Signal Integration and Coincidence Detection in theMitogen-activated Protein Kinase/ExtracellularSignal-regulated Kinase (ERK) CascadeCONCOMITANT ACTIVATION OF RECEPTOR TYROSINE KINASES AND OF LRP-1 LEADS TOSUSTAINED ERK PHOSPHORYLATION VIA DOWN-REGULATION OF DUAL SPECIFICITYPHOSPHATASES (DUSP1 AND -6)*

Received for publication, January 15, 2011, and in revised form, May 23, 2011 Published, JBC Papers in Press, May 24, 2011, DOI 10.1074/jbc.M111.221903

Nishamol Geetha‡, Judit Mihaly‡, Alexander Stockenhuber‡, Francesco Blasi§, Pavel Uhrin‡, Bernd R. Binder‡†,Michael Freissmuth¶1, and Johannes M. Breuss‡

From the ‡Department of Vascular Biology and Thrombosis Research and ¶Insitute of Pharmacology, Center of Physiology andPharmacology, Medical University of Vienna, Vienna 1090, Austria and the §Department of Molecular Biology and functionalGenomics, Dipartimento di Biotechnolgia (DiBiT), San Raffale Scientific Research Institute, 20132 Milan, Italy

Diverse stimuli can feed into the MAPK/ERK cascade; thisincludes receptor tyrosine kinases, G protein-coupled recep-tors, integrins, and scavenger receptors (LDL receptor-re-lated protein (LRP)). Here, we investigated the consequenceof concomitant occupancy of the receptor tyrosine kinases(by EGF, basic FGF, VEGF, etc.) and of LRP family members(by LDL or lactoferrin). The simultaneous stimulation of areceptor tyrosine kinase by its cognate ligand and of LRP-1(by lactoferrin or LDL) resulted in sustained activation ofERK, which was redirected to the cytoplasm. Accordingly,elevated levels of active cytosolic ERK were translated intoaccelerated adhesion to vitronectin. The sustained ERKresponse was seen in several cell types, but it was absent incells deficient in LRP-1 (but not in cells lacking the LDLreceptor). This response was also contingent on the presenceof urokinase (uPA) and its receptor (uPAR), because it wasabsent in uPA�/� and uPAR�/� fibroblasts. Combined stim-ulation of the EGF receptor and of LRP-1 delayed nuclearaccumulation of phosphorylated ERK. This shift in favor ofcytosolic accumulation of phospho-ERKwas accounted for byenhanced proteasomal degradation of dual specificity phos-phatases DUSP1 and DUSP6, which precluded dephosphory-lation of cytosolic ERK. These observations demonstrate thatthe ERK cascade can act as a coincidence detector to decodethe simultaneous engagement of a receptor tyrosine kinaseand of LRP-1 and as a signal integrator that encodes thisinformation in a spatially and temporally distinct biologicalsignal. In addition, the findings provide an explanation ofwhy chronic elevation of LRP-1 ligands (e.g. PAI-1) can pre-dispose to cancer.

The LDL receptor-related protein LRP-12 is a scavengerreceptor that mediates the endocytotic uptake of a large collec-tion of ligands (�30) and thus their clearance from the extra-cellular space (1). The intracellular carboxyl terminus of LRP-1comprises 100 amino acids and is subject to regulatory phos-phorylation by both serine/threonine and tyrosine kinases.These phosphorylation reactions promote the interaction withadapter proteins and thus nucleate the formation of signalingplatforms. In fact, LRP-1 enhances the capacity of the endocy-tosed PDGF� receptor to activate the MAPK/ERK cascade (2,3). These observations raise several interesting questions,namely concerning (i) themechanism underlying the enhancedactivation of the ERK cascade; (ii) whether this is unique for thePDGF receptor; and (iii) the extent to which this enhancedresponse is of pathophysiological relevance in vivo and thustranslates into modification in the course of human diseases.Enhanced signaling through the ERK cascade is likely to

result from a change in the dynamic (feed forward and feed-back) loops that regulate its activity. There aremultiple layers offeedback loops that regulate signaling through the MAPK cas-cade. If the linear cascade RAS 3 RAF 3 MEK1 3 ERK isexamined, there are at least three points atwhich feedback inhi-bition can be exerted: (i) desensitization of the entry point (i.e.RAS activation by phosphorylation of the exchange factor SOS)(4), (ii) a decline in the activation of MEK1 by the RAF kinasedue to phosphorylation by the downstream target ERK (5), and(iii) dephosphorylation of ERK by induction of dual specificityphosphatases (DUSP/MPKP isoforms, MAPK phosphatases)(6). A recent analysis concluded that the most important com-ponent in shaping the response of murine fibroblasts to PDGFwas negative feedback at the RAF/MEK1 level (7). Here, weexamined the action of LRP-1 ligands on signaling via severalreceptor tyrosine kinases (i.e. the receptors for EGF, PDGF,FGF, and VEGF).We observed that signal integration occurred

* This work was supported by the Cell Communication in Health and disease(CCHD) doctoral program, which is funded by the Fonds zur Forderung derWissenschaftlichen Forschung (Austrian Science Fund) and the MedicalUniversity of Vienna.Author’s Choice—Final version full access.

† Deceased August 28, 2010.1 To whom correspondence should be addressed: Center of Physiology and

Pharmacology, Wahringer Str. 13a, 1090 Vienna, Austria. Tel.: 43-1-4277-64171; Fax: 43-1-4277-9641; E-mail: michael.freissmuth@meduniwien.ac.at.

2 The abbreviations used are: LRP, LDL receptor-related protein; DUSP, dualspecificity phosphatase; IGF, insulin-like growth factor; MEF, murineembryonic fibroblast; PMA, phorbol 12-myristate 13-acetate; RAP, recep-tor-associated protein (a ligand of LRP-1); uPA, urokinase plasminogenactivator; uPAR, urokinase plasminogen activator receptor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 29, pp. 25663–25674, July 22, 2011Author’s Choice © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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This article has been withdrawn by Nishamol Geetha, Judit Mihaly, Alexander Stockenhuber, Francesco Blasi, Pavel Uhrin, Michael Freissmuth, and Johannes M. Breuss. The withdrawing authors regret that Bernd R. Binder passed away. In Fig. 3A, The EGF/LF lanes did not accurately represent the experimental results. The tERK lanes were duplicated in Fig. 3C. The LF lanes were derived from a different immunoblot in Fig. 3D. The pERK and tERK immunoblots from PDGF/LF treatment in Fig. 4B were reused in Fig. 4C for EGF/LF treatment. The DUSP1 and actin immunoblots from untreated cells in Fig. 7A were reused in Fig. 8C. The DUSP6 immunoblot from untreated cells in Fig. 7B was reused in Fig. 8D. In addition, the actin immunoblot from untreated cells in Fig. 7B was reused in Figs. 7C and 8D for the untreated samples. The corrected blots are available upon request.

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at the third level (i.e. the regulation ofDUSPdegradation); stim-ulation of various receptor tyrosine kinases caused an earlypeak in ERK phosphorylation. This was converted into a sus-tained rise if cells received concomitant input from LRP-1 andthe urokinase/plasminogen activator receptor uPAR. Engage-ment of both receptors stimulated proteasomal degradation ofDUSP1 and DUSP6, which changed not only the temporal butalso the spatial pattern of ERK activation.

MATERIALS AND METHODS

Proteins and Antibodies—Fibronectin, vitronectin, andactive rhPAI-1 were from Technoclone (Vienna, Austria), andthe proteasome inhibitorMG132was fromSigma-Aldrich. Theantibodies recognizing holo-ERK1/2 and phospho-ERK, phos-pho-SRC (Tyr416), holo-SRC (L4A1,mousemonoclonal), phos-pho-AKT (Ser473), phospho-AKT (Thr308), and pan-AKT werefrom Cell Signaling Technologies (Beverly, MA). Antibodiesagainst DUSPs were from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA) or Abcam (Cambridge, UK). Antibodiesdirected against the integrin subunits �1, �3, �5, and �v werefrom Chemicon (Temecula, CA). The R3 and R2 antibodies foruPAR domains were provided by Dr. Gunilla Høyer-Hansen(Finsen Laboratory, Copenhagen). EGF was purchased fromPromoKine/PromoCell (Heidelberg, Germany), and other tyro-sine kinases, like FGF, PDGF, VEGF, and IGF, were from R&DSystems (Minneapolis, MN). Lactoferrin, phorbol 12-myristate13-acetate (PMA), and crystal violet were purchased from Sig-ma-Aldrich. The chemiluminescence system used was SuperSignal West Femto Substrate from Thermo Scientific (Rock-ford, IL).Cell Culture—For cell culture, RPMI 1640 (Hyclone, Logan,

UT), Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich), and fetal calf serum (HyClone) were used. Primarycultures of mouse skin fibroblasts were prepared from the der-mis of urokinase-deficient and of urokinase receptor-deficientmice backcrossed for 8 generations into C57/Bl6 backgroundand from control C57/Bl6 mice by an explant culture protocol(8). The HT1080 fibrosarcoma cell line (ATCC,Manassas, VA)andmurine cells were grown in RPMI 1640 supplemented with10% fetal calf serum (FCS). Murine embryonic fibroblasts(MEFs) were kindly provided by Dr.Wolfgang Schneider (Uni-versity of Vienna).MAPK Stimulation—Cells (1 � 106 cells/ml) were seeded in

12-well plates in medium containing 10% FCS and allowed toadhere by overnight incubation. After serum starvation for 4 h,they were incubated in the absence and presence of EGF (25ng/ml, �4 nM) or additional growth factors and/or lactoferrin(70 �g/ml, 0.9 �M) for different time periods from 0 to 60 min.The phorbol ester PMA (10 �M) was used as the positive con-trol. At the indicated time points, the medium was aspirated,and the reaction was stopped by immersion in liquid nitrogen.Cells were lysed in sample buffer for SDS-PAGE containing 4%SDS and�-mercaptoethanol. Proteins (equivalent to 15% of thelysate) were separated by denaturing electrophoresis (10%monomer concentration in the running gel) and transferredonto nitrocellulose membranes. Immunoreactive bands wererevealed with secondary antibodies linked to horseradish per-oxidase using chemiluminescence. Signals were recorded on an

AlphaImager HP (Alpha Innotech), which allows for linear sig-nal detection over a large dynamic range. ERK1/2 activationwas detected by sequential probing of membranes with anti-bodies to holo- and phospho-p44/42MAPK. Ratios of densito-metric values for each sample were recorded and comparedwith the control. DUSP protein levels were determined usingthe respective specific antibodies. Values were normalized toactin by reprobing the membranes with antibody against �-ac-tin (Sigma-Aldrich).Cell Adhesion Assay—96-well plates were coated with vit-

ronectin (5 �g/ml) in Hanks’ buffered saline for 2 h at 37 °C.Nonspecific sites were blocked by the addition of 0.1 ml of 3%BSA in Hanks’ buffered saline for 30 min at 37 °C, followed bytwo washes in Hanks’ buffered saline solution. Thereafter,HT1080 cells (2 � 105/well) were seeded in RPMI containing0.1% BSA in the presence and absence of stimuli. Cells wereincubated for 10, 30, and 60 min at 37 °C and 5% CO2. There-after, the non-adherent cells were aspirated, and the wells wererinsed twice with RPMI containing 0.1% BSA. Attached cellswere quantified by the addition of crystal violet (0.5% in 25%methanol, 50 �l/well). After 10 min, excess crystal violet wasremoved by washing three times with distilled water. Plateswere allowed to dry overnight, and crystal violet pigment wasextracted in 0.1ml of 0.1 M sodium citrate in 50% ethanol for 20min. Cell adhesion was quantified by determining the absorb-ance at 540 nm in a Victor ELISA plate reader (Wallac, Turku,Finland) with the reference wavelength set at 405 nm.Recording of Random Migration by Time Lapse Microscopy—

Glass coverslips were coated with vitronectin (5 �g/ml) inHanks’ buffered saline for 2 h at 37 °C. HT1080 cells (1 � 105)were seeded on vitronectin-coated glass coverslips and werestimulatedwith either EGF (25 ng/ml) or lactoferrin (70�g/ml)or a combination thereof. The coverslips were mounted ontocell culture chambers on a heated stage insert on an OlympusAX-70microscope for time lapse recording of cellmigration for12–15hwith 10 images recorded/h (F-Viewdigital camera, SoftImaging System). Recorded time lapse sequenceswere analyzedby assessing the length of the track of every individual cell.Transfections of Cells with siRNA—The ON-TARGETplus

siRNA targeting DUSP6 and siGENOME non-targetingsiRNAs were purchased from Dharmacon (Lafayette, CO).For siRNA transfection, HT1080 cells were plated in 12-wellplates in complete medium. After 12 h, cells were transfectedwith siRNA at a final concentration of 50 pM using the Xtremegene transfection reagent (RocheApplied Science) according tothe manufacturer’s instructions. 48 h after siRNA transfection,the medium was removed and replaced by serum-free mediumcontaining 0.1% BSA. The cells were serum-starved for 4 h andsubsequently stimulated with EGF, lactoferrin, or a combina-tion thereof. The level of phospho-ERKwas determined as out-lined above.Confocal Microscopy—After the pertinent incubation, cells

were fixed in 4% paraformaldehyde and processed as describedpreviously (9). To label ERK, mouse monoclonal antibody tophospho-ERK and rabbit polyclonal antibodies to total ERKwere used (Cell Signaling Technology, Beverly, MA) Secondaryantibodies used were labeled with Alexa Fluor-488 (anti-rabbitIgG) or Alexa Fluor-568 (anti-mouse IgG). Slides were

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mounted in Permafluor (Lab Vision Corp., Fremont, CA).Images were captured at 40- and 100-fold magnification on aZeiss LSM 510 META confocal microscope in multitrackmode, using pinhole sizes between 0.7 and 1.5 �m and theappropriate standard laser-filter combinations and were digi-tized using the built-in software.Statistics—Values are reported as means � S.E. of three

experiments done in duplicate (ERK phosphorylation) or intriplicate to quintuplicate (adhesion assay). Statistically signif-icant differences were verified by paired or unpaired t test andby analysis of variance followed by a Bonferroni post hoc test orDunnett’s test for multiple comparison, as appropriate.

RESULTS

Combined Stimulation of Cells with EGF and LactoferrinInduces Sustained ERK2 Activation—The shape and durationof receptor-induced ERK stimulation are highly variable. Wesurmised that the upstream cascade can integrate additionalsignals that are translated into distinct time-dependent activityprofiles. This conjecture was tested by incubating HT1080 cells

(a human fibrosarcoma endowedwith several receptor tyrosinekinases) in the presence of the LRP ligand lactoferrin, which perse does not stimulate ERK phosphorylation (Fig. 1A, top, laneson the right). EGF-induced stimulation of ERK phosphoryla-tion peaked at 5–10 min and then declined to a low residualactivity that could be still detected after 2 h (Fig. 1A, top, laneson the left). However, when EGF was combined with lactofer-rin, a sustained activation was observed over �60 min (Fig. 1A,bottom, lanes on the left). In contrast to incubations with EGFalone, the combination of EGF and lactoferrin resulted in ERKphosphorylation that was still detectable after 4 and 16 h. Infact, the time course was comparable with that seen with per-sistent stimulation of protein kinase C isoforms by the phorbolester PMA (Fig. 1A, bottom, lanes on the right).

This response was not unique to the fibrosarcoma cell linebecause it was recapitulated in primary cultures of human skinfibroblasts (Fig. 1B). This suggests that the ERK cascade actedas a coincidence detector and signal integrator that translatedthe simultaneous occupancy of the EGF receptor and an LDLreceptor-related protein into a sustained response. We verified

FIGURE 1. Combined stimulation of HT1080 cells with EGF and with ligands of LRP induces sustained activation of ERK. Serum-starved HT1080 fibro-sarcoma cells (A and C) and normal human skin fibroblasts (B) were stimulated with either EGF (25 ng/ml) or lactoferrin (LF; 70 �g/ml) alone or in combinationfor the indicated times. PMA (10 �M) was used as a positive control. Thereafter, the cells were lysed, and the samples were processed as outlined under“Materials and Methods” to detect the active dually phosphorylated ERK (p ERK) and total ERK (t ERK). The blots shown are representative of three independentexperiments. The graphs summarize three experiments (error bars, S.E.) in which the chemiluminescence was directly quantified (AlphaImagerTM) and plottedas normalized values of phospho-ERK (i.e. ratio of phospho-ERK to total ERK as a percentage of control).

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that this coincidence detection was not limited to the pair EGFand lactoferrin; the combination of lactoferrin and basic fibro-blast growth factor (FGF2) also resulted in sustained ERKphos-phorylation (Fig. 1C, cf. left-hand bFGF-labeled laneswith laneslabeled bFGF/LF). Similarly, incubation in the presence of LDL(100 �g/ml) augmented the levels of phospho-ERK regardlessof whether cells were treated concomitantly with EGF or FGF;LDL per se did not change the levels of ERK phosphorylation(Fig. 1C). Comparable findings were also obtained with PDGF(10 ng/ml), IGF2 (100 ng/ml), and VEGF 165 (50 ng/ml) (datanot shown).EGF activatesmany additional pathways other than ERK.We

determined whether the amplifying effect of lactoferrin waslimited to EGF-dependent activation of the MAPK pathway byassessing other pathways (i.e. PI3K-dependent activation ofAKT and stimulation of the non-receptor tyrosine kinase SRC).EGF induced phosphorylation of AKT both on Ser473 (Fig. 2A)and Thr308 (Fig. 2B). The combination of EGF and lactoferrinneither caused any additional stimulation nor affected the timecourse of AKT phosphorylation (Fig. 2, A and B). Similarly, thephosphorylation of SRC elicited in response to the combinationof EGF and lactoferrin was comparable with that caused by the

addition of EGF alone (data not shown). Although we did notexamine additional signaling pathways (e.g. activation of phos-pholipase C�), these observations suggest that the signal ampli-fication caused by lactoferrin does not indiscriminately involveall possible pathways but appears to be confined to MAPKactivation.Sustained ERK Activation Depends on LRP-1—The associa-

tion of ligands with LRP can be blocked by receptor-associatedprotein (RAP) (10). If the action of lactoferrin and of LDL weremediated via interaction with an LRP family member, it oughtto be blunted by preincubation of the cells in the presence ofRAP. This was the case (Fig. 3A). There are several familymem-bers of the LDL receptor-related protein family. Earlier obser-vations indicated complex formation between LRP-1 and thePDGF receptor (2, 11). Hence, we substantiated the conjecturethat LRP-1 might be required also for supporting the lactofer-rin-promoted augmentation of EGF-induced ERK phosphory-lation by using MEFs isolated from mice deficient in LRP-1and/or LDL receptor. Unlike the wild type MEFs (Fig. 3B),MEFs lacking LRP-1 failed to respond to the combination ofEGF and lactoferrin by sustained ERK activation (Fig. 3C). Infact, in the absence of LRP-1, lactoferrin blunted the responseto EGF (triangles in Fig. 3C); the underlying mechanism is cur-rently not understood. In contrast, the absence of LDL receptordid not impair the sustained response to EGF and lactoferrin(Fig. 3D).SustainedERKActivationDepends on theUrokinase Receptor

Domain 1—Both the EGF receptor and LRP-1 are engaged infunctionally important and context-dependent interactionswith the urokinase receptor (12). We isolated murine skinfibroblasts from animals deficient in either uPA or uPAR toassess the possible contribution of uPAR to the sustained stim-ulation in response to EGF and lactoferrin stimulation. Conflu-ent monolayers of these cells were stimulated with EGF, lacto-ferrin, or a combination thereof for up to 60 min. In wild typemurine skin fibroblasts, ERK was activated in a sustained fash-ion by EGF and lactoferrin in amanner comparable with that inhuman skin fibroblasts or in HT1080 fibrosarcoma cells (Fig.4A). In contrast, sustained ERKphosphorylationwas blunted inuPAR- and uPA-deficient murine skin fibroblasts (Fig. 4A).This indicates a crucial role of both urokinase and its receptorin promoting the action of LRP-1 on the EGF receptor.We defined the required region of uPAR by employing two

monoclonal antibodies, R3 and R2, directed against uPARdomains 1 and 3, respectively. Cells were pretreated with theseantibodies and then challenged with a receptor tyrosine kinasereceptor agonist and lactoferrin. Fig. 3B shows the results forstimulation of cells with PDGF; blockage of the uPAR domain 1with the R3 antibody precluded sustained ERK phosphoryla-tion in response to the combination of PGDF and lactoferrin(Fig. 4B). This is consistent with the fact that the antibodyblocks the association of uPA and uPAR (13). A similar obser-vationwasmadewithHT1080 cells that had been challenged bya combination of EGF and lactoferrin in the presence of the R3antibody (Fig. 4C). In contrast, monoclonal antibody R2, whichdoes not interfere with uPAR-uPA binding, did not interferewith sustained ERK2 activation (Fig. 4D). Similar findings wereobtained with FGF, VEGF, and IGF (data not shown).

FIGURE 2. Combined stimulation of HT1080 cells with EGF and with lacto-ferrin does not induce a sustained activation of AKT. Serum-starvedHT1080 fibrosarcoma cells were stimulated with either EGF (25 ng/ml) orlactoferrin (LF; 70 �g/ml) or in combination for the indicated times. Thereaf-ter, the cells were lysed, and the samples were processed as outlined under“Materials and Methods” to detect the active phosphorylated AKT (A, phos-pho-Ser473 AKT; B, phospho-Thr308 AKT) and total levels of AKT (t Akt). Thegraphs summarize three experiments (error bars, S.E.) in which the chemilu-minescence was directly quantified (AlphaImagerTM) and plotted as normal-ized values of phosphorylated protein (i.e. ratio of phosphorylated to totalprotein as a percentage of control).

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Cytoplasmic Accumulation of Phospho-ERK Translates intoAccelerated Adhesion on Vitronectin—ERK phosphorylationpromotes nuclear translocation of the enzyme (14). This wasalso seen if HT1080 cells were stimulated by EGF (Fig. 5, cf. firstand second row). The sole addition of lactoferrin did not affectthe distribution of ERK (Fig. 5, row 3). Surprisingly, the combi-nation of EGF and lactoferrin resulted in a delayed transloca-tion of active phosphorylated ERK into the nucleus; for the first60 min, there was little appreciable translocation of phospho-ERK into the nucleus (Fig. 5, cf. rows 4–6 with control rows 1and 2), whereas it accumulated in the perinuclear region andmost prominently at submembraneous spots. After prolongedstimulation with EGF and lactoferrin, phosphorylated ERK didaccumulate in the nucleus (Fig. 5, bottom row).

The observations suggested that the stimulation of cells bythe combination of EGF and lactoferrin initially redirected ERKsignaling to cytosolic targets. Cytosolic ERK has many targets,and several of these are involved in actin dynamics (15), such

that ERK plays a prominent role in integrin-dependent adhe-sion (16). Accordingly, we evaluated whether the combinedaddition of EGF and lactoferrin promoted cell adhesion. Thiswas the case; at early time points (10 min), the number of cellsthat adhered to vitronectin-coated dishes was augmented (Fig.6A, white bars). In contrast, neither EGF nor lactoferrin bythemselves affected cell adhesion at this early time point. Thus,increased cytosolic phosphorylated ERK (levels) translated intoa relevant biological response, namely accelerated cell adhesionpromoted by the combination of EGF and lactoferrin. Blockageof the MAPK cascade ought to blunt the response to the com-bination of EGF and lactoferrin if there were a cause-and-effectrelation between cytosolic accumulation of MAPK andenhanced adhesion. This prediction was verified by employingtheMEK1 inhibitor PD98059. Pretreatment of cells with 25 �M

PD98059 for 30 min reduced the number of adherent cells byabout 50% if cells were stimulated by EGF and lactoferrin; incontrast, PD98059 had no appreciable effect on cell adhesion

FIGURE 3. Blockage by RAP of sustained ERK activation induced by EGF and lactoferrin. A, HT1080 fibrosarcoma cells were pretreated with RAP (10 �g/ml)and stimulated with EGF (25 ng/ml) or lactoferrin (LF; 70 �g/ml) as outlined in the legend to Fig. 1 and under “Materials and Methods.” B–D, wild type murineembryonic fibroblasts (B), murine embryonic fibroblasts deficient in LRP-1 (C), and murine embryonic fibroblasts deficient in LDL receptor (LDLR) (D) werestimulated with EGF and lactoferrin for the indicated times. The phosphorylation of ERK was assessed by quantitative immunoblots and normalized to totalERK. The blots shown are representative of three independent experiments. Graphs show the quantification of phospho-ERK levels (normalized to total ERKand given as a percentage of control; error bars, S.E.; n � 3).

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under any of the other conditions tested (Fig. 6A, black bars).EGF (17) and uPAR (18) stimulate cell migration via stimula-tion of the MAPK cascade. Accordingly, we allowed HT1080cells to adhere on vitronectin and subsequently examined overthe next 15 h by time lapse microscopy whether the combina-tion of EGF and lactoferrin had a stronger chemokinetic effectthan the sole addition of EGF. In fact, trajectories of cells weresignificantly longer in the presence of EGF and lactoferrin thanwhen each compound was added separately (Fig. 6B). We alsoexamined directed migration induced by wounding a cellmonolayer. The combination of EGF and lactoferrin alsoresulted in accelerated migration (data not shown).Proteasomal Degradation of DUSPs Accounts for Sustained

ERK Activation in Response to EGF and Lactoferrin—Themag-nitude and duration of MAPK signaling is dependent on thebalance between the activities of upstream activators and deac-tivation by phosphatases. Because phosphorylation of boththreonine and tyrosine residues is required for activity, dephos-phorylation of either is sufficient for inactivation. This is

achieved by dual specificity (threonine/tyrosine) protein phos-phatases (DUSPs) (19). DUSPs differ in their affinity for indi-vidual MAPKs. ERK1/2 can be dephosphorylated by the induc-ible DUSP1, DUSP4, and DUSP5, which are found primarily inthe nucleus, and by the cytosolic isoforms DUSP6, DUSP7, andDUSP9 (20). Sustained activation of ERK2 via the combinedinput from a tyrosine kinase receptor and LRP-1 may be due toa decline in DUSP levels.We tested this hypothesis by assessingthe effect of combined stimulation with EGF and lactoferrin onthe amount ofDUSP in lysates ofHT1080 cells. IncubationwithEGF and lactoferrin led to a pronounced and sustained down-regulation of the nuclear DUSP1 (Fig. 7A) and of the cytosolicDUSP6 (Fig. 7B). The capacity of LRP-1 ligands to synergizewith tyrosine kinase receptor ligands was not limited to lacto-ferrin but was also recapitulated in the presence of LDL (cf. Fig.1C). Thus, when combined with EGF, LDL was predicted tocause a similar decline in DUSP levels if the decline in DUSPlevels and the change in ERK phosphorylation were causallyrelated. This was the case (Fig. 7, A and B). In contrast, stimu-

FIGURE 4. Sustained ERK activation requires the urokinase receptor domain 1. A, serum-starved wild type, uPA�/�, or uPAR�/� murine skin fibroblastswere stimulated with EGF (25 ng/ml) or lactoferrin (LF; 70 �g/ml) for the indicated times. ERK phosphorylation was assessed by immunoblots and quantified(normalized to total ERK (t-ERK)). B–D, HT1080 cells were pretreated with uPAR domain 1-specific R3 antibody (2 �g/ml; C) or uPAR domain 3-specific R2antibody (2 �g/ml; C) for 30 min and subsequently stimulated with PDGF (10 ng/ml) and lactoferrin (70 �g/ml) or EGF (25 ng/ml) and lactoferrin (70 �g/ml). Theblots shown are representative of three independent experiments. The levels of phospho-ERK (p-ERK) were quantified as in Fig. 1 and plotted. The values aremeans � S.E. (error bars).

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lation with EGF alone caused only a transient reduction in thelevels of DUSP1 and DUSP6, whereas lactoferrin alone did notaffect the expression levels of DUSP1 and DUSP6 (open dia-monds in Fig. 7,A andB).We also verified that the combinationof EGF and lactoferrin did not result in an indiscriminate loss of

all DUSP isoforms. There was, for instance, no appreciablechange in the levels of the nuclear isoform DUSP5 (Fig. 7C),DUSP7, and DUSP9 (data not shown).The down-regulation of DUSP1 andDUSP6 occurred swiftly

(i.e. it was detectable within 10 min). An obvious explanation

FIGURE 5. Localization of phosphorylated ERK by immunocytochemistry in cells stimulated by EGF and lactoferrin. Serum-starved HT1080 cells werestimulated with EGF (25 ng/ml), lactoferrin (70 �g/ml), and a combination thereof for the indicated time points. Thereafter, the cells were fixed with paraform-aldehyde (4%), permeabilized with 0.2% Tween 20, and stained for total ERK (green, first column) and phospho-ERK (red, second column). Confocal images werecaptured on a Zeiss LSM 510 microscope using the settings outlined under “Materials and Methods.” The third column shows merged confocal images of thefirst and second columns. Arrowheads mark phosphorylated ERK in the cytoplasm and in basal adhesion contacts. Images were captured, micrographs wereprinted, and cells were scored for the presence of phospho-ERK in the cytosol and in the nucleus. 90 cells from three independent experiments were scored foreach condition. Error bars, S.E.

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for this rapid decline was proteolytic degradation via the pro-teasome. In fact, when cells had been pretreated with the pro-teasome inhibitor MG132 (10 �M), DUSP levels were notdecreased upon combined stimulation with EGF and lactofer-rin or LDL (Fig. 7,A andB). Similar findings were obtainedwithcells that had been incubated with FGF and lactoferrin or LDL(Fig. 8, A and B). We also observed that the MEK1 inhibitorPD98059 increased DUSP6 protein levels and suppressed theDUSP6 degradation triggered by the combination of EGF andlactoferrin (data not shown). The LRP-1-induced switch to sus-tained ERK phosphorylation was contingent on concomitantinput via the uPA/uPAR system (Fig. 4). This provided an addi-tional test for the relation between DUSP down-regulation andsustained ERK signaling; blockage of uPAR by the antibody R3directed against domain 1 of uPAR precluded the down-regu-lation of DUSP1 and DUSP6 (Fig. 8, C and D).

FIGURE 6. A, enhanced adhesion on vitronectin of EGF- and lactoferrin-stim-ulated HT1080 cells. A, serum-starved HT1080 cells were incubated in theabsence (vehicle) and presence of PD98059 (25 �M) for 30 min and subse-quently stimulated with EGF (25 ng/ml) and/or lactoferrin (70 �g/ml) andplated on vitronectin-coated plastic dishes. After 10 min, the non-adherentcells were removed by aspiration, and the adherent cells were stained withcrystal violet. Cell adhesion was quantified by determining the absorbance at540 nm in an ELISA plate reader. Values represent mean absorbance � S.E.(error bars) (n � 4). Statistical significance was assessed by analysis of variancefollowed by Dunnett’s multiple comparison test, which documented a statis-tically significant effect of EGF � lactoferrin versus control and a statisticallysignificant inhibition of this stimulation by PD98059 (p � 0.01). B, long termstimulation with EGF and lactoferrin enhances migration of HT1080 cells onvitronectin. HT1080 cells were seeded on vitronectin-coated glass coverslipsand were stimulated with either EGF (25 ng/ml) or lactoferrin (LF; 70 �g/ml)alone or in combination. The coverslips were mounted onto cell culturechambers on a heated stage insert on an Olympus AX-70 microscope forrecordings by time lapse microscopy of cell migration for over 15 h with 10images recorded per hour (F-View digital camera, Soft Imaging System).Recorded time lapse sequences were analyzed by assessing the length of thetrack of individual cells. Data are based on the analysis of the trajectories of 90cells recorded in three independent experiments. The effect of lactoferrin �EGF was statistically significant in relation to all other values (p � 0.05, anal-ysis of variance followed by Dunnett’s test for multiple comparisons).

FIGURE 7. Changes in the levels of DUSP1 (A), DUSP6 (B), and DUSP5 (C) incells stimulated by the combined addition of EGF and lactoferrin. Serum-starved HT1080 cells were pretreated with vehicle or MG132 (10 �M) for 30 minand subsequently stimulated with EGF (25 ng/ml) and lactoferrin (70 �g/ml) orLDL (100 �g/ml) for the indicated time periods. Expression of DUSP1 (A), DUSP6(B), and DUSP5 (C) was assessed from immunoblots, quantified densitometrically,and normalized to actin. The blots shown are representative of three indepen-dent experiments; relative levels (initial control � 100%) are shown in the graphs(error bars, S.E.).

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The combination of EGF and lactoferrin increased DUSPdegradation and triggered sustained ERK activation. Thus,down-regulation of DUSPs ought to lead to a sustained EGFresponse. We focused on DUSP6 because this is the cytosolicisoform, the down-regulation of which ought to phenocopy thecombined effect of lactoferrin and EGF. DUSP6 levels werereduced by using an appropriate siRNA (Fig. 9A). Silencing ofDUSP6 led to a sustained activation of ERK with EGF alone;there was not any appreciable difference in the time course ofphospho-ERK accumulation in cells that had been stimulatedby the sole addition of EGF or by the combination of EGF andlactoferrin (Fig. 9B). This was not the case in cells transfectedwith a control siRNA (Fig. 9C) or untransfected cells, whichwere examined in parallel (Fig. 9D).If sustained ERK activation induced by combined stimula-

tion with EGF and lactoferrin were indeed the result of protea-somal degradation of DUSP1 and/or DUSP6, blockage of theproteasome ought to prevent sustained activation. This predic-tion was verified by treating cells with the proteasome inhibitorMG132 prior to stimulation by EGF, lactoferrein, or a combi-nation thereof. Inhibition of the proteasome byMG132 did not

affect the response to EGF (Fig. 10, cf.A and B, left). In contrast,in cells challenged by the combined addition of EGF and lacto-ferrin, pretreatment with MG132 precluded sustained ERKphosphorylation (Fig. 10B, right). Phospho-ERK levels declinedmore rapidly after the initial peak than in untreated cells (Fig.10A, right).

DISCUSSION

The ERK cascade plays a central role in mitogenic signaling.It has attracted interest because input via extracellular signal istranslated into both a spatial and a temporal dimension. Thisconcept was originally developed with the study of PC12 differ-entiation (14) and probed rigorously by rewiring the signalingnetwork (21); the rat pheochromocytoma cell line PC12 is dif-ferentiated by NGF because this agonist causes a very long last-ing stimulation of ERK activity. In contrast, the kinetics of EGF-induced ERK phosphorylation supplies a stimulus for celldivision. In addition, the ERK cascade displays hysteresis andbistability. It is therefore capable of storing information andconverting an analog signal (the graded concentration of mito-gen) to a digital output; regardless of whether differentiation or

FIGURE 8. Degradation of DUSP1 (A and C) and DUSP6 (B and D) induced by the combination of FGF and lactoferrin (A and B) or of EGF and lactoferrin(C and D) in the absence and presence of MG132 (A and B) and of the uPAR-antibody R3 (C and D). Serum-starved HT1080 cells were pretreated withvehicle or MG132 (10 �M) for 30 min (A and B) or of the domain 1-specific uPAR antibody R3 (2 �g/ml) and subsequently stimulated with FGF (20 ng/ml), EGF(25 ng/ml), lactoferrin (70 �g/ml), or LDL (100 �g/ml) or a combination thereof for the indicated intervals. The levels of DUSP1 (A and C) and DUSP6 (B and D)were assessed by immunoblotting and quantified. The blots shown are representative of three independent experiments; relative levels (initial control �100%) are shown in the graphs (error bars, S.E.).

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cell division is examined, if the activity has reached a criticalthreshold, an irreversible decision is made. The cell undergoesreplication or differentiates. It is therefore interesting to under-stand how the kinetics of ERK activation is controlled. Becausethe ERK signaling pathway receives input from several path-ways, it allows for signal integration; irrespective of the sourceof the upstream input, it is converted into an analog signal,namely the enzymatic activity of the dually phosphorylatedERK. Thus, the integral of the input is encoded in the strengthof the output signal. Because ERK is subject to deactivation bydephosphorylation and because it drives irreversible decisions,

it also allows for coincidence detection; thresholds may only bereached if two signals are present at the same time. Thus, inthe originalmodel of PC12 cell differentiation, the ERK cascadealso acted as a coincidence detector; the simultaneous presenceof cAMP converted the EGF-driven output from proliferationto differentiation (14).Previous investigations have focused on the cross-talkwithG

protein-coupled receptors. These studies have provided evi-dence for trans-activation (22), for release of growth factors viathe release of latent membrane-bound growth factors (23), andfor second messenger-driven loops (14, 24). More recently, the

FIGURE 9. Silencing of DUSP6 by siRNA transfection (A) in HT1080 converts EGF-induced transient activation of ERK to a sustained stimulation (B–D).HT1080 cells were transfected with an siRNA against DUSP6 and an irrelevant control siRNA; untransfected cells were used as an additional control. After 48 h,an aliquot of the serum-starved cells was used to determine DUSP6 levels by immunoblotting (A). A second aliquot of the cells that had been transfected withthe DUSP6-specific siRNA (B) or the control siRNA (C) or of the untransfected control cells (D) was stimulated with either EGF (25 ng/ml) or lactoferrin (LF; 70�g/ml) alone or in combination for the indicated times. Thereafter, the cells were lysed, and the samples were processed as outlined under “Materials andMethods” to detect the active phosphorylated ERK (p ERK) and total ERK (t ERK). The blots shown are representative of three independent experiments. Thegraphs summarize three experiments (error bars, S.E.) in which the chemiluminescence was directly quantified (AlphaImagerTM). Data were plotted as normal-ized values of phosphorylated protein (i.e. ratio of phosphorylated to total protein) as a percentage of control.

Occupancy of LRP-1 Promotes Sustained ERK Activation

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cooperation between two receptor tyrosine kinases has beensubjected to a rigorous analysis (25). Here we show that inputvia LRP-1 and uPAR can substantially alter the temporal shapeof the ERK response. These observations underscore the func-tion of the cascade as a signal integrator and coincidence detec-tor; our experiments demonstrated that the absence or pres-ence of two additional signals (i.e. engagement of the LRP-1receptor and input via the urokinase receptor uPAR) produceda temporally and spatially distinct pattern of enzyme activation(i.e. sustained phosphorylation of ERK that was initially con-fined to the cytoplasm). Although these findings are consistentwith the predictions arising from the analysis of the networkproperties (26), there are, to the best of our knowledge, no ear-lier reports that document this type of cooperation betweenLRP-1 and receptor tyrosine kinases. However, the results from

our experiments are unequivocal. The simultaneous activationof the EGF receptor and the engagement of LRP-1 profoundlyaffected the temporal and spatial pattern of ERK phosphoryla-tion. This response was not confined to the ErbB family ofreceptor kinases but was found with all receptor tyrosinekinases examined, indicating that it is a universal phenomenon.The very mechanism by which this effect is brought about isalso consistent with the following generalization. The occu-pancy of LRP-1 triggers degradation of DUSP1 and DUSP6 viaproteasomal degradation. This effect is predicted to enhanceERK phosphorylation by receptor tyrosine kinases regardless ofthe nature of the upstream input.Interestingly, the sustained response required input via both

the urokinase/plasminogen activator receptor uPAR andLRP-1; although we did not study the detailed mechanismunderlying this cooperation, our experiments document thatneither sustained ERK phosphorylation nor LRP-1-triggereddegradation of DUSP1 and DUSP6 occurred if signaling eithervia uPAR or via LRP-1 was abrogated. Both DUSP1 (MAPKphosphatase-1, MKP-1) and DUSP6 (MKP-3) are subject toregulation by the ERK cascade; this regulation provides for aninitial feed-forward activation of the cascade because phosphor-ylation of DUSP1 (27, 28) and DUSP6 (29, 30) by MEK1/ERKrenders them susceptible to ubiquitination and hence to pro-teasomal degradation. Thus, the response to phosphorylatedERK is enhanced. In addition, activation of ERK and its nucleartranslocation results in enhanced transcription of DUSP1 andof DUSP6, which gives rise to feedback inhibition (27, 30). Thebalance between these two events shapes, at least in part, thespatiotemporal wave of ERK activation. Closely related recep-tor tyrosine kinases have been noted to differ in their ability totrigger the feed-forward and the feedback limb; PDGFR� elic-ited a less pronounced decline in DUSP6 levels and thus amoresustained ERK activation than PDGFR� (30). Our experimentsare in linewith these observations and highlight the importanceof the two limbs for regulating the steady state levels of DUSP1and DUSP6. (i) Coincident input via LRP-1 and uPAR led to arapid degradation of DUSP1 and DUSP6, which accounted forthe sustained ERK phosphorylation. (ii) Blockage of the protea-some unmasked the concomitant induction of DUSP1 andDUSP6 expression. Accordingly, DUSP levels exceeded thoseseen in control cells. Surprisingly, the decline inDUSP1was notmatched by enhanced nuclear accumulation of phospho-ERKin cells stimulated by the combined addition of EGF and lacto-ferrin. In fact, ERKwas efficiently translocated into the nucleus,but it appeared to be subject to rapid dephosphorylation, result-ing in the nuclear accumulation of ERK rather than phospho-ERK immunoreactivity. As noted, the levels of DUSP5, anothernuclear phosphatase, did not change, and this may be the phos-phatase that deactivates ERK in cells subjected to combinedstimulation of receptor tyrosine kinases and LRP-1. This possi-bility is currently being explored.Sustained ERK phosphorylation predisposes to dysregulated

cell growth; in fact, persistent activation of the ERK cascade,mostly via RAS and RAF mutations, is found in many cancers(31). There are also some instances where the low expression orloss of DUSP isoforms has been observed in various humancancer forms (e.g.DUSP1 in cells derived from esophagogastric

FIGURE 10. Proteasome inhibition switches the EGF- and lactoferrin-me-diated sustained ERK activation to a transient one. A, serum-starvedHT1080 fibrosarcoma cells were stimulated with EGF (25 ng/ml) or lactoferrin(LF; 70 �g/ml) alone or in combination for the indicated times. The phorbolester PMA (10 �M) was used as a control. B, prior to stimulation with EGF andlactoferrin, the cells were treated with MG 132 (10 �M) for 30 min. The cellswere lysed, and the samples were processed to detect the active dually phos-phorylated ERK (p-ERK) and total ERK (t-ERK) by immunoblotting as outlined inthe legend to Fig. 1 and outlined under “Materials and Methods.” The blotsshown are representative of three independent experiments. The graphsshow the quantification of chemiluminescence from three independentexperiments (error bars, S.E.) using an AlphaImagerTM. Data were plotted asnormalized values of phosphorylated protein (i.e. ratio of phosphorylated tototal protein as a percentage of control).

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cancermetastases (32),DUSP2 in leukemia (33),DUSP4 in pan-creatic (34) and breast cancer (35), and DUSP6 in pancreaticintraepithelial neoplasia and/or intraductal papillary-muci-nous neoplasms (36)). Ligands that are cleared by LRP-1 areabundantly present in human body fluids; these include, amongothers, LDL and PAI-1 (the plasminogen activator-inhibitor).Obesity is known to be an independent risk factor for cancer(37), and this may, in part, be mediated by elevated LDL cho-lesterol (38). Similarly, elevated levels of PAI-1 are an indepen-dent prognostic marker for cancer progression; this is also truefor uPA (39). It is attractive to speculate that the current obser-vations may also be of relevance to the in vivo situation; in thismodel, elevated levels of LRP-1 ligands in conjunction withuPA/uPARmay also enhanceMAPK signaling by growth factorand thus promote the emergence of clinically manifest cancer.

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Occupancy of LRP-1 Promotes Sustained ERK Activation

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Bernd R. Binder, Michael Freissmuth and Johannes M. BreussNishamol Geetha, Judit Mihaly, Alexander Stockenhuber, Francesco Blasi, Pavel Uhrin,

DUAL SPECIFICITY PHOSPHATASES (DUSP1 AND -6)TO SUSTAINED ERK PHOSPHORYLATION VIA DOWN-REGULATION OF ACTIVATION OF RECEPTOR TYROSINE KINASES AND OF LRP-1 LEADSKinase/Extracellular Signal-regulated Kinase (ERK) Cascade: CONCOMITANT

Signal Integration and Coincidence Detection in the Mitogen-activated Protein

doi: 10.1074/jbc.M111.221903 originally published online May 24, 20112011, 286:25663-25674.J. Biol. Chem. 

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