Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells

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Research Article

Eps8 is recruited to lysosomes and subjected tochaperone-mediated autophagy in cancer cells

Thilo Welscha,⁎, Alexander Younsia, Andrea Disanzab, Jose Antonio Rodriguezc,Ana Maria Cuervoc, Giorgio Scitab, Jan Schmidta

aDepartment of General, Visceral and Transplant Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, GermanybIFOM, FIRC Institute of Molecular Oncology, Milan, ItalycDepartment of Developmental and Molecular Biology, Marion Bessin Liver Research Center Institute for Aging Research,Albert Einstein College of Medicine, New York, USA

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +49 6221 56 578E-mail address: [email protected]

0014-4827/$ – see front matter © 2010 Elseviedoi:10.1016/j.yexcr.2010.02.020

A B S T R A C T

Article Chronology:

Received 5 January 2010Revised version received13 February 2010Accepted 18 February 2010Available online 23 February 2010

Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity,and indirectly by regulating Rac-activation when engaged into a trimeric complex with Eps8-Abi1-Sos1. Recently, Eps8 has been associated with promotion of various solid malignancies, butneither its mechanisms of action nor its regulation in cancer cells have been elucidated. Here, wereport a novel association of Eps8 with the late endosomal/lysosomal compartment, which isindependent from actin polymerization and specifically occurs in cancer cells. Endogenous Eps8localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such asAsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8increased the size of lysosomes. Structure–function analysis revealed that the region encompassingthe amino acids 184–535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, thisfragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA).

Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elementsfor the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to(11.9±5.1%) and was incorporated into (5.3±6.5%) lysosomes. Additionally, Eps8 binding tolysosomes was competed by other known CMA-substrates. Fluorescence recovery afterphotobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic.Collectively, these results indicate that Eps8 in certain human cancer cells specifically localizes tolysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8is regulated and contributes to tumor promotion in human cancers.

© 2010 Elsevier Inc. All rights reserved.

Keywords:

Eps8LysosomeAutophagyPancreatic cancerActin

LAMP

Introduction

The epidermal growth factor receptor pathway substrate 8 (Eps8)is a 97 kDa protein that was originally identified as a substrate forthe kinase activity of the epidermal growth factor receptor (EGFR).Early results demonstrated that Eps8 increases epidermal growth

1.lberg.de (T. Welsch).

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factor (EGF) responsiveness, thus contributed to malignanttransformation in tumor cells [1,2]. There is now growing evidencethat Eps8 plays an important role in promotion of various solidmalignancies, including colon, squamous cell, thyroid and cervicalcancer [3–7]. We recently reported that Eps8 expression isenhanced in metastatic pancreatic cancer cells when compared

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to cells from the primary tumor [8]. Moreover, high Eps8expression in metastatic cells was associated with increased cellmotility and accumulation of Eps8 at actin dynamic sites, e.g. theleading edge of the cells.

The Eps8 molecule controls actin remodeling through multipleinteractions. The C-terminal “effector region” directly binds F-actinand caps actin barbed ends [9]. In addition, Eps8 participates in theactivation of the small GTPase Rac in a tri-protein complex withAbi1 and Sos1, and synergizes with IRSp53 to bundle actinfilaments [10]. A number of recent evidences indicate that actindynamics and endocytic machineries are intimately related. Thisnotion appears to apply also in the case of Eps8. Rac-signalingthrough Eps8 can be hampered by binding of RN-tre, a GTPaseactivating protein (GAP) for Rab5, to the src homology-3 (SH3)domain of Eps8, leading to disruption of the Eps8-Abi1-Sos-1complex and to the de-activation of Rab5, which in turns regulatesthe internalization of EGFR [11]. Intriguingly, Rab5-dependentendocytosis and early endosome trafficking have recently beenshown to be required for Rac-mediated, spatially restricted actinremodeling at the cell periphery [12].

Within this context, Eps8 has all the features to posit that itmight be an important regulatory elementmediating the cross talkbetween membrane and actin dynamics, which ultimately isrequired to generate propulsive force at the leading cell edge.However, whether Eps8 directly participates in membraneinternalization and/or endomembrane trafficking remains unclear.Since there is little known about the degradation of Eps8 [13], itmight be possible that the regulatory function of Eps8 is controlledthrough its expression levels.

Chaperone-mediated autophagy (CMA) is a cellular process forthe selective degradation of cytosolic proteins [14]. Unlike otherforms of autophagy, CMA mediates the direct translocation ofindividual proteins across the lysosome membrane for theirdegradation in the lysosome [15,16]. CMA activity decreaseswith age and has shown to be altered in different humanpathologies [17,18]. Malfunctioning autophagy has been closelylinked to oncogenic signaling [19].

Here, we investigate a novel association of Eps8 to thelysosomal compartment in human cancer cells and describe itsdegradation through CMA.

Fig. 1 – Endogenous Eps8 localized to vesicular structures inthe human pancreatic cancer cells AsPC-1 and Capan-1. (A, B) Inthe pancreatic cancer cell line PANC-1, Eps8 showed a spot-likepattern associated with F-actin structures (merged insert inpanel B). (C, D) In Capan-1 cells, endogenous Eps8 stronglylocalizes to large vesicular structures. These vesicularstructures were negative for F-actin. (E, F) Likewise, Eps8localizes to vesicular structures in AsPC-1 cells. (G)Representative antibody staining of endogenous Eps8 in anAsPC-1 cell. Eps8 stained the lamellipodium (arrow), and largevesicles, that are mostly located at the perinuclear region. (H)Higher magnification (insert in G) clearly demonstrates Eps8positivity at the vesicular membrane. The diameter of theindicated vesicle (arrow) was 0.77 μm. Image widthsrepresent: (A, B) 80 μm, (C-F) 50 μm, (G) 60 μm, (H) 29 μm.

Material and methods

Expression vectors, antibodies, reagents and siRNA

Vectors encoding for GFP fusion proteins were engineered in thepEGFP C1 plasmid (Clontech Laboratories, Inc.) by cloning theappropriate fragments, obtained by recombinant PCR, in framewith the GFPmoiety as described recently [20]. All constructs weresequence verified. Details are available upon request. Celltransfection was performed with Lipofectamine 2000 (Invitrogen,Eugene, OR) according to the manufacturer's instructions.

The antibodies used were: mouse monoclonal anti-Eps8(Transduction Laboratories, Lexington, KY), rabbit polyclonalanti-Eps8 [1], mouse anti-EEA1 (clone 14, BD Biosciences), rabbitanti-EGFR (sc-03, Santa Cruz Biotechnology, Heidelberg,Germany), mouse anti-LAMP-1 and mouse anti-LAMP-2 (clonesH4A3 and H4B4, Developmental Studies Hybridoma Bank,University of Iowa, IA), and mouse anti-Hsc70 (clone 13D3,

Novus Biologicals, Inc., Littleton, CO). The monoclonal anti-Abi1and anti-IRSp53 antibodies were generated as previouslydescribed [9,10].

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LysoTracker (DND-99, Molecular Probes, Invitrogen) wasapplied at 100 nM and added to the cell medium for 2 h beforefurther processing. Rhodamine-EGF (Molecular Probes, Invitro-gen) was used at 30 ng/ml for the indicated time periods underserum-free conditions. To inhibit actin polymerization, cytochala-sin D (Sigma, Deisenhofen, Germany) was added to the cells at1 μg/ml for 30 min. Inhibition of lysosomal acidification andprotein degradation was achieved using bafilomycin A1 (Sigma) at100 nM for 60 min. The effect of bafilomycin was proved byabsence of LysoTracker uptake into the lysosomes.

Fig. 2 – Eps8-positive vesicular structures were identified as lysosomthen stained for markers of the early and late endocytic compartmea full overlap with the GFP-fluorescence. (D–F) Endosomes positivEps8-positive vesicles. (G–I) Eps8 colocalizedwith the lysosomememof the Eps8-positive vesicles was enriched with the LysoTracker dyendogenous Eps8 and in HeLa cells (not shown). Together, these datImage widths represent: (A–C) 69 μm, (D–F) 49 μm, (G–I) 65 μm, (

Cell lines

Human pancreatic cancer cell lines (AsPC-1, Capan-1, PANC-1)and HeLa cells were purchased from ATCC (Rockville, MD).Cells were cultured at 37 °C with 5% CO2 in RPMI 1640 (PAALaboratories GmbH, Cölbe, Germany) without antibiotics andsupplemented with 10% fetal calf serum. For immunofluores-cence analysis, cells were grown on collagen A-coated(Biochrom, Berlin, Germany) glass coverslips for at least 2days.

es. AsPC-1 cells were transfectedwith full-length Eps8-GFP andnt. (A–C) As proof-of-principle, Eps8 antibody staining revealede for the early endosome marker 1 (EEA1) were distinct frombranemarker LAMP-1 to lysosomes. (J–L)Moreover, the lumen

e. The same results were yielded with antibody staining ofa strongly suggest that the Eps8-positive vesicles are lysosomes.J–L) 39 μm.


Immunofluorescence staining and confocal laser scanningmicroscopy

Briefly, cells were fixed with 2% paraformaldehyde for 10 min,permeabilized with 0.3% Triton X-100 for 5 min, and blocked inblocking solution (2% FBS, 2% bovine serum albumin, 0.2% gelatin inPBS) followed by incubation with the primary antibody for 60 min.Colocalization analysis with F-actin was performed using fluo-

Fig. 3 – Increased expression of Eps8 promotes Eps8 recruitmentcancer cell lines overexpressing full-length Eps8-GFP were co-stainEndogenous Eps8 did not localize to lysosomes in untransfected Hlysosomes in HeLa cells. (D–F) Endogenous Eps8 was present at lyslysosomes appeared larger. (G–I) To quantify whether Eps8 overexwith LysoTracker dye and either transfected with full-length Eps8-sizes (according to LysoTracker fluorescence) were measured withlysosomes (50 cells) each, and were significantly larger in Eps8-GFrepresent: (A–C) 36 μm, (D–F) 89 μm, (G–J) 60 μm.

rochrome (Alexa488)-conjugated phalloidin. Antigen-antibodycomplexes were visualized with Cy3 (1:200)- or fluoresceinisothiocynate (FITC, 1:25)-conjugated secondary antibodies fromsheep (Sigma). As negative controls, the primary antibodies werereplaced with rabbit or mouse IgG, respectively, and absence ofimmunoreactivitywas confirmed. Confocal imagingwasperformedon a TCS-SP laser-scanning microscope using the 40× or 63× oil-immersion objectives (Leica Microsystems, Heidelberg, Germany).

to lysosomes and influences lysosomal morphology. Differented with the lysosome membrane marker LAMP-1. (A–C)eLa cells. After overexpression, Eps8-GFP was observed atosomes in Capan-1 cells. In cells overexpressing Eps8-GFP, thepression increases lysosome size, AsPC-1 cells were incubatedGFP (G, H), or with the empty EGFP C1 vector (I, J). Lysosomethe ImageJ software (http://rsb.info.nih.gov/ij/) in >200P transfected cells (p=0.0396, Student t-test, K). Image widths

http://rsb.info.nih.gov/ij/


Measurement of each lysosome size was performed using theconfocal z-plane adjusted to the maximum diameter and fluores-cence gain of the LysoTracker dye. The size of the lysosomes inpixels was quantified with ImageJ outlining each lysosome(National Institutes of Health, Bethesda, MD).

For living cell confocal imaging, cells were grown on 25 mmglass cover slips and transfected with Eps8-GFP and incubatedwith LysoTracker dye for 2 h. The cover slips were then placed inan Attofluor® cell chamber (Molecular Probes, Invitrogen) whichwas mounted on a Leica TCS SP5 confocal microscope and kept at37 °C during imaging.

Fluorescence recovery after photobleaching (FRAP)

FRAP analysis was performed on a Leica TCS SP5 confocalmicroscope using the 63x oil immersion objective. Cells weregrown in 35-mm Petri dishes containing a coverslip insert (MatTekCorporation, Ashland, MA) and transfected with Eps8-GFP plasmid.After three frames (12% laser intensity), a boxed region of interest(ROI) containing a lysosome with Eps8-GFP was bleached using100% laser power at an excitationwavelength of 488 nm. Repetitiveimages were acquired every 6 s (and later every 10 s) as thefluorescence recovered using 12% laser intensity. Fluorescencerecovery was analyzed using the ImageJ software (NationalInstitutes of Health). First, for each frame of a timelapse series, thefluorescence intensity of a ROI containing the area of the cell thatwas photobleached was determined (F1). Next, the fluorescenceintensity of a ROI containing anon-bleached cytoplasmic regionwasdetermined for each frame (F2). A normalized fluorescenceintensity value (Fn) was then obtained using F1/F2=Fn. Thebackground fluorescence intensity (Fb) was set as Fn of the firstframe after photobleaching, and this value was subtracted from allframes such that the fluorescence intensity value for the ROI in agiven frame equals Fn− Fb, and this value is zero for the first frameafter photobleaching. The amount of fluorescence recovery wasthen calculated as the fluorescence intensity value of a given framedivided by the fluorescence intensity value of the frame immedi-ately before photobleaching and was expressed as a percentage.

Cell lysis, co-immunoprecipitation and immunoblot

AsPC-1 or HeLa cells were briefly rinsed with PBS and scraped intoa lysis buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1%glycerol, 1% Triton X-100, 1.5 mM MgCl2, 5 mM EGTA, 1 mM DTT,and protease inhibitors (complete Mini EDTA free, Roche,

Fig. 4 – Identification of the protein region of Eps8, that is sufficieengineered as GFP fusion proteins were checked for colocalization wafter transfection of AsPC-1 cells or HeLa cells (not shown). (A–C) Tpositive for LAMP-1 (insert). Since this fragment lacks the SH3 andactin-associated structures as the endogenous Eps8. (D–F) The N-tefragment 180–821 localized to lysosomes and actin-associated strulocalized to actin-associated structures. Image widths represent 40various Eps8 fragments with their binding capability to lysosomespredicted by analysis of the primary sequence, are indicated: a phoputative epidermal growth factor receptor (EGFR) binding region (mediating the interactionwith Abi1 and RN-tre, and the C-terminalwith Sos1 and F-actin. Binding of the various Eps8 fragments (amilysosome is indicated on the right. Lysosomal binding was mediateKFERQ-like motifs (aa: 226, QVDVR; aa: 470: QEIKR).

Mannheim, Germany). The supernatant was collected aftercentrifugation at 13,200 rpm for 10 min at 4 °C. The proteinconcentration of the lysates was determined using the BCA-Kit(BCA Protein Assay, Thermo Scientific, Rockford, IL). For immuno-precipitation, 1 μl of the primary antibody (polyclonal anti-Eps8)was added to lysates containing 1000 μg protein, and precipitatedover night at 4 °C by adding 25 μl of protein A Sepharose beads (GEHealthcare Bio-Science GmbH, Munich, Germany). The beads werethen centrifuged at 1000 rpm for 5 min at 4 °C, washed with lysisbuffer for three times, and then analyzed by immunoblot.

For immunoblot, protein sampleswere heated to 99 °C for 3minand were separated in a 4–12% gel (NuPAGE, Invitrogen, Carlsbad,CA). After blotting on a nitrocellulose membrane (BioRad),membranes were blocked in 5% non-fat dry milk or Slim Fastpowder (Slim Fast, Allpharm Vertiebs-GmbH, Germany) diluted inTBS-Buffer (Amresco, MoBiTec, Goettingen, Germany) at pH 7.5with 0.05% Tween for 1 h at room temperature. The blotswere thenincubated with the primary antibody (mouse anti-Lamp2 1:1000,mouse anti-eps8 1:5000, mouse anti-Hsc70 IgM 1:1000) inblocking buffer overnight at 4 °C, rinsed for 1 h with washingbuffer and incubated with horseradish peroxidase (HRP)-conju-gated secondary antibodies for 1 h at room temperature. Afteradditional washes, the blots were incubated in chemiluminescencesolution (ECL; Amersham Biosciences, Freiburg, Germany) for2 min and exposed to an X-ray film. Before further primaryantibody incubations, the membrane was exposed to strippingbuffer (Thermo Scientific, Rockford, IL) for 45 min at 37 °C.

Isolation of lysosomes

Rat liver lysosomes were isolated from a light mitochondrial-lysosomal fraction in a discontinuous metrizamide density gradi-ent by the shorter method described previously [21]. Lysosomalintegrity was verified after isolation by measuring the activity ofβ-hexosaminidase, a lysosomal enzyme, in the incubationmedium.Preparations with more than 10% broken lysosomes immediatelyafter isolation or more than 20% at the end of the incubation werediscarded. We did not find significant differences in the stability oflysosomes incubated in the presence or absence of Eps8.

Protein purification and uptake of substrate proteins byisolated lysosomes

His-Eps8 FL was purified from Sf9 insect cells. Briefly, Sf9 cellsinfected with the appropriate virus were lysed in 50 mM Tris–HCl

nt to mediate lysosomal binding. Variou:ss Eps8 fragments, allith the lysosomal markers LAMP-1 or LysoTracker (not shown)

he N-terminal fragment 1–535 localized to lysosomes that werethe “effector region” domain, it did not localize torminal 1–184 fragment did not localize to lysosomes. (G–I) Thectures, e.g. the lamellipodium. (J–L) The fragment 535–821 onlyμm. (M) Domain organization of Eps8 and schematic of the. Human Eps8 is 821 amino acids (aa) long. The domains,sphotyrosine binding protein domain (PTB, aa: 60–197), theaa: 296–362), the src-homology 3 (SH3) domain (aa: 535–586)“effector region” (aa: 648–821) responsible with the interactionno acid boundaries are given on the left of the fragments) tod by the amino acids 184–535. In this region, we identified two


at pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 5%glycerol, protease inhibitor cocktail (Roche, Basel, Switzerland),1 mM NaF and 1 mM NaVO4. His-Eps8 was purified using Ni-NTA-agarose (Qiagen, Venlo, Netherlands) following standard proce-dures. The purified proteins were dialyzed against MOPS buffer

(10 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7.3,0.3 M sucrose) before use for lysosomal uptake assays.

Transport of purified proteins into isolated lysosomes wasanalyzed using a previously described in vitro system [21–23]. Eps8was incubated with freshly isolated intact rat liver lysosomes in


MOPS buffer (10 mM 3-(N-morpholino) propanesulfonic acid(MOPS) pH 7.3, 0.3 M sucrose) and an ATP regenerating system for20 min at 37 °C. Where indicated, lysosomes were pre-incubatedwith a cocktail of proteinase inhibitors for 10 min at 0 °C [21]. Anyadditional protein added to the incubation medium was dissolvedin MOPS buffer and when necessary pH was adjusted again to 7.3.At the end of the incubation, lysosomes were collected bycentrifugation, washed with MOPS buffer and subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and immunoblot with an antibody specific for Eps8. Transport wascalculated by subtracting the protein associated to lysosomes in thepresence (protein bound to the lysosomalmembrane and taken upby lysosomes) and absence (protein bound to the lysosomalmembrane) of inhibitors of lysosomal proteases, as described [24].In the absence of protease inhibitors, the substrate that reaches thelysosomal lumen is rapidly degraded and the only substrateremaining is that bound to the lysosomal membrane. If lysosomalproteases are blocked, both themembrane-bound protein and thattranslocated into the lumen can be detected. Uptakewas calculatedas the difference between the amount of substrate associated withprotease inhibitor-treated lysosomes and that bound to thelysosomal membrane without protease inhibitors.

Results

We recently described that Eps8 localizes to dynamic actinstructures, i.e. to the tips of F-actin filaments in filopodia, and tothe leading edge of pancreatic cancer cells, and that variouspancreatic cancer cell lines expressed different mRNA and proteinlevels of Eps8 according to the following order: PANC-1<Capa-n1<AsPC-1 [8].

Fig. 5 – Eps8 binds Hsc70 and Lamp2. (A) Endogenous Eps8 was pand LAMP-2. Lysate was total AsPC-1 cell lysate (40 μg), mock contwith beads only, and IP was cell lysate (1 mg) with precipitating aImmunofluorescence analysis of full-length Eps8-GFP and LAMP-2(image width represents 58 μm).

In addition to the spot-like, F-actin-associated pattern (such asin PANC-1 cells, Figs. 1A, B), endogenous Eps8 strikingly localizedalso to large vesicular structures in other pancreatic cancer cells.This staining pattern was particular prominent in cells derivedfrom metastasis (Capan-1, Figs. 1C, D), or from malignant ascites(AsPC-1, Figs. 1E, F) that express relatively high levels of Eps8 [8].In these cells, Eps8 was present on the limiting membrane ofvesicular structures that were predominantly located at theperinuclear region (Figs. 1G, H).

To identify the nature of these vesicular compartments, weanalyzed the colocalization of Eps8-GFP with specific markers ofdifferent endocytic organelles (Fig. 2). Eps8-positive vesicles weredevoid of the early endosome marker 1 (EEA1, Fig. 2D) and Rab5(not shown), but enriched in the lysosome marker LAMP-1.Moreover, the vesicular lumen was strongly enriched with theLysoTracker dye, thus defining Eps8-positive vesicular structuresas bona fide lysosomes. Confocal time-lapse imaging of Eps8-GFP inAsPC-1 and HeLa cells revealed that the lysosomes were mostlystationary (see Figs. S1 and 2). Inhibition of actin polymerizationwith cytochalasin D did not change the lysosomal association ofEps8 (see Fig. S3), indicating that actin dynamics is requiredneither for the formation of these structures nor for therecruitment of Eps8. Interestingly, Eps8 recruitment to lysosomalmembranes persisted also after inhibition of lysosomal acidifica-tion with bafilomycin A1 indicating that lysosomal recruitmentof Eps8 is not strictly dependent on functional lysosomes (seeFig. S4).

Since Eps8 is a substrate of the EGFR kinase activity, we nextanalyzed whether Eps8 is directed to lysosomes through its directassociation with EGFR or via receptor-mediated internalization. Tothis end, rhodamine-labeled EGF (rhEGF) ligand was used toinduce EGFR internalization and to track its cognate the receptor

recipitated in AsPC-1 cells and co-immunoprecipitated Hsc70rol was lysis buffer without cells, control was cell lysate (1 mg)ntibody (polyclonal rabbit anti-Eps8) and beads. (B)in AsPC-1 cells. Eps8 and LAMP-2 colocalized at lysosomes


along the endocytic route (see Fig. S5). In the case of the EGFR,ligand remains bound and signaling from the complex remainsactive until receptors can be sequestered in multivesicular bodiesfor subsequent degradation in the lysosome [25]. Notably, only fewvesicles, however, were focally positive for both EGF or EGFR andEps8, while the vast majority of them display only Eps8 along thelysosomal limiting membrane (Fig. S5D–F), arguing that EGFRbinding is presumably not responsible for the recruitment of Eps8to lysosomes. Consistently, most rhEGF was not found incolocalization with late endosomes/lysosomes as indicated bythe LAMP-1 co-staining (Fig. S5G–I).

Likewise, the Eps8 binding partners Abi1 and the insulinreceptor tyrosine kinase substrate of 53 kDa (IRSp53) did notlocalize to Eps8 at lysosomes (Fig. S6).

We previously published that AsPC-1 and Capan-1 cells expressrelatively high levels of the Eps8 protein compared to other

Fig. 6 – Chaperone-mediated autophagy of Eps8. Freshly isolated ras indicated, were incubated with purified Eps8 for 20 min at 37 °Cincubation by centrifugation were subjected to SDS-PAGE and immsubstrate proteins (GAPDH and RNase A) or non-CMA substrate pro(A) Representative immunoblot. (B) The amount of Eps8 associatedthe immunoblots. Values are expressed as percentage of the total EPreactions. Uptake was calculated as the amount of Eps8 associatedafter discounting the amount associated to untreated lysosomes (biand association are the mean values of the 8 experiments and hendifference between themeans. (C) Effect of the addition of increasinlysosomes calculated by densitometric quantification of immunobpercentage of protein added and are mean + S.E. of 4–8 independento lysosomes after addition of the indicated concentrations of prot

pancreatic cancer cell lines, e.g. BxPC-3 or PANC-1 [8]. In theformer cells, endogenous Eps8 displayed a vesicular-lysosomaldistribution. To assess whether the vesicular distribution of Eps8could be detected in other cell types, we examined HeLa cellsectopically expressing Eps8. In these latter cells, which expressrelative low levels of the protein, endogenous Eps8 could not bedetected in lysosomes. Conversely, ectopically expressed Eps8-GFPextensively localized to lysosomes (Figs. 3A–C). In AsPC-1 orCapan-1 cells, Eps8 expression also led to an increase in lysosomesize (Figs. 3D–J). This could be quantified analyzing lysosomal sizein AsPC-1 cells overexpressing either full-length Eps8-GFP(Figs. 3G, H) or the empty pEGFP C1 vector (Figs. 3I, J) as control(Fig. 3K). The enlargement of lysosomes after Eps8-overexpressionwas statistically significant.

To identify the protein region that mediates lysosomal bindingof Eps8, AsPC-1 cells were transfected with various fragments of

at liver lysosomes treated or not with protease inhibitors (+PI)in an isotonic media. Lysosomes collected at the end of theunoblot for Eps8. Where indicated, well-characterized CMAteins (ovalbumin) were added simultaneously to the reaction.to lysosomes was calculated by densitometric quantification ofS8 added to the reaction and are mean + S.E. of 8 independentto lysosomes treated with protease inhibitors (association)nding) for each experiment. Note that values of binding, uptakece the final mean value of uptake does not exactly match theg concentrations of different proteins on the binding of EPS8 tolots as the one shown in the insert. Values are expressed as thet reactions. (D) Percentage of inhibition on the binding of Eps8eins, calculated from the experiments described in C.


Eps8 fused to GFP, and their colocalization with the lysosomemarker LAMP-1 was examined (Fig. 4). This analysis revealed thatthe first 1–535 amino acids were sufficient for lysosomalrecruitment. This fragment lacks both, the SH3 and the “effectorregion” that are responsible for F-actin and Abi1 binding,respectively. We concluded that the “effector region” and theSH3 domain are not required for Eps8-trafficking to lysosomes.The use of additional fragments of Eps8 permitted to restrict theminimal region required for lysosomal targeting to amino acids184 and 535 (Fig. 4M). Notably, this latter region of Eps8, inaddition to include the putative EGFR binding region [13], alsocontains two KFERQ-like pentapeptide motifs, which are com-monly required for chaparone-mediated autophagy (CMA) [14]:QVDVR (amino acids 226–230) and QEIKR (amino acids 470–474).These motifs adhere to the properties defined for CMA targetingmotifs: a pentapeptide flanked in either side by a Q residue andcontaining a positive residue (R or H), a negative (D, E), ahydrophobic residue (F, L, I or V) and being the additional residuepositive or hydrophobic but never negative. These residues canorganize in any order inside the sequence as far as the Q is in one ofthe side. According to this criteria, the Eps8 residues are annotatedas QVDVR (Q[]-[]+) and QEIKR (Q-[]++). These motifs arecharacteristic for cytosolic proteins that undergo lysosomaldegradation through the CMA pathway, and are recognized bythe heat shock cognate protein of 73 kDa (Hsc70) [14]. Theinteraction with Hsc70 targets the substrate to the lysosomalmembrane, where it interacts with one LAMP-2 isoform (LAMP-2A) before being translocated across the lysosomalmembrane intothe lumen [14]. Thus, we predicted that if Eps8were a substrate forCMA, it should interact with Hsc70 and LAMP-2. Consistently,Hsc70 and LAMP-2 co-immunoprecipitated with Eps8 in AsPC-1cells (Fig. 5). Additionally, LAMP-2 colocalized with Eps8 atlysosomes in AsPC-1 cells.

Next, we assessedwhether the association and relocalization ofEps8 to lysosomes could be reconstituted in a cell-free system. Tothis end, we examined lysomal binding and uptake of purifiedEps8 into isolated rat liver lysosomes (Fig. 6A). In this assay, thepotential substrate protein is incubated with lysosomes previous-

Fig. 7 – Fluorescence recovery after photobleaching (FRAP) analysicells, and FRAP analysis was performed. In each experiment (n=10)and the fluorescence recovery monitored over 2 min. (A) Representand after bleaching (time is indicated in the upper left corner). (B) Q2 min (mean±S.E.) suggesting an immobile fraction of about 20%

ly treated or not with protease inhibitors. In the absence ofprotease inhibitors, all the protein that crosses the lysosomalmembrane is rapidly degraded, and consequently the proteinrecovered when lysosomes collected by centrifugation correspondto that bound to the lysosomal membrane. In contrast, the proteinassociate to lysosomes previously treated with protease inhibitorsrepresents both that bound to the lysosomal membrane and theone that reached the lysosomal lumen. Uptake can be calculatedin these assays by discounting the protein bound to themembrane from that associated to lysosomes treated withprotease inhibitors. Using this assay, the amount of lysosomalbinding and uptake of Eps8 was 11.9±5.1% and 5.3±6.5% of thetotal amount of protein added, respectively, and is comparable tothose of other known CMA substrates (Figs. 6A, B). Furthermore,the lysosomal binding of Eps8 was specific since it could be readilycompeted by the well-characterized CMA substrates glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) and ribunuclease A(RNase A) in a concentration-dependent manner, but not byovalbumin (a protein that does not contain a CMA-targetingmotif) (Fig. 6C, D).

The kinetics of Eps8 at the lysosomal membrane was furthercharacterized by fluorescence recovery after photobleaching(FRAP) analysis. Fluorescence recovery asymptotically approached80% after 120 s (mobile fraction), demonstrating that the bulk ofEps8 is recruited to the lysosome membrane in a dynamic fashion(Fig. 7). The diffusion rate τD was 27 s.

Discussion

Here, we describe a novel and unexpected association ofendogenous Eps8 with the lysosomal compartment in humancancer cell lines. Eps8 has been associated with dynamic actin sitesand the early endocytic compartment [1,9–11,20,26,27]. Theseinteractions are mediated by either the SH3-domain (Abi1 andRN-tre binding), or by the C-terminal “effector region” (Sos1 andF-actin binding), respectively. The protein region that wasresponsible for the lysosomal localization of Eps8 (amino acids

s of Eps8 at lysosomes. Full-length Eps8 was expressed in AsPC-1, a small area including an Eps8-positive lysosomewas bleached,ative images before (left image, image width represents 50 μm)uantification of FRAP data indicated a 76.3±2.7% recovery after, and a motile fraction of about 80% at lysosomes.


184–535, target region) lacked these domains, suggesting thatEps8 recruitment does not require a functional SH3 domain and isindependent of actin dynamics-based processes. Indeed, inhibitionof F-actin polymerization with cytochalasin D did not affect Eps8recruitment to lysosomes. Similarly, the interaction of Eps8 withEGFR, a cargo receptor that traffics to the lysosome [28], does notappear to be required for Eps8 recruitment to the limitingendomembranes of these organelles since only a small fractionof Eps8-positive lysosomes also contained EGFR after ligandinduced internalization.

Interestingly, we could identify two KFERQ-like motifs withinthe target region. These pentapeptide sequences are recognized bythe heat shock protein of 73 kDa (Hsc70) and other co-chaperones,and initiate chaperone-mediated autophagy (CMA). This proteincomplex binds to monomers of the LAMP-2A isoform in thelysosomal membrane. After unfolding, the substrate protein istranslocated across the lysosomal membrane, and subjected todegradation [14,24,29]. About 30% of cytosolic proteins undergothis specific lysosomal degradation that is detectable in all tissuesalthough with different activity [14]. In the present study, weprovided evidence that Eps8 binds to isolated lysosomes, translo-cates into the lysosomal lumen, and that lysosomal membranebinding can be competed by known CMA substrates, suggestingthat Eps8 itself is a CMA substrate. The present data furthershowed that elevated expression of Eps8 appears sufficient topromote Eps8 recruitment to lysosomes, however we cannot ruleout that additional molecular interactions may contribute to thislocalization. It is of note that human pancreatic cancer cells withhigh endogenous Eps8 expression (and endogenous Eps8 recruit-ment to lysosomes) could be generated from advanced tumorstages, i.e. from pancreatic cancer metastases (Capan-1) or frommalignant ascites (AsPC-1) [8]. This association to the lysosomalcompartment suggests the possibility that Eps8 may undergoaccelerated targeting to lysosomes using the CMA machinery inmetastatic cancer cells. One alternative possibility is that, althoughwe showed that the “effector region” of Eps8 is not required forlysosomal binding, Eps8 under certain circumstances may linkactin with lysosomal membranes. Under this hypothetical scenar-io, CMA can be seen as a way to regulate lysosomal membrane andactin dynamics through changes in levels of resident Eps8. This, inturn, may affect some key lysosomal functions, such as actin-basedfacilitation of fusion events of the late endosomal/lysosomalcompartment, as previously reported [30]. This latter notionis consistent with the observation that naturally occurring orectopically generated elevated levels of Eps8 correlate with theenlargement of lysosome vesicles, further suggesting that Eps8may not only be routed to these structures for its degradation, butalso promote the lysosomal targeting of yet to be identified cargos.In keeping with this latter possibility, Goebeler et al. recentlyreported that the actin-interacting annexin A8 governs lateendosomal organization and function in HeLa cells. Annexin A8expression resulted in enlarged late endosomes, while itsdepletion caused delayed degradation of endosomal cargos, suchas the EGF receptor [31].

In conclusion, the finding that Eps8 localizes and binds tolysosomes in human cancer cells, and is degraded through CMAprovides novelmolecular interactions to study. Although it remainsunclear how Eps8 and the regulation of CMA are connected incancer cells, the present data indicate that Eps8 may play a moreversatile role in cancer progression than expected. Future experi-

ments have to analyze if Eps8 can regulate CMA, control fusion oflate endosomes, or even impair competitive protein degradation,e.g. EGF receptor degradation in cancer cells.

Conflict of interest

The authors state no conflict of interest in this study.

Acknowledgments

We would like to thank Sonja Bauer for her excellent technicalassistance. This work was partly supported by the NIH GrantAG021904 (to AMC).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.yexcr.2010.02.020.

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Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells