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Pedjcellphysiology2014.Pdf ORIGINAL RESEARCH ARTICLE 1359 JournalJournal ofof Cellular Phosphorylation-Regulated Physiology Degradation of the Tumor- Suppressor Form of PED by Chaperone-Mediated Autophagy in Lung Cancer Cells CRISTINA QUINTAVALLE,1,2 STEFANIA DI COSTANZO,1 CIRO ZANCA,1 IMMACULADA TASSET,3 ALESSANDRO FRALDI,4 MARIAROSARIA INCORONATO,5 PEPPINO MIRABELLI,5 MARIA MONTI,6 ANDREA BALLABIO,4 PIERO PUCCI,6 3 1,2 ANA MARIA CUERVO, AND GEROLAMA CONDORELLI * 1Department of Molecular Medicine and Medical Biotechnology, ‘‘Federico II” University of Naples, Naples, Italy 2Institute of Endocrinology and Experimental Oncology ‘‘G. Salvatore” (IEOS), Consiglio Nazionale delle Ricerche (CNR), Naples, Italy 3Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, New York 4Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 5IRCCS, SDN Foundation Institute of Diagnostic and Nuclear Development (SDN), Naples, Italy 6Department of Chemical Science, ‘‘Federico II” University of Naples, Naples, Italy PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)—which, among other processes, is involved in chaperone-mediated autophagy (CMA)—as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms. J. Cell. Physiol. 229: 1359–1368, 2014. © 2014 Wiley Periodicals, Inc. Phosphoprotein enriched in diabetes (PED), known also as phosphoprotein enriched in astrocytes 15 (PEA-15), is a death effector domain (DED) family member with a variety of effects Contract grant sponsor: Associazione Italiana Ricerca sul Cancro on cell growth and metabolism (Condorelli et al., 1999; Renault (AIRC); Contract grant numbers: 10620, 14046. et al., 2003). It has a broad anti-apoptotic action, being able to Contract grant sponsor: MERIT; inhibit the intrinsic and the extrinsic apoptotic pathways Contract grant number: RBNE08E8CZ_002. (Stassi et al., 2005; Garofalo et al., 2008; Zanca et al., 2008; Contract grant sponsor: POR Campania FSE 2007–2013 Project Incoronato et al., 2010). PED is known to interact with a CRÈME. number of proteins, among which are extracellular signal- Contract grant sponsor: Fondazione Berlucchi. regulated protein kinases 1 and 2 (ERK1/2), sequestering them Contract grant sponsor: NIH/NIA; in the cytosol and, thus, regulating their function (Formstecher Contract grant numbers: AG031782, AG03872. et al., 2001; Romano et al., 2012). PED can be found in an Contract grant sponsor: ‘‘Federazione Italiana Ricerca sul Cancro'' unphosphorylated state, or phosphorylated at Ser104 by (FIRC) Post-Doctoral Research Fellowship. protein kinase C, and at Ser116 by AKT or calcium calmodulin Contract grant sponsor: Fulbright Postdoctoral Fellowship. kinase II (CamKII) (Trencia et al., 2003; Sulzmaier et al., 2012). *Correspondence to: Gerolama Condorelli, Department of Interestingly, PED can act as either a tumor promoter (when Molecular Medicine and Medical Biotechnology, ‘‘Federico II” phosphorylated) or a tumor suppressor (when unphosphory- University of Naples, Via Pansini 5, 80131 Naples, Italy. lated) (Sulzmaier et al., 2012). Moreover, PED has been E-mail: [email protected] reported up-regulated in a number of different cancer types, Manuscript Received: 23 October 2013 but the mechanisms regulating its expression remain poorly Manuscript Accepted: 27 January 2014 understood (Hao et al., 2001; Stassi et al., 2005; Garofalo Accepted manuscript online in Wiley Online Library et al., 2007; Zanca et al., 2008). (wileyonlinelibrary.com): 29 January 2014. To get further insight into the role of PED in cancer, we have DOI: 10.1002/jcp.24569 previously reported on the PED interactome identified © 2014 WILEY PERIODICALS, INC. 1360 QUINTAVALLE ET AL. through tandem affinity purification (TAP) in non-small-cell with washing buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% lung-cancer cells (Zanca et al., 2010): among the interacting Triton, 10% glycerol), 30 ml of sample buffer added, and the proteins found were RAC1 (ras-related C3 botulinum toxin samples boiled at 100°C for 5 min. The supernatants were substrate 1), a mammalian rho GTPase protein family member, resolved on 12% polyacrylamide gels by SDS–PAGE. and HSC70 (heat shock cognate protein of 70 kDa), a chaperone involved in a multitude of housekeeping functions Western blotting and in chaperone-mediated autophagy (CMA) (Kaushik and Cuervo, 2012a,b). CMA is a uniquely selective form of Total proteins from A549 and HEK293 cells were extracted with autophagy in which specific cytosolic proteins are transported RIPA buffer. Fifty micrograms of sample extract were resolved on one-by-one across the lysosomal membrane for degradation 10–15% SDS–polyacrylamide gels using a mini-gel apparatus (Massey et al., 2006). This process is constitutively active in (Bio-Rad Laboratories, Richmond, CA) and transferred to many cell types, but it is maximally activated during stress Hybond-C extra nitrocellulose (GE Healthcare, Milan, Italy). (Kaushik and Cuervo, 2012a). CMA is selective for a subset of Membranes were blocked for 1 h with 5% non-fat dry milk in soluble cytosolic proteins containing a pentapeptide motif— TBS–0.05% Tween-20, incubated overnight with primary antibody, known as the CMA-targeting motif—biochemically related to washed, incubated for 1 h with secondary antibody, and visualized Lys-Phe-Glu-Arg-Gln (KFERQ) (Dice, 1990). In the cytosol, by chemiluminescence. HSC70 recognizes the KFERQ sequence present in CMA substrates, and chaperones them to the lysosomal membrane, Immunofluorescence where they can interact with lysosome-associated protein type 2A (LAMP2A), the CMA receptor. Moreover, HSC70 likely A549 cells were serum starved for 24 h before analysis. To facilitates substrate unfolding, a requirement for substrate enhance the lysosomal localization and to prevent degradation, translocation across the lysosomal membrane (Kaushik and bafilomycin A (Sigma–Aldrich) was added to serum-depleted Cuervo, 2012a,b). media. Cells were fixed with 4% paraformaldehyde for 10 min, In the present report, we describe the interaction of PED permeabilized with Triton 0.1% in PBS for 10 min at room with HSC70, demonstrating that HSC70 targets temperature, and blocked in 10% FBS–0.1 % saponin in PBS. unphosphorylated PED for CMA. This event has a deleterious Primary antibodies were dissolved in 1:10-diluted blocking effect on cell fate by promoting tumorgenesis. solution and incubated at the following dilutions for 1 h at room temperature: anti-PED, 1:100; anti-HSC70, 1:100; anti-LAMP2A, 1:200. Cells were then incubated for 1 h at room temperature as Materials and Methods fl Cell lines and reagents appropriate with an anti-rabbit green uorochrome antibody (Alexa 488-conjugated phalloidin), an anti-rabbit red The human A549 cell line was purchased from American Type fluorochrome antibody (Alexa 594), or an anti-mouse red Culture Collection (Milan, Italy) and maintained in RPMI medium fluorochrome antibody (Alexa 459), all purchased from BD supplemented with 10% heat-inactivated fetal bovine serum (FBS), Bioscience (Milan, Italy) and diluted 1:1,000 in 1:10-diluted blocking 2 mM L-glutamine, and 100 U/ml penicillin–streptomycin. The solution. Cells were then washed four times in cold PBS. Antigen– human embryonic kidney (HEK) 293 cell line was grown in DMEM antibody complexes were visualized by confocal microscopy under supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, a Zeiss LSM 510 microscope equipped with a Zeiss confocal- and 100 U/ml penicillin–streptomycin. Media, sera, and antibiotics scanning laser and a 1.4 objective with a 60Â numerical aperture. for cell culture were from Sigma–Aldrich (Milan, Italy). Protein electrophoresis reagents were from Bio-Rad (Milan, Italy), and Quantification of cell viability Western blotting and ECL reagents were from GE Healthcare (Milan, Italy). HSC70 antibody was from Santa Cruz Biotechnology Cells were seeded in 96-well plates in triplicate and incubated at (Santa Cruz, CA), human LAMP2A antibody was from Abcam (MA), 37°C in a 5% CO2 incubator. SuperKiller TRAIL was used at a final b-actin antibody was from Sigma–Aldrich, and Myc antibody was concentration of 50 ng/ml for 24 h. Cell viability was evaluated with from Cell Signaling Technology (Milan, Italy). The PED antibody was the CellTiter 96 AQueous One Solution Cell Proliferation Assay generated as described before (Condorelli et al., 2002). For (Promega, Milan, Italy), according to the manufacturer’s protocol. transfection, Lipofectamine 2000 was purchased from Life Metabolically active cells were detected by adding 20 ml of MTS to Technologies (Milan, Italy). The plasmids used encoded Myc–PED each well. After 2 h of incubation, plates were analyzed in a (Condorelli et al., 1998) and the phosphorylation mutants HA-PED- Multilabel Counter (BioTek, Milan, Italy). S104/116A and HA-PED-S104/116D (a kind gift from Dr Mark H. Ginsberg, The Scripps Research Institute, La Jolla, San Diego, CA); Recombinant PED protein production Lamp2a shRNA and Lamp2a cDNA were from Dr Ana Maria Cuervo (Massey et al., 2006);) siRNA for Hsc70 was from Santa BL21 cells were transformed with a glutathione-S-transferase Cruz Biotechnology.
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