-3-3 interaction dependence on phosphorylation was tested by dephosphorylating the hVps34 and Beclin-1 immunoprecipitates. Using protein serine/threonine phosphatase 2B (PP2B) and protein phosphatase 1 (PP1), hVps34, isolated from continuous serumgrowing HeLa cells, was dephosphorylated (dP), and its binding to DIG-14-3-3 was abolished (Figure 8B,8C). These data support that a phosphorylation-dependent mechanism is required for hVps34/14-3-3 association.

The HeLa cells used to identify hVps34/14-3-3-binding proteins were cultured in medium with serum that contained a mixture of cell effectors. To identify the cellular stimuli that were responsible for promoting the phosphorylation of hVps34 at sites that facilitate binding to 14-3-3 proteins, cells were deprived of serum and stimulated in different ways. Pull down of proteins using precipitation with 14-3-3-Sepharose were used to identify hVps34. Among the tested stimuli, an increased amount of hVps34 protein bound to 14-3-3 from cells treated with the phorbol ester PMA was found (Figure 8D), while stimulation with IGF-1 or sphingolipid C2-ceramide does not promote hVps34/14-3-3binding.

Moreover, PMA-stimulation promoted 14-3-3-binding of hVps34 and as a result there was decreased hVps34 kinase activity (Figure 8E). It is interesting to note that hVps34 kinase activity in serum-grown HeLa cells increased when cells were exposed to a serum-free medium (where autophagy has been induced, Supplementary Figure 12). Under this circumstance, as was mentioned above, a fast PMA-dependent phosphorylation of hVps34 promotes 14-3-3-binding and as a result a decreased kinase activity of the protein.

In addition to a well characterized ability to activate PKC isoforms [START_REF] Parker | PKC at a glance[END_REF] , PMA was found to stimulate the phosphorylation of p42/p44 MAPK (mitogen-activated protein kinase) [ERK1 (extracellular-signal regulated kinase 1)/ERK2] in HeLa cells (Figure 8D). Lack of SAPK2a activation (stress-activated protein kinase 2a/p38α) was found under stimulation with PMA, unlike C2-ceramide that activates the SAPK2a/p38a pathway [START_REF] Chen | Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway[END_REF] (data not shown). Aimed at analysing in detail kinase responsible for hVps34/14-3-3 interaction, the pull down of proteins with 14-3-3-Sepharose from PMA-treated cells with inhibitors was used to identify 14-3-3/hVps34 interactions. Thus, the binding of hVps34 to 14-3-3 proteins in response to PMA was unaffected by the drug PD 184352, which inhibited the phosphorylation of the p42 and p44 MAPKs [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF] (Fig. 8F). Contrarily, the response of hVps34 to PMA was blocked by H-7, which inhibits a number of other protein kinases, including the PMA-activated PKC [START_REF] Hidaka | Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C[END_REF] (Fig. 8F). Consistent with this result shown for HeLa cells, DIG-14-3-3 assay suggests a PMA-dependent hVps34 interaction with 14-3-3 proteins, H-7 being dependent on HEK293T cells (Supplementary Fig. S13).

Together, these findings suggested that PKC isoforms, or a kinase activated downstream of PKC, were responsible for phosphorylation of at least one 14-3-3-binding site on hVps34, although the involvement of other protein kinases cannot be ruled out. Nevertheless, phosphorylation in vitro with PKC (catalytic subunit from rat brain) of hVps34 immunoprecipitates from serum-free stimulated HeLa cells enhanced hVps34/14-3-3 binding (Figure 8G). Additional analysis focused on investigating the 14-3-3-binding of hVps34 by phosphorylation-dependent mechanisms was carried out. Frequently, 14-3-3 binds to specific Ser(P) and Thr(P) motifs within target proteins and two optimal 14-3-3 phosphopeptide ligands were reported 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). [START_REF] Yaffe | The structural basis for 14-3-3:phosphopeptide binding specificity[END_REF] Some of the 14-3-3s bind to phosphorylated motifs different to the establish consensus sites. [START_REF] Aitken | Functional specificity in 14-3-3 isoform interactions through dimer formation and phosphorylation. Chromosome location of mammalian isoforms and variants[END_REF] Recently, the crystal structure of Vps34 was determined, its composition possessing strong homology with the catalytic subunits of others PI3Ks. [START_REF] Walker | Structural insights into phosphoinositide 3kinase catalysis and signalling[END_REF][START_REF] Miller | Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34[END_REF] Aimed at analysing the sequence domain of hVps34, which probably contains phosphorylated binding sites required for hVps34/14-3-3 association, a series of deletion mutants (GFP-tagged fragments of hVps34) were constructed, expressed in HEK293T cells and examined by specific 14-3-3-binding by DIG-14-3-3 overlay (Fig. 8H,I). All GFP-tagged fragments of hVps34 are able to interact with 14-3-3 proteins by DIG-14-3-3 overlay. Even a recombinant GFP-tagged fragment (MT2) containing the N-terminal region binds 14-3-3s suggesting that one or several phophorylated sites included in GFP-tagged fragment (MT2) is responsible for the binding. In this context, the hVps34 sequence of GFP-tagged fragment MT2

(1-390 aa) was analysed by mode 1 14-3-3-binding consensus sites and PKC phosphorylation consensus site, using the software Scansite. [START_REF] Obenauer | Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs[END_REF] Mode 2 of 14-3-3-binding consensus sequence was also taken into consideration. Using low stringency in Scansite analysis, several possible 14-3-3binding sites emerge such as Ser165 (RTSS 165 TLS) as mode 1 14-3-3-binding site and Ser34 (REQKS 34 Y), Thr166 (RTSST 166 LS), Thr197 (RLT 197 FR) and Ser276 (RS 276 LRSGP) as possible PKC phosphorylated sites. Completely covering hVps34 sequence of GFP-tagged fragment MT2 (1-390 aa) other possible 14-3-3-binding sites emerge such as Ser77 (RTS 77 YK), Ser212 (RSS 212 NF) and Ser279 (RSLRS 279 GP). Taking these data into consideration, green fluorescent proteins (GFP)-hVps34 wild type (WT) and several mutants with specific Serine or Threonine changed by Alanine were generated. Although, other 14-3-3-binding sites cannot be ruled out, after PMA-stimulation GFP-hVps34 WT shows 14-3-3-binding as shown above, while this association is reduced in GFP-hVps34 Thr 197 A and GFP-hVps34 Ser 212 A mutants (Figure 8J). Interestingly, both sites seem to contribute to hVps34/14-3-3 association giving a high level of complexity to hVps34/14-3-3 interaction (see Supplementary Figure S14). Several hVps34 lipid kinases assays were performed analysing the consequence of both mutations in the activity, unfortunately lipid kinase activity levels in GFP-hVps34 WT and mutant immunoprecipitates were extremely low and insignificant in comparison with the endogenous protein. This fact has been reported before when overexpressed hVps34 was only 5% active relative to the endogenous enzyme, presumably due to the lack of hVps15 (p150) and other binding partners. [START_REF] Backer | The regulation and function of Class III PI3Ks: novel roles for Vps34[END_REF] Thus, hVps34 kinase activity of overexpressed protein is likely to be non-physiologically relevant, but the overexpressed proteins were interesting in terms of phosphorylation and 14-3-3-binding site analysis. Nevertheless, these data suggest multiple phosphorylated sites responsible for hVps34/14-3-3 interaction.

DISCUSSION

As shown here, well known anti-apoptotic 14-3-3 proteins are also controlling cellular growth by regulating autophagy in cancer cell lines. Several observations strongly support this idea: (a) The initiator of autophagy, hVps34 (PI3KC3) directly interacts with 14-3-3 proteins under physiological conditions, through a PKC-dependent phosphorylation mechanism (b) 14-3-3/hVps34 association inhibits hVps34 kinase activity (c) Overexpression of 14-3-3 proteins block hVps34 activation and counteracts ceramide-stimulated autophagy, (d) Depletion of 14-3-3 proteins induces hVps34 activity and promotes autophagy and e) Starvation-induced autophagy promote dissociation of 14-3-3/hVps34 complex and activation of hVps34 kinase activity, its activation being physiologically important in autophagy initiation.

These data that connect PKCs and hVps34, are interesting because both activities have been implicated in autophagy regulation. The role of hVps34 in autophagy has recently been well established [START_REF] Petiot | Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells[END_REF] , while inhibitors of PKC such as Safingol (L-threo-sphinganine) induce autophagy in tumour cells [START_REF] Coward | Safingol (L-threo-sphinganine) induces autophagy in solid tumor cells through inhibition of PKC and the PI3-kinase pathway[END_REF] , which implies PKC as a negative regulator of autophagy.

Data obtained from different recombinants GFP-tagged fragments of hVps34 sequence and using site-directed mutagenesis analysis suggests that N-terminal region of the protein seems to be required for the hVps34/14-3-3 binding, while Thr197 (LDRLT 197 FR) and Ser212 (EKRSS 212 NF) are likely involved in the binding. Taking into account the typical 14-3-3-binding motives, it is interesting to mention the presence of the usual arginine preceding the Ser/Thr responsible for the 14-3-3-binding, while they lack the proline residue that is in the +2 position of the canonical 14-3-3binding phosphopeptides. [START_REF] Rittinger | Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding[END_REF] Nevertheless, these data are consistent with recent reports suggesting the +2 proline residue occurs in less than half of the 14-3-3-binding sites of target proteins and raising the possibility that the two short stretch tandem phosphorylated sites on hVps34 may bind to the 14-3-3 dimer conforming a change in N-terminal tail of hVps34 altering its activity, which is the case for other 14-3-3-binding enzymes such as tyrosine hydroxylase. [START_REF] Rubio | 14-3-3s regulate fructose-2,6bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase[END_REF][START_REF] Johnson | Bioinformatic and experimental survey of 14-3-3-binding sites[END_REF] A recent report form Dr. Yuan's lab shows that Vps34 was phosphorylated on Thr159 by Cdk1 during mitosis being a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell-cycle progression. [START_REF] Xiang | 14-3-3 facilitates insulin-stimulated intracellular trafficking of insulin receptor substrate 1[END_REF] This phosphorylated residue has been analysed by its 14-3-3-binding but Thr159 (EPTKT 159 PG) does not seem responsible for hVps34/14-3-3 binding, probably because do not conform even remotely a typical 14-3-3-binding motif.

14-3-3 proteins have been shown to directly interact with insulin receptor substrate 1 (IRS-1), which promotes dissociation of IRS-1/PI3K class I complex during insulin desensitization. [START_REF] Vander Haar | Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40[END_REF] On the other hand, 14-3-3 may contribute to autophagy regulation by association with two regulators of mTOR activation such as TSC2 (tuberous sclerosis complex 2) and PRAS40 (proline-rich Akt/PKB substrate 40 kDa) proteins. The 14-3-3-binding to these proteins promotes activation of mTOR [START_REF] Maiuri | Self-eating and self-killing: crosstalk between autophagy and apoptosis[END_REF] .

Nutrient or serum deprivation promotes inactivation of mTOR through dissociation of TSC2/14-3-3 and PRAS40/14-3-3 complexes. It is interesting to note, that overexpression of 14-3-3ζ impaired C2-ceramide-induced autophagy even if mTOR is inhibited (by substrate P-p70S6K) suggesting that interaction of 14-3-3 with proteins involved in autophagosomes formation during autophagy, such as hVps34, may suppose a later control of autophagy. These data give an idea of how finely regulated autophagy is and how important it is that the cell does not embark on a cellular degradation process if not strictly necessary. Beyond the regulation by 14-3-3 proteins of Vps34 kinase activity, certainly other 14-3-3-binding proteins involved in regulating autophagy possibly remain to be identified.

Although autophagy and apoptosis use fundamentally different molecular mechanisms to

execute protective functions, they also seem to be highly interconnected, recently found in the case of Bcl-2 and cFLIP. [START_REF] Brunet | Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor[END_REF] 14-3-3 proteins have been generally involved in survival mechanisms by regulation of essential proapoptotic proteins such as Bad, FOXO and ASK1 that lead the blockade of apoptosis. [START_REF] Zha | Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)[END_REF][START_REF] Zhang | Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins[END_REF][START_REF] Rubinsztein | Potential therapeutic applications of autophagy[END_REF] Thus, the 14-3-3 proteins role show similarities to Bcl-2 [START_REF] Pattingre | Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy[END_REF] and cFLIP [START_REF] Lee | FLIP-mediated autophagy regulation in cell death control[END_REF] functions, 14-3-3 proteins not only possessing anti-apoptotic activity but additionally serve as anti-autophagy molecules through their interaction and inhibition of hVps34. Thus, the autophagy signal transduction pathway is under tight surveillance, for example, by the mTOR kinase in autophagy induction [START_REF] Panaretou | Characterization of p150, an adaptor protein for the human phosphatidylinositol (PtdIns) 3-kinase. Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex[END_REF] , by cFLIP in autophagosome elongation or by Bcl-2 and 14-3-3 proteins in autophagosome generation. 14-3-3 is also part of the hVsp34/Beclin-1 complex and joins the pool of proteins that mediate regulation of hVps34 kinase activity such as hVps15 [START_REF] Hoyer-Hansen | Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death[END_REF] , Atg 14L or Rubicon. [START_REF] Zhong | Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex[END_REF] Thus, 14-3-3 binds to hVps34 that leads the rapid inactivation of the enzyme under physiological condition. The dynamic change in protein composition between different functional Beclin-1/hVps34 complexes may have a central role in mediating the Beclin-1/hVps34 activity, which governs autophagy.

In summary, a new link between 14-3-3ζ isoform and autophagy is identified. These data shown here, contribute also to the understanding as to how regulators of autophagosome formation, such as hVps34, respond to survival signals in cells during physiological condition or antagonistic to growth and survival, such as Ceramide or nutrients starvation, through association/dissociation with 14-3-3 proteins. The identification of this regulation will help to understand the molecular events that lead to activation of autophagy by anticancer drugs and may contribute to the design of new therapeutic strategies for inhibiting tumor growth.

MATERIAL AND METHODS

Cell lines

HeLa cells were maintained in DMEN media supplemented with 10% fetal bovine serum, at 37 o C in a humidified 5% CO 2 , 95% air incubator. HeLa cells stably transfected with GFP-14-3-3ζ were maintained in DMEN media supplemented with 10% fetal bovine serum and 500 μg/ml Geneticin sulphate (G-418). Human embryonic kidney 293 (HEK293T) cells were maintained in DMEN media supplemented with 10% fetal bovine serum and 10% Glutamine. The MCF-7 breast epithelial cells expressing GFP-LC3 fusion protein described previously [START_REF] Moorhead | Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin[END_REF] and generously provided by Dr. M Jäättelä and Dr. A Lopez-Rivas were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10% Glutamine, and 500 μg/ml G-418 at 37 o C in a humidified 5% CO 2 , 95% air incubator. Then appropriate cells where starved using Earle`s balanced salt solution (EBSS; starvation condition). All chromatographic columns were from Amersham-Pharmacia Biotech (Uppsala, Sweden). GFP-LC3 and TRAIL (TNF-related apoptosis-inducing ligand) was generously provided by Dr. J Oliver and Dr A. López-Rivas. HA-14-3-3ζ, Protein Phophatase 1 (PP1) and GFP-14-3-3ζ were a gift from Dr. C MacKintosh. TLC coated silica gel 60 was purchased from Merk (Whitehouse Station, USA).

Reagents

The catalytic subunit of PKC from rat brain and Protein Phosphatase 2B (PP2B) were from Calbiochem (Nottingham, UK).

Antibodies

Anti-14-3-3 polyclonal, 14-3-3ζ, 14-3-3θ, 14-3-3σ, 14-3-3ε, 14-3-3γ, p150, GFP, p62/SQSTM1, GAPDH, P-p70S6K, p70S6K, Atg-1, P-S6, S6 and Beclin-1 (D-18, used for immunoprecipitation) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Monoclonal antibody to alpha-tubulin was purchased from Sigma Chemical Corp. (St.

Louis, MO, USA). LC3 from NanoTools (Teningen, Germany) and Novus Biologicals (Littleton, CO, USA) and Beclin-1 antibodies used by Western-blot was from BD Biosciences (San Jose, CA, USA). Atg16 and Atg12-Atg5 antibodies were from Novus Biologicals (Littleton, CO, USA). PIST, LAMP1 and LAMP 2 were purchased from ProSci Incorporates (Poway, CA, USA). hVps34 antibody used for immunoprecipitation was purchased from Echelon Biosciences (Salt Lake City, UT, USA) and hVps34 antibody for Western-blot was from Zymed (San Francisco, CA, USA). P-ERK1/2, ERK1/2 and P-PKB (Ser473) were from Cell Signalling (Danvers, MA, USA). Total PKB was a gift from DSTT (Univ. of Dundee, UK). Appropriate peroxidase-conjugated secondary antibodies from DAKO (Glostrup, Denmark) and ECL immunoblotting reagents were from Millipore corporation (Billerica, MA).

Immunoblotting and 14-3-3 overlay assay

Total cell lysate (30-50 μg) were heated at 90°C for 5 minutes in SDS sample buffer, and subjected to polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose membrane. The membrane was blocked for 1 hour in TBS-Tween buffer containing 5% (w/v) skimmed milk. The membrane was probed with 1 μg/ml of indicated antibodies in TBS-Tween, 5% (w/v) skimmed milk for 16 hours at 4°C. Detection was performed using horseradish peroxidase-conjugated secondary antibodies and exposed using the ECL® system. For the immunoblot detection of LC3-I and LC3-II, the samples were treated with 10 μg/ml pepstatin A and 10 μg/ml E64d for the last 4 h before the lysis. Quantification of Western blot signals was performed by densitometry with ImageQuant TL software after scanning the films on ImageScanner II (GE Healthcare (Buckinghamshire, UK)). The relative expression of proteins was normalized to that of loading controls. The values are expressed as arbitrary units of the expression levels in control cells. For 14-3-3 overlays, DIG-labelled 14-3-3s were used in place of a primary antibody. The membrane was incubated with 5 μg/ml total His-BMH1 and His-BMH2 (yeast 14-3-3 isoforms), in TBS-Tween containing 1 mg/ml BSA for 2 hours at room temperature. After several washes, the membrane was incubated with an anti-DIG-horseradish peroxidise secondary antibody and exposed using the ECL® system (Amersham Biosciences, Sweden).

RNA interference

Cells were transfected with indicated siRNAs at 20 nM for 14-3-3ζ, 14-3-3σ and 14-3-3θ siRNAs employing DharmaFECT transfection agent (Dharmacon Research, Lafayette, CO, USA) according to manufacturer's guidelines. After 48 hours of transfection, cells were used for further analysis. siRNAs corresponding to control siRNA (5´-CGACCGAGACAAGCGCAAGtt-3´) from Dharmacon Research (Lafayette, CO, USA). siRNAs against 14-3-3ζ (sc-29583), siRNAs against 14-3-3σ (sc-29590) and siRNAs against 14-3-3θ (sc-29586) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Affinity purification of 14-3-3 binding proteins

Clarified lysates of HeLa cells containing 5 mg total protein, were incubated at 4°C for 2 hour on a vibrating platform with 50 μl of BMH1/2 CH-Sepharose (14-3-3-Sepharose). The beads were washed ten times with 1 ml of lysis buffer containing 0.15 M NaCl, and five times with Buffer A (50 mM Tris-HCl pH 7,4 plus 1mM EGTA). The 14-3-3-binding proteins were treated with sample buffer and subjected to electrophoresis and immunoblotting.

14-3-3 affinity chromatography of human HeLa cell extracts

HeLa cells were thawed and extracted in 20 ml lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 0,2% (w/v) NP-40, 1 mM sodium orthovanadate, 10 mM sodium-βglycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM dithiothreitol (DTT) and a complete proteinase inhibitor cocktail). The broken cells were centrifuged at 15 000 g for 60 min, and the supernatant was collected, filtered and processed for affinity chromatography as previously described. [START_REF] Rubio | 14-3-3s regulate fructose-2,6bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase[END_REF] Briefly, the clarified solution was mixed end-over-end for 1 h at 4°C with 5 ml Sepharose linked to BMH1/BMH2 (the Saccharomyces cerevisiae 14-3-3 isoforms). [START_REF] Chen | Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA[END_REF] The mixture was poured into a column, flow-through collected, and the column washed with 600 column volumes of 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT (buffer A) and then 12 ml of a control synthetic phosphopeptide that does not bind 14-3-3s (1 mM RSRTRTDpSYSAGQSV in buffer A). Proteins that bind to the phosphopeptide binding site of 14-3-3s were eluted with 12 ml of 1 mM ARAApSAPA phosphopeptide in buffer A. Samples (12 ml) from the beginning, middle and end of the 0.5 NaCl wash, the control peptide mock-elution pool (12 ml) and the ARAApSAPA elution pool (12 ml) were each concentrated to ~400 ml, desalted in Vivaspin concentrators, and analysed as indicated in Figure 2. AAAGCCCGGGCAAGCTTGTG). All constructs were confirmed by restriction analysis and sequencing and purified using Quiagen Plasmid Maxi according to the manufacturer's protocol.

Transfection of HEK293T and stable HeLa/GFP-14-3-3ζ cell line

Clones (GFP-hVps34 constructs, GFP-LC3, GFP-14-3-3ζ and HA-14-3-3ζ) were used to transfect HEK293T cells which were cultured on density of 2x 10 6 per 10 cm dish in DMEM containing 10% fetal calf serum for 24 h. Medium was change into fresh medium and cells were transfected by a modified calcium phosphate method with 2 μg/ml DNA/plate then added 0,5 ml 2X HBS (HBS: 50 mM N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid-HCl, pH 6.96, 0.28 M NaCl and 1.5 mM Na2HPO4) and 150 mM NaCl. [START_REF] Mizushima | Methods in mammalian autophagy research[END_REF] The resulting mixture was vortexed for 1 min, allowed to stand at room temperature for 20 min and then added dropwise to a 10 cm dish of HEK293T cells. The cells were placed in an atmosphere of 5% CO 2 for 16 h at 37 o C, then the medium was aspirated and replaced with fresh DMEM containing 10% fetal calf serum.

To generate stable GFP-14-3-3ζ HeLa, the cells were cultured in 10 cm diameter dishes to 50-70% confluence and transfected with 3 μg pGFP-14-3-3ζ construct using Fugene 6 reagent (Roche)

according to the manufacturer's instructions. After 24 hours, G-418 was added to the medium to a final concentration of 3 μg/ml, and the medium was changed every 24 hours maintaining G-418.

After 14-20 days, individual surviving colonies expressing low levels of GFPfluorescence were selected and expanded. FACS analysis was also performed to ensure uniform expression of GFP in the selected cell lines and anti-14-3-3ζ immunoblotting analysis of lysed cells was performed to ensure a band at the expected apparent molecular mass. To generate transient expression of GFP-LC3 in HeLa cells 3 μg pGFP-LC3 and Fugene 6 reagent were used.

Immunoprecipitation assay

Cells were lysed in lysis buffer and the lysate was centrifuged at 4°C for 10 min at 13 000 g and an aliquot of the supernatant (0,5-2 mg protein) was incubated with 1 μg of Beclin-1 (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)), hVps34 (Echelon Biosciences (Salt Lake City, UT, USA)) or 10 μg GFP antibodies (DSTT (Division of Signal Transduction Therapy)) coupling to 20 μl protein G-Sepharose for 3 h at 4 o C. The suspension was centrifuged for 1 min at 13 000 g, the protein G-Sepharose/antibody/protein complex was washed twice with 1 ml lysis buffer containing 150 mM NaCl and twice with buffer A and the immunoprecipitate resolves in SDS-PAGE for DIG-14-3-3 or Western-blot analysis.

Autophagy detection

Several methods were used to assess autophagy according to [START_REF] Mizushima | Methods in mammalian autophagy research[END_REF] such as monitoring phagophore and autophagosome formation using fluorescence microscopy by checking increase in punctuate of fluorescence highly specific autophagy marker GFP-LC3 (Atg8 in yeast)

previously transfected in cells. [START_REF] Mizushima | In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[END_REF][START_REF] Ichimura | A ubiquitin-like system mediates protein lipidation[END_REF] In yeast Atg8, an ubiquitin-like protein, is present on autophagic membranes as a phosphatidylethanolamine (PE)-conjugated form (Atg8-PE) to expand the autophagic membranes. [START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF] Lipid conjugation leads to the conversion of the soluble 18 kDa form of Atg8 (mammalian LC3-I) to an autophagic associated vesicle, fast migrating the 16 kDa form onto SDS-PAGE gels (mammalian LC3-II). Fluorescence analysis of GFP-LC3/Atg8 indicates a change from a difuse cytoplasmic or nuclear distribution to punctate dots which reflect autophagic vesicles.

Thus, cells cultured on coverslips expressing GFP-LC3 were fixed in 3.7% formaldehyde and applied to a Leica CTR 6000 confocal microscope. Cells presenting a mostly diffuse distribution in the cytoplasm and nucleus were considered as non-autophagic, whereas, cells with several intense LC3-GFP punctae were classified as autophagic. The percentage of cells with GFP-LC3 translocation or the number of GFP-LC3 punctae per cell were quantified (600 cells/sample on duplicated samples of three different experiments). Cross-sections with over five dots on LC3 were considered positive. Other methods of monitoring autophagosome formation were measuring turnover of LC3-II, GFP-LC3 processing, levels of a general marker of autophagy such as p62/SQSTM1 and mTOR activity (by analysing P-p70S6K) by inmunoblot analysis. p62/SQSTM1 binds LC3 and it is selectively recognized and degraded by autophagy becoming a autophagy marker. 57 hVps34 kinase assay.

hVps34 kinase assay was performed as described previously. [START_REF] Zhong | Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex[END_REF] Hela and HEK293T cells were lysed in 1% Nonidet P-40 lysis buffer (20 mM Tris-pH 7.5, 137 mM NaCl, 1 mM MgC1 2 , 1 mM CaC1 2 , 100 mM NaF, 10 mM sodium pyrophosphate, 100 μM Na 3 VO 4 , 10% glycerol, 0.35 mg/ml PMSF, protease and phosphatase inhibitor cocktails). Immunoprecipitation was performed with 1 μg/IP of anti-hVps34 (Echelon Biosciences (Salt Lake City, UT, USA)) or anti-Beclin (D-18) (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)) affinity gel beads, according to the manufacturer's protocol. Beads (associated with purified proteins) were washed three times in lysis buffer, followed by three washes in washing buffer (100 mM Tris-HCl pH7.4 and 500 mM LiCl) and two washes in reaction buffer (10 mM Tri-HCl-pH7.4, 100 mM NaCl and 1 mM EDTA). Beads were resuspended in 60 μl of reaction buffer, MnCl 2 (10 μl of 100 mM) and sonicated phosphatidylinositol (10 μl of 2 μg/μl) were added. The reaction was started by the addition of ATP (10 μl of 440 μM) containing γ-32 P-ATP (10 μCi); beads were incubated for 20 min at room temperature. The reaction was terminated by adding HCl (20 μl of 8 M), and the organic phase was extracted with 160 μl chloroform:methanol (1:1). Extracted phospholipid products were resolved by TLC using TLC coated silica gel and a solvent composed of chloroform:methanol:H2O:ammonium acetate (v/v/v/v, 20:15:5:3), followed by visualization with Fujifilm FLA-5100 imaging system.

Software used for activity quantification was Multi Gauge (Lifescience/Fujifilm, USA).

Detection of cell death

The Crystal Violet assay as has been previously described was used to determine cell viability. Anti-GFP

TITTLES AND LEGENDS TO FIGURES

  Optimen, protease-inhibitor cocktail tablets and Geneticin (G-418) were purchased from Invitrogen (Paisley, UK). Peptide (ARAApSAPA) was synthesized by Graham Bloomberg at the University of Bristol and generously provided by Dr. C MacKintosh. N-Acetyl-D-sphingosine (C2ceramide), Phosphatidylinositol, E64d, 12-O-Tetradecanoylphorbol 13-acetate (PMA), IGF-1, H-7 inhibitor, 3-Methyladenine, Etoposide, Rapamacyn and Propidium iodide (PI) were obtained from Sigma Chemical Corp. (St. Louis, MO, USA). PD 184352 inhibitor was from Axon Medchem (Groningen, The Netherlands). Frozen human HeLa cell pellets from 4c Biotech (Ghent, Belgium).

  cDNA cloning and transfection of HEK293T cDNA cloning and sequencing human hVps34 (catalytic phosphatidylinositol 3-kinase 3 [Homo sapiens] NP_002638.2) was cloned by PCR using EST source (IMAGE 5517878). Primers PI3KC3 33: (AAAAGAATTCGCGCTGGGGGAAGCAGAGAAGTTTC AC) and PI3KC3 34 (TTTTGGATCCTTTTCTCCAGTACTGGGCAAACTTGTGAATCT GTTC) were used to amplify the open reading frame and add a GFP-tag at the 5¢-terminus. The resulting 2719-bp cDNA fragment was digested with BamH1 and EcoRI and ligated into the same sites in pEGFP-C2 (Clontech) to produce pGFP-hVps34 WT. Fragment MT1 was amplify using primers PI3KC3 33: and the MT2 fragment was amplify using primers PI3KC3 33: (AAAAGAATTCGCGC TGGGGGAAGCAGAGAAGTTTCAC) and PI3KC3 37: (GCGGATCCCTCATCATCGGC CTGTCGCAACCG). The resulting MT1 (2040-bp cDNA) and MT2 (1170-bp cDNA) fragments were digested with BamH1 and EcoRI and ligated into the same sites in pEGFP-C2 (Clontech) to produce pGFP-hVps34 MT1 and pGFP-hVps34 MT2.

Figure 1 .

 1 Figure 1. Overexpression of 14-3-3ζ proteins block C2-ceramide induced autophagy

Figura 4 .

 4 Figura 4. hVps34 dissociates from 14-3-3 and is activated during C2-ceramide-induced

Figura 5 .

 5 Figura 5. Overexpression of 14-3-3ζ decreases hVps34 activity meanwhile blocks C2-

Figure 6 .Figure 7 .

 67 Figure 6. Downregulation of endogenous 14-3-3ζ increase hVps34 activity.

Figure 8 .

 8 Figure 8. hVps34 binds 14-3-3 proteins by a phosphorylation-dependent mechanism.

  Figure 1

  The mutant at Ser34 (GFP-hVps34 Ser 34 A) was created by PCR (QuikChange® II Site-

	Directed Mutagenesis Kits, Stratagene) using primers PI3KC3mut 41: (GGAAGAGAGA
	ACAAAAGGCTTATAAAGCTGTCCTGG) and PI3KC3mut 42: (CCAGGACAGCTTT
	ATAAGCCTTTTGTTCTCTCTTCG). The mutant at Thr163, Ser164, Ser165 and Thr166 (GFP-
	hVps34 Thr 163 A, Ser 164 A, Ser 165 A, Thr 166 A) was created using primers PI3KC3mut 45
	(CAAAAACTCCTGGCAGAGCAGCTGCCGCTCTCTCAGAAGATCAG) and PI3KC3mut 46:
	(CTGATCTTCTGAGAGAGCGGCAGCTGCTCTGCCAGGAGTTTTTG). The mutant at Thr197
	(GFP-hVps34	Thr 197 A)	was	created	using	primers	PI3KC3mut	47:
	(GGCTGGATAGATTGGCATTTAGAGAAATAGAAATG)	and	PI3KC3mut	48:
	(CATTTCTATTTCTCTAAATGCCAATCTATCCAGCC). The mutant at Ser212, (GFP-hVps34
	Ser 212 A) was created using primers PI3KC3mut 49:	(GAGAGTGAAAAACGA
	ACTGCTAATTTCATGTACCTG) and PI3KC3mut 50:	(CAGGTACATGAAATTAGC
	AGTTCGTTTTTCACTCTC). The mutant at Ser276 and Ser279, (GFP-hVps34 Ser 276 A, Ser 279 A)
	was created using primers PI3KC3mut 51: (CACAAGCTTGCCCGGGCTTTAAGA
	GCTGGACCTTCTGACCAC) and PI3KC3mut 52: (GTGGTCAGAAGGTCCAGCTCTT

Cells were seeded at approximately 10 000 cells per well in 48-well plates and allowed to adhere overnight. After treatment, cells were fixed in 100% methanol for 10 minutes, stained with 0.5% crystal violet solution in 25% methanol for 10 minutes, washed three times with 1X PBS to remove excess dye and allowed to dry overnight. The following morning, crystal violet containing cells was solubilized in 1 ml 1% SDS and an absorbance reading was taken at 560 nm. Hypodiploid death cells (sub-G1) were detected using PI by flow cytometry as described previously.[START_REF] Ruiz-Ruiz | Mitochondria-dependent and -independent mechanisms in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis are both regulated by interferon-gamma in human breast tumour cells[END_REF] Flow cytometry

ACKNOWLEDGEMENTS

Grant support: "Ministerio de Educación y Ciencia" grant BFU2006-01088/BMC and "Programa Ramón y Cajal" contract (B.O.E. 17/02/2004 ORDEN CTE/351/2004) to Mercedes Pozuelo Rubio. I thank. J. Oliver, A. López-Rivas and M. Jäättelä for tools provided and G. Herrero and C. Fumarola for helpful discussion. Thanks to C. MacKintosh for antibodies, constructs and other helpful tools, P. Chacón for mouse lung tissue extraction and P. Domínguez, M.J. Quintero, A. Vela, N. Leslie and J.C. Ostos for helpful assistance. Thanks to Diane Haun for spell checking.

was performed on a FACScan cytometer using Cell Quest software (Becton Dickinson, Mountain View, CA, USA).

Statistical analysis

Independent experiments were pooled when the coefficient of variance could be assumed identical. Statistical significance was evaluated by using t-test Student (N=number of independent experiments). P values of under 0.05 were considered significant.

ONLINE SUPPLEMENTARY INFORMATION

Supplementary information is available at the Cell Death and Differentiation´s website

Supplementary Figure S1. C2-ceramide 10 μM for 24 h does not induce cell death. S2. Over expression of 14-3-3ζ proteins block C2-ceramide cell death with autophagy. S3. Downregulation of endogenous 14-3-3ζ sensitizes cells to autophagy. S4. Downregulation of endogenous 14-3-3ζ does not affect levels of expression of autophagy-related proteins Supplementary Figure S5. Downregulation of endogenous 14-3-3σ and 14-3-3θ sensitizes cells to autophagy. Supplementary Figure S6. hVps34 dissociates from 14-3-3 and is activated during C2-ceramide-induced autophagy in HEK293T cell.

Supplementary Figure

Supplementary Figure

Supplementary Figure

Supplementary Figure S7. Overexpression of 14-3-3ζ decreases hVps34 activity meanwhile blocking C2ceramide-dependent autophagy in HEK293T cells

Supplementary Figure S8. Downregulation of endogenous 14-3-3ζ increase hVps34 activity in MCF-7 cells.

Supplementary Figure S9. hVps34 dissociates from 14-3-3 and is activated during Starvation-induced autophagy in MCF-7 cells.

Supplementary Figure S10. hVps34 dissociates from 14-3-3 during Etoposide and Rapamycin-induced autophagy.

Supplementary Figure S11. hVps34 from lung mouse tissue bind 14-3-3 proteins.

Supplementary Figure S12. Serum-free medium increases hVps34 lipid kinase activity

Supplementary Figure S13 Amino acid sequence of hVps34 with the 14-3-3-binding domains.

CONFLICT OF INTEREST

The authors declare no conflict of interest. HA-14-3-3ζ. Tubulin was used as a loading control. A full-colour version of this figure is available at Cell Death and Differentiation Online.

Figure 2. Downregulation of endogenous 14-3-3ζ sensitizes cells to autophagy. (A)

HeLa cells expressing GFP-LC3 were transfected either with siRNA oligonucleotide targeting 14-3-3ζ or with a scrambled RNA oligonucleotide as described in Material ad Methods.

After 48 hours, untransfected cells (Control) or transfected either with siRNA 14-3-3ζ (14-3-3ζ) or scrambled siRNA (SC) were analysed using green fluorescence GFP-LC3 by microscopy. Bars=10 extracts from untransfected cells (Control) or transfected either with siRNA 14-3-3ζ (14-3-3ζ) or scrambled siRNA (SC) were harvested after 48 hours for immunoblot analysis of LC3-I processing into LC3-II, GFP-LC3 processing into GFP, p62/SQSTM1 and endogenous 14-3-3ζ isoform.

Tubulin was used as a protein loading control. Endogenous LC3-II/Tubulin levels were quantified and the ratio presented as arbitrary units (N=3, * P=0.0003, ** P=0.0004, Student t-test). A fullcolour version of this figure is available at Cell Death and Differentiation Online.

Figure 3. 14-3-3 affinity chromatography of human HeLa cell extracts

Clarified HeLa cell extract was chromatographed on 14-3-3-Sepharose, as described in Material and Methods. Column fractions were run on SDS/PAGE using 10% Tris-Glycine gels, and transferred to nitrocellulose membranes. Amounts of protein run on SDS/PAGE were as follows:

extract, flow through and beginning of salt wash (1st Wash), 40 μg of each; middle and end of salt wash (2nd Wash and 3rd Wash respectively), protein undetectable; control (phospho)peptide pool,<1 μg; and ARAApSAPA elution pool, 2 μg. Membranes were checked for binding to DIG-14-3-3 (top panel). Western blots were probed with antibodies against the indicated proteins related to autophagy (Bottom panels).