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SIGNAL INTEGRATION AND COINCIDENCE DETECTION IN THE MITOGEN-ACTIVATED PROTEIN KINASE/ERK-

CASCADE: CONCOMITANT ACTIVATION OF RECEPTOR TYROSINE KINASES AND OF LRP-1 LEADS TO SUSTAINED ERK PHOSPHORYLATION VIA DOWNREGULATION OF

DUAL SPECIFICITY PHOSPHATASES (DUSP-1 AND -6)

Nishamol Geetha1, Judit Mihaly1, Alexander Stockenhuber1, Francesco Blasi3, Pavel Uhrin1, Bernd R. Binder1,*, M Freissmuth2,†, Johannes M. Breuss1

1Department of Vascular Biology and Thrombosis Research and 2Insitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna 1090, Austria

3Department of Molecular Biology and functional Genomics, DIBIT , San Raffale Scientific Research Institute, 20132 Milan.

Running title: Occupancy of LRP1 promotes sustained ERK-activation

* deceased August 2010 † To whom correspondence should be addressed at the Center of Physiology and Pharmacology, Währinger Str. 13a, 1090 Vienna, Austria; Ph: +43-1-4277 64171; Fax: +43-1-4277 9641 email: michael.freissmuth@meduniwien.ac.at Abstract Diverse stimuli can feed into the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) cascade: this includes receptor-tyrosine kinases, G protein-coupled receptors, integrins and scavenger receptors (LRP=LDL-receptor related protein). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, bFGF, VEGF etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL-receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts.

Combined stimulation of the EGF-receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK-cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal intergrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation why chronic elevation of LRP1 ligands (e.g., PAI-1) can predispose to cancer. Introduction The LDL-receptor related protein LRP-1 is a scavenger receptor that mediates the endocytotic uptake of a large collection of ligands (~30) and thus their clearance from the extracellular space (1). The intracellular carboxyl terminus of LRP1 comprises 100 amino acids and is subject to regulatory phosphorylation by both

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.221903The latest version is at JBC Papers in Press. Published on May 24, 2011 as Manuscript M111.221903

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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serine/threonine and tyrosine kinases. These phosphorylation reactions promote the interaction with adapter proteins and thus nucleate the formation of signaling platforms. In fact, LRP1 enhances the capacity of the endocytosed PDGFβ receptor to activate the MAP kinase/ERK cascade (2, 3). These observations raise several interesting questions, namely (i) on the mechanism underlying the enhanced activation of the ERK cascade; (ii) whether this is unique for the PDGF-receptor; (iii) the extent to which this enhanced response is of pathophysiological relevance in vivo and thus translates into the 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 feedback) loops that regulate its activity. There are multiple layers of feedback loops that regulate signaling through the MAP kinase cascade. If the linear cascade RAS → RAF → MEK1 → ERK is examined, there at least three points at which feedback inhibition 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-kinase due to phosphorylation by the downstream target ERK (5) and (iii) dephosphorylation of ERK by induction of dual specificity phosphatases (DUSP /MPKP-isoforms, MAP kinase phosphatases) (6). A recent analysis concluded that the most important component in shaping the response of murine fibroblasts to PDGF was negative feedback at the RAF/MEK1 level (7). Here, we examined the action of LRP-1 ligands on signaling via several receptor tyrosine kinases (i.e., the receptors for EGF, PDGF, FGF, and VEGF). We observed that signal integration occurred at the third level, i.e., the regulation of DUSP-degradation: stimulation of various receptor tyrosine kinases caused an early peak in ERK phosphorylation. This was converted into a sustained rise if cells received concomitant input from LRP-1 and the urokinase/plasminogen activator receptor uPAR. Engagement of both receptors stimulated proteasomal degradation of DUSP1 and DUSP6 which not only changed the temporal but also the spatial pattern of ERK activation.

Materials & Methods

Proteins and antibodies- Fibronectin, vitronectin, active rhPAI-1, were from Technoclone (Vienna, Austria) and the proteasome inhibitor MG132 (Sigma, St. Louis, MO) The antibodies recognizing holo ERK1/2 and phospho-ERK, phospho-SRC (Y416) and holo-SRC (L4A1, mouse monoclonal), phospho-AKT (S473) and phospho-AKT (T308) and pan-AKT were from Cell Signalling Technologies (Beverly, MA), antibodies against DUSPs from Santa Cruz Biotechnologies (Santa Cruz, CA) or Abcam (Cambridge, UK). Antibodies directed against the integrin subunits β1,β3, β5 and αv were from Chemicon (Temecula, CA). The R3 and R2 antibodies for uPAR domains were provided by Dr Gunilla Høyer-Hansen (Finsen Laboratory, Copenhagen). EGF was purchased from PromoKine/PromoCell (Heidelberg, Germany) and other tyrosine kinases like, FGF, PDGF, VEGF, IGF, were from R&D systems (Minneapolis, MN). Lactoferrin, phorbol myristate (PMA) and crystal violet were purchased from Sigma–Aldrich (St. Louis, MO). The chemiluminescence system used was Super Signal West Femto Substrate from Thermo Scientific (Rockford, IL). Cell culture - For cell culture RPMI-1640 (Hyclone, Logan, USA), Dulbecco´s modified Eagle medium (DMEM) (Sigma–Aldrich St. Louis, MO) and fetal calf serum (HyClone, Logan, USA) were used. Primary cultures of mouse skin fibroblasts were prepared from the dermis of urokinase-deficient and of urokinase receptor-deficient mice backcrossed for 8 generation into C57/Bl6 background and from control C57/Bl6 mice by explant cultures protocol (8) HT-1080 fibrosarcoma cell-line (ATCC, Rockville, MD) and murine cells were grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS). Murine embryonic fibroblasts (MEFs) were kindly provided by Dr. Wolfgang Schneider (University of Vienna, Austria). MAP kinase stimulation -Cells (1x10 6 /ml) were seeded in 12 well plates in medium containing 10% FCS and allowed to adhere by overnight incubation. After serum starvation for 4 h, they

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were incubated in the absence and presence of EGF (25 ng/mL, ~ 4 nM) or additional growth factors and/or lactoferrin (70 µg/mL, 0.9 µM) for different time points from 0 to 60 min. The phorbol ester PMA (10 µM) was used as the positive control. At the indicated time points, the medium was aspirated and the reaction stopped by immersion in liquid nitrogen. Cells were lysed in sample buffer for SDS-polyacrylamide gel electrophoresis containing 4% SDS and β-mercaptoethanol. Proteins (equivalent to 15% of the lysate) were separated by denaturing electrophoresis (10% monomer concentration in the running gel) and transferred onto nitrocellulose membranes. Immunoreactive bands were revealed with secondary antibodies linked to horse radish peroxidase using chemiluminescence. Signals were recorded on an AlphaImager HP (Alpha Innotech, CA), which allows for linear signal detection over a large dynamic range. ERK1/2 activation was detected by sequential probing of membranes with antibodies to holo- and phospho-p44/42 MAPK. Ratios of densitometric values for each sample were recorded and compared to the control. DUSP protein levels were determined using the respective specific antibodies. Values were normalized to actin by reprobing the membranes with antibody against β-actin (Sigma–Aldrich St. Louis, MO). Cell adhesion assay - Ninety six well plates were coated with vitronectin (5 µg/mL) in Hank's buffered saline for 2 h at 37º C. Non-specific sites were blocked by addition of 0.1 mL 3% BSA in Hank's buffered saline for 30 min at 37º C followed by two washes in Hank's buffered saline solution. Thereafter, HT1080 cells (2x105/well) were seeded in RPMI containing 0.1 % BSA in the presence and absence of stimuli. Cells were incubated for 10, 30 and 60 min at 37ºC and 5% CO2. Thereafter the non-adherent cells were aspirated and the wells were rinsed twice with RPMI containing 0.1% BSA. Attached cells were quantified by addition crystal violet (0.5 % in 25% methanol, 50µL/well). After 10 min, excess crystal violet was removed by washing 3 times with distilled water. Plates were allowed to dry overnight and crystal violet pigment was extracted in 0.1 mL 0.1 M sodium citrate in 50% ethanol for 20 minutes. Cell adhesion was quantified by

determining the absorbance 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 µg/mL) in Hank´s buffered saline for 2 h at 37º C. HT-1080 cells (1 x 105) were seeded on vitronectin-coated glass coverslips and were stimulated with either EGF (25 ng/mL) or lactoferrin (70 µg/mL) or the combination thereof. The coverslips were mounted onto cell culture chambers on a heated stage insert on an Olympus AX-70 microscope for time lapse-recording of cell migration for 12 to 15 h with 10 images recorded per hour (F-View digital camera, Soft Imaging System). Recorded time-lapse sequences were analyzed by assessing the length of the track of every individual cell. Transfections of cells with siRNA - The ON-TARGETplus siRNA targeting DUSP 6 and siGENOME non-targeting siRNAs were purchased from Dharmacon (Lafayette, CO). For siRNA transfection, HT-1080 cells were plated in 12-well plates in complete medium. After 12 h, cells were transfected with siRNA at the final concentration of 50 pM using the Xtreme gene transfection reagent (Roche, Mannheim Germany) according to the manufacturer’s instructions. Forty-eight hours after siRNA transfection, the medium was removed and replaced by serum-free medium containing 0.1% BSA. The cells were serum starved for 4 h and subsequently stimulated with EGF, lactoferrin or the combination thereof. The level of phospho-ERK was determined as outlined above. Confocal microscopy - After the pertinent incubation, cells were fixed in 4% paraformal-dehyde and processed as described previously (9). To label ERK, mouse monoclonal antibody to phospho ERK and rabbit polyclonal antibodies to total ERK were used (Cell Signaling Technology Beverly, MA) Secondary antibodies used were labeled with Alexa Fluor-488 (anti-rabbit IgG) or Alexa Fluor-568 (anti-mouse IgG). Slides were mounted in Permafluor (Lab Vision Corp., Fremont, CA). Images were captured at 40- and 100-fold magnification on a Zeiss LSM 510

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META confocal microscope in multitrack mode, using pinhole sizes between 0.7 and 1.5 µm, and the appropriate standard laser-filter combinations and were digitized using the built-in software. Statistics - Values are reported as means ± S.E.M. of three experiments done in duplicate (ERK-phosphorylation) or in triplicate to quintuplicate (adhesion assay). Statistically significant differences were verified by paired or unpaired t test and by ANOVA followed by a Bonferroni posthoc-test or Dunnett's test for multiple comparison, as appropriate. Results Combined stimulation of cells with EGF and lactoferrin induces sustained ERK2 activation - The shape and duration of receptor-induced ERK-stimulation is highly variable. We surmised that the upstream cascade can integrate additional signals which are translated into distinct time-dependent activity profiles. This conjecture was tested by incubating HT-1080 cells (a human fibrosarcoma endowed with several receptor tyrosine kinases) in the presence of the LRP-ligand lactoferrin, which per se does not stimulate ERK-phosphorylation (Fig. 1A, upper panel, lanes on the right hand side). EGF-induced stimulation of ERK phosphorylation peaks at 5 to 10 min and then declines to a low residual activity that can be still detected after 2 h (Fig. 1A, upper panel, lanes on the left hand side). However, when EGF is combined with lactoferrin as sustained activation is observed over > 60 minutes (Fig. 1A, bottom panel, lanes on the left hand side). In contrast to incubations with EGF alone, the combination of EGF and lactoferrin resulted in ERK phosphorylation that was still detectable after 4 h and 16 h. In fact, the time course was comparable to that seen with persistent stimulation of protein kinase C isoforms by the phorbol ester PMA (Fig. 1A, bottom panel, lanes on the right hand side). This response was not unique to the fibrosarcoma cell line because it was recapitulated in primary cultures of human skin fibroblasts (Fig. 1B). This suggests that the ERK cascade acted as coincidence detector and signal integrator that translated the simultaneous occupancy of the EGF-receptor and an LDL-

receptor related protein into a sustained response. We verified that this coincidence detection was not limited to the pair EGF and lactoferrin: the combination of lactoferrin and bFGF (FGF2) also resulted in sustained ERK phosphorylation (Fig. 1C, cf. left hand bFGF-labeled lanes with lanes labeled bFGF/LF). Similarly, incubation in the presence of LDL (100 µg/mL) augmented the levels of phospho-ERK regardless of 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) (data not shown). EGF activates many additional pathways other than ERK. We determined whether the amplifying effect of lactoferrin was limited EGF-dependent activation of MAP kinase pathway by assessing other pathways, i.e. PI3 kinase-dependent activation of AKT and stimulation of the non-receptor tyrosine kinase SRC. EGF induced phosphorylation of AKT both on S473 (Fig. 2A) and T308 (Fig. 2B). The combination of EGF and lactoferrin did neither cause any additional stimulation nor affect the time course of AKT phosphorylation (Fig. 2A & B). Similarly, the phosphorylation of SRC elicited in response to the combination of EGF and lactoferrin was comparable to that caused by the addition of EGF alone (data not shown). While we did not examine additional signaling pathways (e.g., activation of phospholipase Cγ), these observations suggest that the signal amplification caused by lactoferrin does not indiscriminately involve all possible pathways but appears to be confined to MAP kinase activation. Sustained ERK activation depends on LRP-1 - The association of ligands with LRP can be blocked by receptor-associated protein (RAP) (10). If the action of lactoferrin and of LDL were mediated via interaction with an LRP-family member, it ought to be blunted by preincubation of the cells in the presence of RAP. This was the case (Fig. 3A). There are several family members of the LDL-receptor related protein family. Earlier observations indicated complex formation between LRP-1

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and the PDGF-receptor (2,11). Hence, we substantiated the conjecture that LRP-1 might be required also for supporting the lactoferrin-promoted augmentation of EGF-induced ERK phosphorylation by using MEFs (murine embryonic fibroblasts) isolated from mice deficient in LRP-1 and/or LDL-receptor. Unlike the wild type MEFs (Fig. 3B), MEFs lacking LRP-1 failed to respond to the combination of EGF and lactoferrin by sustained ERK activation (Fig. 3C). In fact, in the absence of LRP-1, lactoferrin blunted the response to EGF (triangles in Fig. 3C); the underlying mechanism is currently not understood. In contrast, the absence of LDL-receptor did not impair the sustained response to EGF and lactoferrin (Fig. 3D). Sustained ERK activation depends on the urokinase receptor domain-1 - Both, the EGF-receptor and LRP-1 are engaged in functionally important and context-dependent interactions with the urokinase receptor (12). We isolated murine skin fibroblasts from animals deficient in either uPA or uPAR to assess the possible contribution of uPAR to the sustained stimulation in response to EGF- and the lactoferrin-stimulation. Confluent monolayers of these cells were stimulated with EGF, lactoferrin or the combination thereof for up to 60 minutes. In wild type murine skin fibroblasts ERK was activated in a sustained fashion by EGF and lactoferrin in a manner comparable in human skin fibroblasts or in HT-1080 fibrosarcoma cells (Fig 4A). In contrast, sustained ERK phosphorylation was blunted in uPAR and uPA-deficient murine skin fibroblasts (Fig. 4A). This indicates a crucial role of both, of urokinase and its receptor, in 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 uPAR domains 1 and 3, respectively. Cells were pretreated with these antibodies and then challenged with a receptor tyrosine kinase receptor agonist and lactoferrin. Fig. 3B shows the results for stimulation of cells with PDGF: blockage of the uPAR domain-1 with the R3 antibody precluded sustained ERK phosphorylation in response to the combination of PGDF and lactoferrin (Fig. 4B). This is

consistent with the fact that the antibody blocks the association of uPA and uPAR (13). A similar observation was made with HT-1080 cells that hade been challenged by the combination of EGF and lactoferrin in the presence of the R3 antibody (Fig 4C). In contrast, monoclonal antibody R2, which does not interfere with uPAR-uPA binding, did not interfere with sustained ERK2 activation (Fig 4D). Similar findings were obtained with FGF, VEGF and IGF (data not shown). Cytoplasmic accumulation of phospho-ERK translates into accelerated adhesion on vitronectin - ERK phosphorylation promotes nuclear translocation of the enzyme (14). This was also seen if HT-1080 cells stimulated by EGF (Fig. 5, cf. first and second row). Sole addition of lactoferrin did not affect the distribution of ERK (Fig. 5, row 3). Surprisingly, the combination of EGF and lactoferrin resulted in a delayed translocation of active phosphorylated ERK into the nucleus: For the first 60 min there was little appreciable translocation of phospho-ERK into the nucleus (Fig. 5, cf. rows 4 to 6 with control rows 1 and 2), while it accumulated in the perinuclear region and most prominently at submembra-neous spots. After prolonged stimulation with EGF and lactoferrin, phosphorylated ERK did accumulate in the nucleus (Fig. 5, bottom row). The observations suggested that the stimulation of cells by the combination of EGF and lactoferrin initially redirected ERK signaling to cytosolic targets. Cytosolic ERK has many targets, several of these are involved in actin dynamics (15) such that ERK plays a prominent role in integrin-dependent adhesion (16). Accordingly, we evaluated whether the combined addition of EGF and lactoferrin promoted cell adhesion. This was the case: at early time points (10 minutes), the number of cells that adhered to vitronectin-coated dishes was augmented (Fig. 6A, white bars). In contrast, neither EGF nor lactoferrin by themselves affected cell adhesion at this early time points. Thus, increased cytosolic phospho-rylated ERK (levels) translated into a relevant biological response, namely accelerated cell adhesion promoted by the combination of EGF and lactoferrin. Blockage of the MAP kinase

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cascade ought to blunt the response to the combination of EGF and lactoferrin, if there were a cause-and-effect relation between cytosolic accumulation of MAP kinase and enhanced adhesion. This prediction was verified by employing the MEK1 inhibitor PD98059. Pretreatment of cells with 25 µM PD98059 for 30 minutes reduced the number of adherent cells by about 50%, if cells were stimulated by EGF and lactoferrin; in contrast, PD98059 had no appreciable effect on cell adhesion under any of the other conditions tested (Fig. 6A, black bars). EGF (17) and uPAR (18) stimulate cell migration via stimulation of the MAP kinase cascade. Accordingly, we allowed HT-1080 cells to adhere on vitronectin and subsequently examined over the next 15 h by time lapse microscopy, if the combination of EGF and lactoferrin had a stronger chemokinetic effect than sole addition of EGF. In fact, trajectories of cells were significantly longer in the presence of EGF and lactoferrin than when each compound was added separately (Fig. 6B). We also examined directed migration induced by wounding a cell monolayer. The combination of EGF and lactoferrin also resulted in accelerated migration (data not shown). Proteasomal degradation of DUSPs accounts for sustained ERK activation in response to EGF and lactoferrin- The magnitude and duration of MAP kinase signaling is dependent on the balance between the activities of upstream activators and deactivation by phosphatases. Since phosphorylation of both threonine and tyrosine residues is required for activity, dephosphorylation of either is sufficient for inactivation. This is achieved by dual specificity (threonine/tyrosine) protein phosphatases (DUSPs) (19). DUSPs differ in their affinity for individual MAP kinases. ERK1/2 can be dephosphorylated by the inducible DUSP1, DUSP4 and DUSP5, which are found primarily in the nucleus, and by the cystosolic isoforms DUSP6, DUSP7 and DUSP9 (20). Sustained activation of ERK2 via the combined input from a tyrosine kinase receptor and LRP-1 may be due to decline in DUSP levels. We tested this hypothesis by assessing the effect of combined stimulation with EGF and lactoferrin on the amount of DUSP in lysates of HT1080 cells. Incubation with EGF and

lactoferrin led to a pronounced and sustained down-regulation of the nuclear DUSP1 (Fig. 7A) and of the cytosolic DUSP6 (Fig. 7B). The capacity of LRP-1 ligands to synergize with tyrosine kinase receptor ligands was not limited to lactoferrin, but was also recapitulated in the presence of LDL (cf. Fig. 1C). Thus, when combined with EGF, LDL was predicted to cause as similar decline in DUSP levels, if the decline in DUSP levels and the change in ERK phosphorylation were causally related. This was the case (Fig. 7A & B). In contrast, stimulation with EGF alone caused only a transient reduction in the levels of DUSP1 and DUSP6, while lactoferrin alone did not affect the expression levels of DUSP1 and DUSP6 (open Diamonds in Fig. 7A & B). We also verified that the combination of EGF and lactoferrin did not result in an indiscriminate loss of all DUSP isoforms. There was, for instance, no appreciable change in the levels of the nuclear isoform DUSP 5 (Fig. 7C), DUSP 7 and DUSP 9 (data not shown). The downregulation of DUSP1 and DUSP6 occurred swiftly, i.e., it was detectable within 10 min. An obvious explanation for this rapid decline was proteolytic degradation via the proteasome. In fact, if cells had been pretreated with the proteasome inhibitor MG132 (10 μM). DUSP levels were not decreased upon combined stimulation with EGF and lactoferrin or LDL (Fig. 7A & B). Similar findings were obtained with cells that had been incubated with FGF and lactoferrin or LDL (Fig. 8A & B). We also observed that the MEK1 inhibitor PD98059 increased DUSP 6 protein levels and suppressed the DUSP6 degradation triggered by the combination of EGF and lactoferrin (data not shown). The LRP1-induced switch to sustained ERK phosphorylation was contingent on concomitant input via the uPA/uPAR-system (Fig. 4). This provided an additional test for the relation between DUSP down-regulation and sustained ERK signaling: blockage of uPAR by the antibody R3 directed against the domain-1 of uPAR precluded the down-regulation of DUSP1 and DUSP6 (Fig. 8C & D). The combination of EGF and lactoferrin increased DUSP degradation and triggered sustained ERK activation. Thus, downregulation

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of DUSPs ought to lead to a sustained EGF response. We focused on DUSP6 because this is the cytosolic isoform, the downregulation of which ought to phenocopy the combined effect of lactoferrin and EGF. DUSP6 levels were reduced by using an appropriate siRNA (Fig. 9A). Silencing of DUSP6 led to a sustained activation of ERK with EGF alone; there wasn't any appreciable difference in the time course of phospho-ERK accumulation in cells that had been stimulated by sole addition of EGF or the combination of EGF and lactoferrin (Fig 9B). This was not the case in cells transfected with a control siRNA (Fig. 9C) or untransfected cells, which were examined in parallel (Fig 9D). If sustained ERK activation induced by combined stimulation with EGF and lactoferrin were indeed the result of proteasomal degradation of DUSP1 and/or DUSP6, blockage of the proteasome ought to prevent sustained activation. This prediction was verified by treating cells with the proteasome inhibitor MG132 prior to stimulation by EGF, lactoferrein or the combination thereof. Inhibition of the proteasome by MG132 did not affect the response to EGF (cf. Fig. 10A & B, left hand panel). In contrast, in cells challenged by the combined addition of EGF and lactoferrin, pretreatment with MG132 precluded sustained ERK phosphorylation (Fig. 10B, right hand side) phospho-ERK levels declined more rapidly after the initial peak than in untreated cells (Fig. 10A, right hand side). Discussion The ERK cascade plays a central role in mitogenic signaling. It has attracted interest because input via extracellular signal is translated into both, a spatial and a temporal dimension. This concept was originally developed with the study of PC12 differentiation (14) and probed rigorously by rewiring the signaling network (21): the rat pheochromocytoma cell line PC12 is differentiated by NGF, because this agonist causes a very long lasting stimulation of ERK activity. In contrast, the kinetics of EGF-induced ERK phosphorylation supplies a stimulus for cell division. In addition, the ERK-cascade displays hysteresis and bi-stability. It is therefore capable of storing information and converting an

analog signal (the graded concentration of mitogen) to a digital output: regardless whether differentiation or cell division is examined, if the activity has reached a critical threshold, an irreversible decision is made. The cell undergoes replication or differentiates. It is therefore interesting to understand how the kinetics of ERK activation is controlled. Because the ERK signaling pathways receives input from several pathways, it allows for signal integration: irrespective of the source of the upstream input, it is converted into an analog signal, namely the enzymatic activity of the dually phosphorylated ERK. Thus, integral of the input is encoded in the strength of the output signal. Because ERK is subject to deactivation by dephosphorylation and because it drives irreversible decisions, it also allows for coincidence detection: thresholds may only be reached, if two signals are present at the same time. Thus, in the original model of PC12 cell differentiation, the ERK cascade also acted as a coincidence detector: the simulta-neous presence of cAMP converted the EGF-driven output from proliferation to differentia-tion (14). Previous investigations have focused on the cross-talk with G protein-coupled receptors which have provided evidence for trans-activation (22), release of growth factors via the release of latent membrane-bound growth factors (23) and for second messenger driven loops (14, 24). More recently, the cooperation between two receptor tyrosine kinases has been subjected to a rigorous analysis (25). Here we show that input via LRP-1 and uPAR can substantially alter the temporal shape of the ERK response. These observations underscore the function of the cascade as a signal integrator and coincidence detector: our experiments demonstrated that the absence or presence of two additional signals, i.e., engagement of the LRP-1 receptor and input via the urokinase receptor uPAR produced a temporally and spatially distinct pattern of enzyme activation, i.e. sustained phosphorylation of ERK that was initially confined to the cytoplasm. While these findings are consistent with the predictions arising from the analysis of the network properties (26), there are - to the best of our knowledge – no earlier reports that document this type of cooperation between LRP-1 and receptor tyrosine kinases. However, the results

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from our experiments are unequivocal. The simultaneous activation of the EGF receptor and the engagement of LRP-1 profoundly affected the temporal and spatial pattern of ERK phosphorylation. This response was not confined to the ErbB family of receptor kinases but was found with all receptor tyrosine kinases examined indicating that it is a universal phenomenon. The very mechanism by which this effect is brought about is also consistent with this generalization: the occupancy of LRP-1 triggers degradation of DUSP1 and DUSP6 via proteasomal degradation. This effect is predicted to enhance ERK phosphorylation by receptor tyrosine kinases regardless of the nature of the upstream input. Interestingly, the sustained response required input via both the urokinase/plasminogen activator receptor uPAR and LRP-1: while we did not study the detailed mechanism underlying this cooperation, our experiments document that neither sustained ERK phosphorylation nor LRP-1 triggered degradation of DUSP1 and DUSP6 occurred if either signaling via uPAR or via LRP-1 was abrogated. Both DUSP1 (MAP-kinase phosphatase-1, MKP-1) and DUSP6 (MKP-3) are subject to regulation by the ERK-cascade: this regulation provides for an initial feed-forward activation of the cascade, because phosphorylation of DUSP1 (27,28) and DUSP6 (29,30) by MEK1/ERK renders them susceptible to ubiquitination and hence to proteasomal degradation. Thus, the response to phosphorylated ERK is enhanced. In addition, activation of ERK and its nuclear translocation results in enhanced transcription of DUSP1 and of DUSP6, which gives rise to feedback inhibition (27,30). The balance between these two events shapes, at least in part, the spatiotemporal wave of ERK activation. Closely related receptor tyrosine kinases have been noted to differ in their ability to trigger the feed-forward and the feedback limb: PDGFRβ elicited a less pronounced decline in DUSP6 levels and thus a more sustained ERK activation than PDGFRα (30). Our experiments are in line with these observations and highlight the importance of the two limbs for regulating the steady state levels of DUSP1 and DUSP6: (i) Coincident input via LRP-1 and uPAR led to a

rapid degradation of DUSP1 and DUSP6, which accounted for the sustained ERK phosphorylation. (ii) Blockage of the proteasome unmasked the concomitant induction of DUSP1 and DUSP6 expression. Accordingly, DUSP levels exceeded those seen in control cells. Surprisingly, the decline in DUSP1 was not matched by enhanced nuclear accumulation of phospho ERK in cells stimulated by the combined addition of EGF and lactoferrin. In fact, ERK was efficiently translocated into the nucleus but it appeared to be subject to rapid de-phosphorylation resulting in the nuclear accumulation of ERK rather than phospho-ERK immunoreactivity. As noted, the levels of DUSP5 – another nuclear phosphatase – did not change and may be the phosphatase that deactivates ERK in cells subjected to combined stimulation of receptor tyrosine kinases and LRP-1. This 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 or loss of DUSP-isoforms has been observed in various human cancer forms, e.g. DUSP1 in cells derived from esophagogastral cancer metastases (32), DUSP2 in leukemia (33) DUSP4 in pancreatic (34) and breast cancer (35), and DUSP6 in pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms (36). Ligands that are cleared by LRP-1 are abundantly present in human body fluids, these include among others and LDL, 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-cholesterol (38). Similarly, elevated levels of PAI-1 are an independent prognostic marker for cancer progression; this is also true for uPA (39). It is attractive to speculate that the current observations may also be of relevance to the in vivo situation: in this model, elevated levels of LRP-1 ligands in conjunction with uPA/uPAR may also enhance MAP kinase signaling by growth factor and thus promote the emergence of clinically manifest cancer.

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.

FOOTNOTES

This work was supported by the CCHD doctoral program, which is funded by the FWF (Austrian Science Fund) and the Medical University of Vienna.

The abbreviations used are: BSA, bovine serum albumin; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's Modified Eagle Medium; DUSP, dual specificity phosphatase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase (=MAPK, mitogen-activated protein kinase); HRP, horseradish peroxidase; IGF, insulin like growth factor; LDL, low density lipoprotein; LF, lactoferrin; LRP, low density lipoprotein receptor related protein; LSM, laser scanning microscopy; MEK 1, MAPK/ERK kinase 1; MEF, murine embryonic fibroblasts; MKP, MAP-kinase phosphatase; MSF, murine skin fibroblasts; NGF, nerve growth factor; PAI 1, plasminogen activator inhibitor 1; PMA, phorbol 12,myristate 13 acetate; PDGF, platelet-derived growth factor; RAP, receptor-associated protein (a ligand of LRP-1). RPMI, Roswell Park Memorial Institute; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor; VEGF, vascular endothelial growth factor.

FIGURE LEGENDS FIGURE 1. Combined stimulation of HT1080 cells with EGF and with ligands of LRP induces sustained activation of ERK.. Serum starved HT1080 fibrosarcoma cells (A,C) and normal human skin fibroblasts (B) were stimulated with either EGF (25 ng/mL)or lactoferrin (LF; 70 µg/mL) alone or in combination for the indicated times. PMA (10 µM) was used as positive control. Thereafter the cells were lysed and the samples were processed as outlined under Materials and Methods to detect the active dually phoshporylated ERK (p-ERK) and total ERK (t-ERK). The blots shown are representative of 3 independent experiments. The graphs summarize three experiments (error bars are s.e.m.) in which the chemoluminescence was directly quantified (Alpha-ImagerTM) and plotted as normalized values of phospho-ERK (i.e., ratio of phospho-ERK to total ERK in as % of control). FIGURE 2. Combined stimulation of HT 1080 cells with EGF and with lactoferrin does not induce a sustained activation of AKT. Serum starved HT1080 fibrosarcoma cells were stimulated with either EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) or in combination for the indicated times. Thereafter the cells were lysed and the samples were processed as outlined under Materials and Methods to detect the active phosphorylated AKT (Panel A: phospho-S473 AKT; Panel B: phospho-T308AKT) and total levels of AKT (t-Akt). The graphs summarize three experiments (error bars are s.e.m.) in which the chemoluminescence was directly quantified (Alpha-ImagerTM) and plotted as normalized values of phosphorylated protein (i.e., as ratio of phosphorylated to total protein in as % of control).

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FIGURE 3. Blockage by RAP of sustained ERK activation induced by EGF and lactoferrin. A: HT1080 fibrosarcoma cells were pre-treated with RAP (10 µg/mL) and stimulated with EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) as outlined in the legend to Fig. 1 and in Materials and Methods. B,C & D: Wild type murine embryonic fibroblasts (B), murine embryonic fibroblasts deficient in LRP-1 (C) and deficient in LDLR (D) were stimulated with EGF and lactoferrin for the indicated times. The phosphorylation of ERK was assessed by quantitative immunoblots and normalized to total ERK. The blots shown are representative of 3 independent experiments. Graphs show the quantification of phospho-ERK levels (normalized to total ERK and given in % of control; means ± s.e.m; n = 3). FIGURE 4. Sustained ERK activation requires the urokinase receptor domain-1. A: Serum starved wild type, uPA-/- or uPAR-/- murine skin fibroblasts were stimulated with EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) for ERK phosphorylation was assessed by immunoblots and quantified (normalized to total ERK). B, C & D: HT1080 cells were pretreated with uPAR domain-1 specific R3 antibody (2 µg/mL, Panel B) or uPAR domain-3 specific R2 antibody (2 µg/mL, Panel C) for 30 min and subsequently stimulated with PDGF (10 ng/mL) and lactoferrin (LF; 70 µg/mL) or EGF (25ng/mL) and lactoferrin (LF; 70 µg/mL). The blots shown are representative of 3 independent experiments. The levels of phospho-ERK were quantified as in Fig. 1 and plotted. The values are means ± s.e.m.. FIGURE 5. Localization of phosphorylated ERK by immunocytocehmistry in cells stimulated by EGF and lactoferrin. Serum-starved HT1080 cells were stimulated with EGF (25 ng/mL), lactoferrin (70 µg/mL) and the combination thereof for the indicated time points. Thereafter the cells were fixed with paraformaldehyde (4 %), permeabilized with 0.2% Tween20 and stained for total ERK (green, 1st column), phospho-ERK (red, 2nd column). Confocal images were captured on a Zeiss LSM510 using the settings outlined under Materials and Methods. The 3rd column shows merged confocal images of panel 1st and 2nd column. Arrow heads mark phosphorylated ERK in the cytoplasm and in basal adhesion contacts. Images were captured, micropgraphs were printed and cells were scored for the presence of phospho-ERK in the cytosol and in the nucleus. Ninety cells from three independent experiments were scored for each condition. FIGURE 6. Panel A: Enhanced adhesion on vitronectin of EGF- and lactoferrin-stimulated HT 1080 cells. Panel A, Serum starved HT1080 cells were incubated in the absence (vehicle) and presence of PD98059 (25 µM) for 30 min and subsequently stimulated with EGF (25 ng/mL) and/or lactoferrin (70 µg/mL) and plated on vitronectin-coated plastic dishes. After 10 min, the non-adherent cells were removed by aspiration and the adherent cells were stained with crystal violet. Cell adhesion was quantified by determining the absorbance at 540 nm in an ELISA plate reader. Values represent mean absorbance ± s.e.m (mean ± s.e.m.) (n=4). Statistical significance was assessed by ANOVA followed by Dunnett's multiple comparison test, which documented a statistically significant effect of EGF + lactoferrin vs. control and a statistically significant inhibition of this stimulation by PD98059 (p< 0.01). Panel B: Long term stimulation with EGF and lactoferrin enhances migration of HT 1080 cells on vitronectin. HT 1080 cells were seeded on vitronectin coated glass coverslips and were stimulated with either EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) alone or in combination. The cover slips were mounted onto cell culture chambers on a heated stage insert on an Olympus AX-70 microscope for recordings by time lapse microscopy of cell migration for over 15 h with 10 images recorded per hour (F-View digital camera, Soft Imaging System). Recorded time-lapse sequences were analyzed by assessing the length of the track of individual cells. Data are based on the analysis of the trajectories of 90 cells recorded in 3 independent experiments. The effect of lactoferrin + EGF was statistically significant from all other values (p<0.05, ANOVA followed by Dunnett's test for multiple comparisons)

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FIGURE 7. Changes in the levels of DUSP1 (A), DUSP6 (B) and DUSP5 (C) in cells stimulated by the combined addition of EGF and lactoferrin. Serum-starved HT1080 cells were pretreated with vehicle or MG132 (10 µM) for 30 min and subsequently stimulated with EGF (25 ng/mL) and lactoferrin (70 µg/mL) or LDL (100 µg/mL) for the indicated time points. Expression of DUSP 1 (Panel A), DUSP 6 (Panel B) and DUSP5 (C) was assessed from immunoblots, quantified densitometrically and normalized to actin. The blots shown are representative of 3 independent experiments; relative levels (initial control = 100%) are shown in the graphs (error bars are s.e.m.). FIGURE 8. Degradation of DUSP1 (A,C) and DUSP6 (B,D) induced by the combination of FGF and lactoferrin (A,B) or of EGF and lactoferrin (C,D) in the absence and presence of MG132 (A,B) and of the uPAR-antibody R3 (C,D). Serum-starved HT1080 cells were pretreated with vehicle or MG132 (10 µM) for 30 min (A,B) or of the domain-1 specific uPAR antibody R3 (2 µg/mL) and subsequently stimulated with FGF (20 ng/mL), EGF (25 ng/mL), lactoferrin (70 µg/mL) or LDL (100 µg/mL) and the combination thereof for the indicated intervals. The levels of DUSP1 (Panel A,C) and DUSP6 (Panel B,D) were assessed by immunoblotting, quantified and The blots shown are representative of 3 independent experiments; relative levels (initial control = 100%) are shown in the graphs (error bars are s.e.m.). FIGURE 9. Silencing of DUSP6 by siRNA transfection (A) in HT1080 converts EGF-induced transient activation of ERK to a sustained stimulation (B-D). HT 1080 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 with the DUSP6-specific siRNA (B), the control siRNA (C) or of the untransfected control cells (D) were stimulated with either EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) alone or in combination for the indicated times. Thereafter the cells were lysed and the samples were processed as outlined under Materials and Methods to detect the active phosphorylated ERK and total ERK. The blots shown are representative of 3 independent experiments. The graphs summarize three experiments (error bars are s.e.m.) in which the chemoluminescence was directly quantified (Alpha-ImagerTM). Data were plotted as normalized values of phosphorylated protein (i.e., ratio of phosphorylated to total protein in as % of control. FIGURE 10. Proteasome inhibition switches the EGF and lactoferrin mediated sustained ERK activation to a transient one. A, Serum starved HT1080 fibrosarcoma cells were stimulated with EGF (25 ng/mL) or lactoferrin (LF; 70 µg/mL) alone or in combination for the indicated times. The phorbol ester PMA (10 µM) was used as a control. B, Prior to stimulation with EGF and lactoferrin, the cells were treated with MG 132 (10 µM) for 30 min. The cells were lysed and the samples were processed to detect the active dually phoshporylated ERK (p-ERK) and total ERK (t-ERK) by immunoblotting as outlined in the legend to Fig. 1 and outlined under Materials and Methods. The blots shown are representative of 3 independent experiments. The graphs show the quantification of chemiluminescence from three independent experiments (error bars are s.e.m.) using an Alpha-ImagerTM. Data were plotted as normalized values of phosphorylated protein (i.e., ratio of phosphorylated to total protein in as % of control.

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

phosphatases (DUSP-1 and -6)LRP-1 leads to sustained ERK phosphorylation via downregulation of dual specificity

kinase/ERK-cascade: Concomitant activation of receptor tyrosine kinases and of Signal integration and coincidence detection in the mitogen-activated protein

published online May 24, 2011J. Biol. Chem. 

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