Calmodulin modulates H-Ras mediated Raf-1 activation

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Cellular Signalling 20 (2008) 1092 – 1103 www.elsevier.com/locate/cellsig

Calmodulin modulates H-Ras mediated Raf-1 activation Jemina Moretó a,b , Anna Lladó a,b,c , Maite Vidal-Quadras b , Maria Calvo b,c , Albert Pol b,d , Carlos Enrich a,b,⁎, Francesc Tebar a,⁎ b

a Departament de Biologia Cel·lular, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain c Unitat de Microscopia Confocal, Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain d Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010-Barcelona, Spain

Received 11 October 2007; received in revised form 22 January 2008; accepted 23 January 2008 Available online 5 February 2008

Abstract We have previously demonstrated that, in COS-1 cells, inhibition of calmodulin increases Ras-GTP levels although it decreases Raf-1 activity and consequently MAPK. The present study analyzes the role of calmodulin in the regulation of Raf-1. First we show, using FRET microscopy, that inhibition of Raf-1 was not a consequence of a decreased interaction between H-Ras and Raf-1. Besides, the analysis of the phosphorylation state of Raf-1 showed that calmodulin, through downstream PI3K, is essential to ensure the Ser338-Raf-1 phosphorylation, critical for Raf-1 activation. We also show that the expression of a dominant negative mutant of PI3K impairs the calmodulin-mediated Raf-1 activation; in addition, both calmodulin and PI3K inhibitors decrease phospho-Ser338 and Raf-1 activity from upstream active H-Ras (H-RasG12V) and this effect is dependent on endocytosis. Importantly, in H-Ras depleted COS-1 cells, calmodulin does not modulate MAPK activation. Altogether, the results suggest that calmodulin regulation of MAPK in COS-1 cells relies upon H-Ras control of Raf-1 activity and involves PI3K. © 2008 Elsevier Inc. All rights reserved. Keywords: PI3K; H-Ras; Raf-1; MAPK; EGF–EGFR; Endocytosis; Signalling; Calmodulin

1. Introduction The mitogen-activated protein kinase (MAPK) controls a variety of cellular responses including cell cycle progression and differentiation [1,2]. Stimulation of the epidermal growth factor receptor (EGFR) leads to MAPK activation through the small GTPase Ras via recruitment of the guanine nucleotide

Abbreviations: CD, β-Methyl-cyclodextrin; CFP, cyan fluorescent protein; EGF, epidermal growth factor; FRET, Fluorescence Resonance Energy Transfer; GST, glutathione-S-transferase; MAPK, Mitogen-activated protein kinase; Mek, MAPK/ERK kinase; Pak, p21-activated protein kinase; PI3K, phosphoinositide 3-kinase; RBD, Ras-binding domain; W-13, N-(4-aminobutyl)-5-chloro-2naphthalenesulfonamide; YFP, yellow fluorescent protein. ⁎ Corresponding authors. Departament de Biologia Cel·lular, Facultat de Medicina, Universitat de Barcelona, C/Casanova 143, 08036-Barcelona, Spain. Tel.: +34 932275400x3337; fax: +34 934021907. E-mail addresses: [email protected] (C. Enrich), [email protected] (F. Tebar). 0898-6568/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.01.022

exchange factor Sos. Activated Ras, bound to GTP, recruits and activates the serine/threonine kinase Raf, which then phosphorylates Mek and this, in turn, phosphorylates and activates the p42/p44 MAPKs (ERK1 and ERK2) [3,4]. The regulation of this MAPK signal pathway is very complex and involves scaffolding proteins as well as many other molecules [5]. Calmodulin is a small (148 amino acids), highly conserved and the most abundant calcium-binding protein in non-muscle cells, and it regulates basic cellular processes including cell proliferation, growth and movement as well as cellular membrane trafficking [6–8]. Through its binding to calmodulin binding-proteins (CaMBPs), calmodulin is involved in the regulation of MAPK [9,10] and this regulation has been observed at different levels of the MAPK signaling pathway; for instance, calmodulin binds and modulates EGFR activation [11,12]. Calmodulin also binds to K-Ras and controls its cellular localization and activation via PKC [13–17] and, one step forward in the MAPK cascade, it also regulates Raf-1 activity.

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Interestingly, we have shown that in COS-1 cells, inhibition of calmodulin inhibits MAPK due to reduced Raf-1 activity, despite the fact that it increases EGFR phosphorylation and Ras activity [18]. Raf proteins play a central role in MAPK activation. However, regulation of Raf is incompletely understood due to its high complexity (see reviews: [19–22]). The Raf family consists of three isoforms, A-Raf, B-Raf and Raf-1 (C-Raf), that are highly conserved within three regions (CR1, CR2 and CR3) [23]. CR1 and CR2 encode regulatory functions, while CR3 contains the kinase domain. CR1 has the Ras-binding domain (RBD) and the cysteine rich domain (CRD) implicated in Raf-1 activation via Ras-GTP-mediated translocation to the plasma membrane, where different events take place [24–26]. In the plasma membrane, Raf-1 is regulated by a complex interplay of phosphatases and kinases, and stimulatory and inhibitory phosphorylated sites have been described. Among them, Ser43, Ser259 and Ser621 are inhibitory sites [27–30] while Ser338 and Tyr341 are stimulatory ones [25,31–33]. Furthermore, phosphorylation of Thr491 and Ser494 in the activation loop has been reported to be necessary but not sufficient for Raf-1 activation [34]. It is also noteworthy that endocytosis and phosphoinositode 3-kinase (PI3K) activity are involved in the modulation of Raf-1 activity by H-Ras [35]. In addition, PI3K modulates Ras-GTP levels by acting on GEFs [36,37] and H-Ras also activates PI3K [38]. Moreover, activation of Pak proteins, through PI3K, might phosphorylate Ser338 and it activates Raf-1 [33,39,40]. In addition, calmodulin is also involved in order to ensure PI3K activation [41–43]. The complexity of this crosstalk between calmodulin and signaling molecules upstream of Raf-1 prompted us to further study the critical role of calmodulin in Raf-1 and MAPK activation in COS-1 cells. In the present study we show that despite enhancing the Ras–Raf-1 interaction, inhibition of calmodulin suppresses MAPK activation in COS-1 cells due to the impediment of Raf-1-Ser338 phosphorylation by activated H-Ras. Overall, our results suggest that calmodulin, through the downstream PI3K effector, modulates H-Ras activation of Raf1 in the endosomal compartment. 2. Experimental procedures 2.1. Reagents Mouse receptor-grade EGF and W-13 were from Sigma Chemical Co. (Madrid, Spain). Wortmannin (KY 12420) was from Calbiochem (Merck KGaA, Darmstadt, Germany) and LY294002 from Stressgen Bioreagents (Victoria, Canada). EGF conjugated with TRITC (EGF–TRITC) was from Molecular Probes (Eugene, OR). Monoclonal antibodies to EEA1 and Raf-1 were from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against phosphorylated MAPK, phosphorylated Mek, phospho-Akt (Ser473 or Thr308), MAPK or phospho-Raf (Ser259) were from Cell Signaling, New England Biolabs (Beverly, MA), the monoclonal antibody against phospho-Raf1 (Ser338), monoclonal anti-myc Tag clone 9E10 and polyclonal antibody antiPI3 kinase (p85) were from Upstate (New York, USA). Monoclonal anti-actin antibody was from ICN. Monoclonal anti-GFP antibody was from Stressgen (Victoria, Canada) while the polyclonal one was from Abcam (Cambridge, UK), as was the monoclonal anti-HA antibody. Polyclonal H-Ras (C-20) and

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monoclonal K-Ras (F234) antibodies were from Santa Cruz Biotechnology, Inc. (California, USA). Peroxidase-labeled antibodies and SDS-PAGE molecular weight markers were from Bio-Rad (Hercules, CA).

2.2. Cell culture Green monkey kidney cells (COS-1) and Chinese Hamster Ovary (CHO) cells were grown in DMEM and Ham's F-12 respectively, containing 10% fetal calf serum (FCS), pyruvic acid, antibiotics and glutamine. DMEM, Ham's F-12 and FCS were purchased from Biological Industries (Beit Haemek, Israel). Cells were grown to 90% confluence for cellular fractionation, immunoprecipitation, pull-down or radioactivity experiments, and 60% confluence for immunofluorescence and FRET experiments.

2.3. Plasmid and siRNA transfection H-Ras and Raf-1 cDNAs were kindly provided by Dr. Richard Marais (Institute of Cancer Research, London, UK) and subcloned into living color vectors (Clontech, Palo Alto, CA). Fluorescent proteins were tagged at the Nterminus of the full length proteins. Raf-1-S259A (also provided by Dr. Richard Marais), HA-Dyn-K44A (ATCC) and pSG5-p85deltaSH2 (kindly provided by Dr. Julian Downward, Institute of Cancer Research, London, UK) and GFP-HRasG12V were transfected with Effectene (QIAGEN, Valencia, CA) in p-100 dishes of COS-1 cells to perform immunoprecipitation or Raf-kinase assay after 24–48 h of expression. siRNAs duplexes were synthesized and purified by Invitrogen. The siRNA sequences for targeting H-Ras in COS-1 cells were H-Ras siRNA1 (5′CCUUCUACACGUUGGUGCGUGAGAU-3′) and H-Ras siRNA2 (5′AUCUCACGCACCAACGUGUAGAAGG-3′). After resuspending both duplex RNAs (20 μM solution), 14 μl of these was transfected in p-60 plates of COS-1 cells (40–50% confluence) with 22.5 μl of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in 500 μl of Opti-MEM medium for 6 h following protocols provided by the manufacturer. GFP-siRNA was also transfected as a control. After 24 h of transfection each p-60 dish was split into 6-well plates. Treatments and cell lysis were conducted 72 h after transfection.

2.4. Cellular extracts Cells were lysed in a buffer containing 2% SDS, 67 mM Tris–HCl, pH 6.8, and 10 mM EDTA, and sonicated twice for 10 s. Equal amounts of protein [44] were electrophoresed and immunoblotted. To perform cellular fractionation cells were grown in 100-mm dishes and, after different treatments, were rinsed with PBS and mildly permeabilized by scraping with a rubber policeman in buffer A (150 mM KCl, 2 mM MgCl2, 20 mM HEPES, 10% glycerol, pH 7,2, 1 mM dithiothreitol, 1 mM EGTA, 1 mM EDTA, 1 mM NaVO4, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin) supplemented with 0.02% saponin and then incubated for 15 min at 4 °C to allow the release of cytosolic proteins. The saponin homogenate was then centrifuged at 14,000 ×g for 20 min. Pellets were solubilized with buffer A supplemented with 1% TX100. Aliquots of equal amount of protein (determined by Bradford method) of soluble and insoluble saponin fraction (corresponding to cytosolic and membrane fractions) were processed by electrophoresis and Western blot analysis.

2.5. Fluorescence Resonance Energy Transfer (FRET) microscopy, sensitized emission method FRET analysis was based on the sensitized emission method previously described [45,46] with slight modification for the confocal microscope. A Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems Heidelberg GmbH, Manheim, Germany) equipped with an argon laser, 63× oil immersion objective lens (NA 1.32) and a double dichroic filter (458/514 nm) was used. CFP was used as the donor fluorochrome paired with YFP, as the acceptor fluorochrome. To measure FRET, three images were acquired in the same order in all experiments through 1) CFP channel (Abs. 458 nm, Em. 465– 510 nm), 2) FRET channel (Abs. 458 nm, Em. 525–600 nm) and 3) YFP

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channel (Abs. 514 nm, Em. 525–600 nm). Background was subtracted from images before performing FRET calculations. Control and experimental images were taken under the same conditions of photomultiplier gain, offset and pinhole aperture. In order to calculate and eliminate non-FRET-components from the FRET channel, images of cells transfected with either CFP-x or YFP-x alone were taken under the same conditions as for the experiments. The fraction of cross-over of CFP (A) and YFP (B) fluorescence was calculated for the different experimental conditions. Areas with unusually high or low CFP:YFP ratios (i.e.: outside the 1:1 to 1:4 stoichiometric range) were excluded from analysis. Corrected FRET (FRETc) was calculated on a pixel-by-pixel-basis for the entire image by using the equation: FRETc ¼ FRET  ð A  CFPÞ  ð B  YFPÞ;

ð1Þ

where FRET, CFP and YFP correspond to background-subtracted images of cells expressing CFP and YFP acquired through the FRET, CFP and YFP channels, respectively. Images of FRETc intensity were rescaled according to a lookup table (LUT) where the minimum and maximum values are displayed in each color bar (12 bit depth). Mean FRETc values were calculated from mean fluorescence intensities for each selected region of interest (ROI) according to Eq. (1), and normalized (FRETN) values for membrane regions were calculated according to the following equation: FRETN ¼ FRETc =YFP

ð2Þ

All calculations were performed using the FRET sensitized emission wizard from Leica Confocal Software and Microsoft Excel.

2.6. Immunoprecipitation Starved cells were grown on 100-mm dishes and treated or not with EGF and W-13 or wortmannin in binding medium (DMEM, 0.1% bovine serum albumin, 20 mM HEPES, pH 7.3). Cells were then washed in phosphate-buffered saline (PBS) and solubilized by scraping with a rubber policeman in TGH buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM HEPES, pH 7.3, 5 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin) followed by gentle rotation for 10 min at 4 °C. Lysates were then centrifuged at 14,000 ×g for 10 min at 4 °C. Supernatants were incubated with anti-Raf-1 for 2 h at 4 °C and for 30–60 min with Protein G-Sepharose 4B (Sigma). SDS-polyacrylamide gels, 8%, were used to separate proteins of the immunoprecipitates, previously washed and solubilized with Laemmli loading buffer. Proteins were then transferred to Immobilon-P (Millipore, Bedford, MA) and immunoblotted using anti-phosphoRaf-1 (Ser338) and anti-Raf-1 followed by the appropriate peroxidase-conjugated secondary antibody and ECL detection (Amersham Pharmacia Biotechnology, Buckinghamshire, UK). COS-1 cells transfected with GFP-H-RasG12V (and also Dyn-K44A) were grown in 100-mm dishes and treated and processed as described above. Immunoprecipitation was performed incubating supernatant with polyclonal anti-GFP antibody for 2 h and then for 30–60 min after the addition of ImmunoPure Immobilized Protein A-Agarose (Pierce, Rockford, IL). Immunoprecipitates were washed three times in TGH. Electrophoresis and immunoblotting were performed as previously described.

2.7. Immunofluorescence staining COS-1 cells transiently expressing different proteins, grown on coverslips and after different treatments were fixed with freshly prepared 4% paraformaldehyde for 12 min at room temperature and mildly permeabilized with PBS containing 0.1% Triton X-100, 0.1% BSA at room temperature for 3 min; they were then incubated in the same buffer, in which Triton X-100 was omitted, at room temperature for 1 h with the primary antibody, washed intensively, and incubated with adequate secondary antibodies labeled with Cy3 (Alexa 594, Molecular Probes, Invitrogen) or Cy5 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After staining, the coverslips were mounted in Mowiol (Calbiochem) and the images were collected using an inverted epifluorescence Axiovert 200 M microscope (Carl Zeiss, Göttingen, Germany) equipped with a Photometric Cool Snap HQ camera, all controlled by Slide-Book 3.0.10.5 software (Intelligent Imaging Innovation, Denver, CO). Final analysis of all images was performed using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).

2.8. Calmodulin-sepharose pull-down assay Cleared TGH-cell lysates of control or EGF-stimulated COS-1 cells were split and incubated for 2 h at 4 °C with 20 μl of CaM-Sepharose (Sigma) in the presence of 1 mM CaCl2 or 1 mM EGTA. Samples were washed 5 times in lysis buffer containing 1 mM CaCl2 or 1 mM EGTA, resolved by SDS-PAGE, and immunoblotted with anti-PI3 kinase-p85 and anti-Raf-1 antibodies.

2.9. Raf-1 kinase activity assays To measure Raf-1 activity in COS-1 cells, kinase assays were performed after immunoprecipitation, as previously described [18,47]. Transiently expressed myc-Raf-1-S259A was immunoprecipitated with anti-myc Tag antibody. Endogenous Raf-1 was immunoprecipitated with anti-Raf-1 monoclonal antibody and Raf-1 was also co-immunoprecipitated in cells expressing GFP-H-RasG12V using anti-GFP polyclonal antibody.

2.10. Internalization and recycling of

125

I-EGF

125

I-EGF was purchased from Amersham Biosciences UK (Little Chalfont, Buckinghamshire, England). 125I-EGF internalization and recycling were measured as described previously [48]. Briefly, to measure internalization, COS-1 cells cultured in 12-well dishes were incubated with 125I-EGF (1 ng/ml) and W-13 or wortmannin at 37 °C for 2, 4 and 6 min. After that, medium was aspirated and the monolayers were rapidly washed three times in cold DMEM to remove unbound ligand and then incubated for 5 min with 0.2 M acetic acid (pH 2.8) containing 0.5 M NaCl at 4 °C. The acid wash was used to determine the amount of surface-bound 125I-EGF. Finally, cells were lysed in 1 N NaOH to measure internalized radioactivity. The ratio of internalized to surface radioactivity was plotted against each time point. To measure recycling, cells in 35-mm culture dishes were incubated with 5 ng/ml 125I-EGF for 7 min at 37 °C and washed in cold DMEM. Noninternalized 125I-EGF was removed from the cell surface by a 2.5 min acid wash (0.2 M sodium acetate, 0.5 M NaCl, pH 4.5). At this point cells are referred to as “125I-EGF-loaded cells.” Trafficking of 125I-EGF-receptor complexes in these loaded cells was then initiated by incubating cells in fresh binding medium containing 100 ng/ml of unlabeled EGF and other reagents such as DMSO, W13 and wortmannin at 37 °C for 30 min. At the end of the chase incubation, the medium was collected to measure the amount of recycled 125I-EGF by precipitation with trichloroacetic acid (TCA), and cells were incubated for 5 min with 0.2 M acetic acid (pH 2.8) containing 0.5 M NaCl at 4 °C to determine the amount of surface-bound 125I-EGF. Finally, cells were solubilized in 1 N NaOH to measure the amount of intracellular 125I-EGF. The amount of recycled 125 I-EGF was estimated by adding the radioactivity counted on the cell surface and the TCA-precipitated radioactivity in the medium during chase incubation; the recycling rate was expressed as the ratio of this amount to the total EGF molecules.

3. Results and discussion In a previous study we demonstrated that inhibition of calmodulin leads to tyrosine phosphorylation of EGFR and Ras activation in COS-1 cells. However, downstream in the MAPK pathway, Raf-1 activity and the subsequent levels of P-Mek and P-MAPK were significantly reduced [18]. 3.1. Calmodulin regulates Ser338 phosphorylation of Raf-1 and its activity To examine the role of calmodulin in the regulation of Raf-1 signaling we treated COS-1 cells with the calmodulin antagonist W-13. The use of W-13 has been shown to be highly specific for calmodulin and it does not mimic the effect shown by CaMPK or PKC inhibitors [6,49,50].

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Fig. 1. W-13 increases Ras–Raf-1 interaction at the plasma membrane. Wild type CFP-H-Ras and YFP-Raf-1 were transiently expressed in COS-1 cells grown in glass coverslips. After serum starvation cells were treated with W-13 for 7 min and/or with EGF for the last 2 min at 37 °C. (A) Images were acquired with a Leica TCS SL laser scanning confocal spectral microscope (CFP channel – left column and YFP channel – central column); FRETc (FRET corrected) was presented as a quantitative pseudocolor image (right column). FRET emission was quantified in different membrane and endomembrane areas from 10–15 cells per condition from two different experiments and normalized by YFP (B). (C) COS-1 cells transiently transfected with CFP-H-Ras (blue channel) and YFP-Raf-1 (green channel) were treated with W-13 for 7 min, fixed and stained with anti-EEA1 followed by anti-mouse secondary antibody labeled with Cy3 (red channel). Images were acquired with inverted epifluorescence microscope Axiovert 200M (Carl Zeiss). Both CFP-H-Ras and YFP-Raf-1 can be observed co-localizing with the early endosomal marker, EEA-1. Bar is 10 μm. (D) Serum-starved COS-1 cells were pre-incubated with W-13 for 5 min and then with EGF for 10 min. Saponin (cytosol) and TX100 (membrane) soluble fractions were obtained and processed by electrophoresis and Western blot analysis to detect total Raf-1 (see Experimental procedures). Histogram shows the membrane/cytosol ratio of the densitometry quantification (n = 5) of Raf-1 signals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Raf-1 is regulated at different levels. A first step towards its activation is the binding to Ras-GTP through its Ras-binding domain and the subsequent Raf-1 translocation/recruitment to plasma membrane. This process releases its auto-inhibitory conformation and allows access of the kinases and phosphatases involved in its activation. First, to study whether the Ras–Raf-1 interaction was affected by W-13, the capability of H-Ras to bind to Raf-1 was analyzed by the FRET (Fluorescence Resonance Energy Transfer) technique. Cyan-H-Ras and Yellow-Raf-1 were transiently co-expressed in COS-1 cells. FRET between CFP and YFP typically requires direct interaction of tagged proteins, and therefore the efficiency of energy transfer between CFP and YFP was measured as described in Experimental procedures. Normalized FRET (FRETN) values were used to compare FRET efficiencies in the selected membrane regions of interest. After stimulation of cells with W-13 and/or EGF, YFP-Raf-1 was recruited and co-localized with CFP-H-Ras in plasma membrane (Fig. 1A). Fig. 1B shows that the mean value of FRETN, calculated from several membrane areas, was significantly higher in EGF-stimulated cells than in control cells. Importantly, W-13 treatment also increased significantly the H-Ras and Raf-1 interaction, and did not interfere with the FRET obtained with EGF. Interestingly, in cells treated with W-13, co-localization and FRET between CFP-HRas and YFP-Raf-1 were also detected in vesicular structures, positive for EEA-1, suggesting that these structures are indeed early endosomes (Fig. 1C). The increased interaction of Ras–Raf-1 in membranes, upon treatment with W-13 was also validated biochemically (Fig. 1D) and the analysis showed slight but significantly increased levels of Raf-1 in the membrane fraction with W-13 compared to control. This result reinforces the previous FRET analysis and correlates with the increased translocation of Raf-1 to membranes through Ras-GTP binding seen when calmodulin is inhibited. These results thus indicate that the inhibition of Raf-1 described in COS-1 cells [18], when calmodulin is inhibited, is not a consequence of an impaired interaction between Raf-1 and H-Ras. Once Raf-1 is bound to Ras-GTP, its activity is regulated by a complex and not completely understood interplay of kinases and phosphatases [19,20]. Phosphorylation at Ser-259 is indicative of an inactive Raf-1 and phosphorylation at Ser-338 stands for an active protein. Fig. 2A shows an increased phosphorylation in the inhibitory Ser-259 residue of Raf-1 in membrane fraction after W-13 treatment, compared to control and EGF-stimulated COS-1 cells. However, when the activity of an ectopically expressed myc-tagged Raf-1 mutant (Ser-259 replaced by Ala) was analyzed by immunoprecipitation, W-13 significantly decreased basal or EGF-stimulated activity of this Raf-1-Ser259Ala mutant (Fig. 2B). This indicates that the inhibitory effect of W-13 is not due to this increased level of phospho-Ser259, which most likely is a consequence of the increased levels of inactive Raf-1 in the membrane fraction (Fig. 1D). Therefore, we next examined whether calmodulin was required for the phosphorylation of Ser338 in Raf-1Ser259Ala mutant. Fig. 2C shows that W-13 inhibited Ser338

Fig. 2. Calmodulin affects Raf-1 phosphorylation state. (A) As explained in Fig. 1 panel D, saponin and TX100 soluble fractions were obtained and processed by electrophoresis and Western blot analysis to detect phosphorylated Ser259 and total Raf-1. One representative Western blot is shown. (B) COS-1 cells expressing myc-Raf-1-S259A were treated with W-13 and/or EGF. Kinase activity of immunoprecipitated myc-Raf-1-S259A was analyzed using radioactive kinase assay (see Experimental procedures). Statistical significances between controls and corresponding W-13 treatments were determined using the Student's t test (*p b 0.05, ***p b 0.001). (C) Phosphorylation of Ser338 site of immunoprecipitated myc-Raf-1-S259A was also tested after W-13 treatment, using anti-phospho-Ser338-Raf-1 antibody. (D) Raf-1 from COS-1 lysates was immunoprecipitated with anti-Raf-1 monoclonal antibody after W-13 and EGF treatment and analyzed by Western blotting with anti-phospho-Ser338-Raf-1 antibody.

phosphorylation in the immunoprecipitated mutant. Also, endogenous Raf-1 was immunoprecipitated from EGF and/or W-13 treated cells and P-Ser338 detected by Western blotting. Fig. 2D shows that W-13 inhibited Ser338 phosphorylation of endogenous Raf-1. 3.2. Calmodulin regulates Raf-1 activity through PI3K Although several calmodulin effectors could mediate the activation of Raf-1 in COS-1 cells, including CaMPKII, PKC

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and PI3K, we found that only inhibition of PI3K had a similar effect to W-13. To elucidate the involvement of PI3K in the downstream effects upon Raf-1 activity, we first analyzed the capacity of calmodulin to interact with PI3K by means of an affinity binding assay using calmodulin-sepharose beads. Fig. 3A shows a

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pull-down experiment where p85 (regulatory subunit of PI3K) and Raf-1 bind to calmodulin in a calcium dependent and EGF independent manner; other studies have also described Raf-1 and PI3K interaction with calmodulin [41,42]. In order to determine the role of calmodulin in PI3K activity, the activation of its effector Akt (PKB) was analyzed by its phosphorylation

Fig. 3. PI3K and calmodulin are involved in the regulation of Raf activity. (A) COS-1 cells lysates of control and EGF-stimulated cells were incubated with sepharose4B-calmodulin in the presence or absence of Ca2+ (EGTA). The amount of PI3K and Raf-1 in the bound fraction was analyzed by Western blotting using anti-p85 and anti-Raf-1. (B, C) COS-1 cells were incubated with W-13 or wortmannin and EGF. Equal amounts of protein from cell lysates were electrophoresed in different polyacrylamide gels and each one was analyzed by Western blotting with anti-phospho-Akt (Ser 473 or Thr308) and anti-Akt (n = 3) (B) or anti-phospho MAPK and anti-MAPK antibodies (n = 10) (C). (C) Raf-1 from COS-1 lysates was immunoprecipitated with anti-Raf-1 monoclonal antibody after W-13, wortmannin and EGF treatment and analyzed by Western blotting with anti-phospho-Ser338-Raf-1 antibody (n = 3). Histograms in panel B and C show the densitometry quantification of each phosphorylation (value averaged ± SD) from different independent experiments as indicated. The statistical differences between controls and corresponding W-13 and wortmannin treatments were determined by Student's t test (*p b 0.05, **p b 0.01, ***p b 0.001). (D) Endogenous Raf-1 kinase activities were measured from lysates of treated cells by immunoprecipitation with anti-Raf-1. The figure shows one representative experiment from triplicate samples. Statistical significances were determined using the Student's t test (*p b 0.05).

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on Ser-473- and Thr-308-Akt in lysates of W-13 or wortmannin (specific P13K inhibitor) treated cells. Statistical analysis of densitometry measurements from 3 independent experiments showed that inhibition of calmodulin, or PI3K, significantly inhibited Akt activation in COS-1 cell (Fig. 3B). As shown in Fig. 3C, wortmannin as well as W-13, significantly inhibited Raf-1-Ser338 phosphorylation and subsequent MAPK activation in COS-1 cells. The same result was obtained with the specific PI3K inhibitor LY249002 (data not shown). Both PI3K and calmodulin are necessary to activate Raf-1, as shown by quantitative activity analysis in Fig. 3D.

The results indicate that PI3K may function downstream of calmodulin to activate Raf-1. Indeed, we showed that both W13 and wortmannin inhibit one of the most well characterized targets of PI3K, the protein kinase Akt (PKB). However, it is not known how calmodulin modulates PI3K or its phospholipid products and effectors. It has been reported that calmodulin antagonists do not significantly inhibit PI3K activity but prevent generation of PI(3,4,5)P3, which eventually affects the activation of Akt [51]. It remains unclear whether Akt is involved and could therefore explain the effects we observed in Raf-1 regulation with W-13 or wortmannin. Therefore, we analyzed

Fig. 4. Calmodulin and PI3K increase H-Ras mediated Raf activation. (A) COS-1 cells transiently expressing GFP-H-RasG12V were starved and treated with W-13 or wortmannin and EGF. Activation of Raf-1 was assayed by immunoprecipitation with anti-GFP antibody followed by Western blotting with anti-phospho-Ser338. (B) Raf-kinase assay was performed for endogenous Raf protein co-immunoprecipitated with H-RasG12V-GFP in COS-1 cells under W-13 and wortmannin treatment. One of two representative experiments (with 3 different samples each) is shown. (C) The same procedure as panel A was performed in COS-1 cells expressing GFP-HRasG12V and p85deltaSH2. (D) COS-1 cells overexpressing GFP-H-RasG12V were treated with W-13 and wortmannin at different doses, first at 10 μg/ml and 5 μM respectively and secondly, at 15 μg/ml and 15 μM (maximal doses). After immunoprecipitation with monoclonal anti-GFP antibody, the effect on phosphorylation of Ser338 site and total amount of co-immunoprecipitated Raf-1 were determined by Western blot. Statistical significances were determined using the Student's t test, n = 3, *p b 0.05, **p b 0.01, ***p b 0.001.

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the P-Ser338-Raf-1 levels in Akt-1 knock-down cells after W13 or wortmannin treatment. We observed an increased basal levels of P-Ser338 in Akt-1-RNAi cells compared to GFPRNAi control cells, without modification of the inhibitory effects of W-13 and wortmannin (data not shown). Hence, Akt

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is probably more involved in the inhibition of Raf-1 through its phosphorylation at Ser259 as described by Zimmermann and Moelling [52]. On the other hand, it has been described that PI3K increases Ras-GTP levels by acting on GEFs [44,45] and that calmodulin

Fig. 5. H-Ras-mediated Raf-1 activation is dependent on endocytosis but not on recycling. (A) COS-1 cells transiently expressing GFP-H-RasG12Vand HA-DynK44A were treated with EGF and W-13 or wortmannin for 15 min. After treatment, GFP-H-RasG12V was immunoprecipitated and the phosphorylation of Raf-1 bound to this GFP-H-RasG12V was visualized by Western blotting with anti-phospho-Raf-1(Ser338). Membranes were also blotted with anti-Raf-1 and anti-GFP. DynK44A expression was confirmed using anti-HA antibody in cell lysates (n = 4). Cells co-expressing GFP-H-RasG12V (green channel) and DynK44A (detected with anti-HA antibody followed by Cy5-secondary antibody, blue channel), showed inhibition of EGF–TRITC (red channel) internalization after 15 min of incubation. Bar is 10 μm. (B) COS-1 transiently expressing GFP-H-RasG12V were pre-incubated for 1 h at 37 °C or at 18 °C and then treated with W-13 or wortmannin for 15 min. After immunoprecipitation with anti-GFP antibody and electrophoresis of the immunoprecipitates, membranes were blotted with anti-phospho-Ser338-Raf-1, anti-Raf-1 and anti-GFP antibodies (n = 3). (C) COS-1 cells were incubated in the presence of W-13 or wortmannin with 1 ng/ml of 125I-EGF for the indicated time to measure internalization. Cells were incubated at 37 °C and 18 °C for 1 h and afterwards incubated in the presence of W-13 or wortmannin with 5 ng/ml of 125I-EGF for 30 min (see Experimental procedures) to determine recycling of EGF at both temperatures (n = 3). The statistical significance of differences was determined using the Student's t test, *p b 0.05, **p b 0.01, ***p b 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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interferes EGFR and PKC-mediated Ras activation [17,18]. In fact, wortmannin, and not W-13, reduced Ras-GTP levels in COS-1 cells, as analyzed by the GST-RBD pull-down assay (data not shown). Then, to further study the role of calmodulin and PI3K in Raf-1 stimulation and to avoid the differential effects of both inhibitors upstream of Raf-1, COS-1 cells transiently expressing H-RasG12V were treated for 15 min with W-13 or wortmannin and for the last 10 min with or without EGF. Neither W-13 nor wortmannin interfered with the association between active H-Ras and Raf-1 shown by coimmunoprecipitation experiments. However, both inhibitors significantly reduced phosphorylation of Raf-1-Ser338 from active H-Ras (Fig. 4A). Moreover, assays of Raf-1 specific activity showed that W-13 or wortmannin treatment significantly inhibited H-RasG12V-mediated Raf-1 activation (Fig. 5B). These results prompted us to further study whether PI3K was actually downstream of calmodulin in the stimulation of Raf-1 from activated H-Ras. Then, a dominant negative mutant of

PI3K, p85deltaSH2, was co-expressed with GFP-H-RasG12V in COS-1 cells and the phospho-Ser338-Raf-1 was analyzed after W-13 or wortmannin treatments. Fig. 4C shows that overexpression of the dominant negative mutant of PI3K almost completely inhibited the effect of W-13 and wortmannin in phosphorylation of Ser338-Raf-1. In addition, when we used W-13 and wortmannin at maximal concentrations, no additive effect was found in the inhibition of phospho-Ser338-Raf-1 activated by H-RasG12V (Fig. 4D). To ascertain whether the inhibitory effect of W-13 and wortmannin on Ser-338-Raf-1 phosphorylation could also be observed in an other cell type, we analyzed the effects of both inhibitors in CHO cells. W-13 and wortmannin inhibited Raf-1-Ser338 phosphorylation and subsequent MAPK activation in CHO cells (Supplemental information, Fig. S1 panels A and B). Likewise, as observed in COS-1 cells, W-13 as well as wortmannin inhibited phosphorylation of Ser473- and Thr308Akt in CHO cells (Supplemental information Fig. S1C).

Fig. 6. The calmodulin modulation of MAPK observed in COS-1 cells depends on the activated H-Ras isoform. (A) COS-1 starved cells were treated with β-methylcyclodextrin (5 mM) for 60 min and subsequently with W-13 for 10 min and EGF for 5 min more. The phosphorylation of MAPK was tested by Western blot from equal amount of protein lysates. (B and C) Small interference RNA was used to specifically inhibit H-Ras expression in COS-1 cells and for GFP expression as a control. The inhibition of H-Ras expression was confirmed by Western blot with antibodies against H-Ras (and K-Ras as a control) after 96 h of siRNA transfection. Cells were treated with W-13 or wortmannin for 15 min and EGF for the last 10 min at 37 °C. Equal amounts of protein were electrophoresed and analyzed by Western blot with anti-phospho MAPK (B) and anti-phospho-Akt (Ser 473) (C). Anti-actin antibody was used as a loading control. The statistical significance of differences was determined using the Student's t test, n = 3, *p b 0.05, **p b 0.01.

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Therefore, these results indicate that calmodulin regulates PI3K activity and the Raf-1/MAPK activation in CHO cells. Moreover, both inhibitors, W-13 and wortmannin, also impaired the Raf-1-Ser338 phosphorylation co-precipitated from ectopically expressed active H-Ras (GFP-H-RasG12V) (Supplemental information Fig. S1D). These results seem to suggest a general mechanism by which calmodulin, through PI3K, regulates Raf-1 activity from activated H-Ras. Therefore, calmodulin and PI3K-mediated regulation of the phosphorylation of Ser-338 residue, one of the most important key phosphorylation sites that control Raf-1 activity [32], was confirmed in this study. Ser338 phosphorylation on endogenous Raf-1 is increased by ligands/signals and this leads to Raf-1 activation. Mutation of S338 to alanine significantly impairs the response to growth factors showing that ligand-mediated activation of Raf-1 requires Ser338 phosphorylation [31]. Moreover, calmodulin is necessary to activate PI3K and this also seems to be linked to the regulation of Raf-1 activity. Here we show that both W-13 and wortmannin impair the Ser338 phosphorylation of Raf-1, which is required to fully activate this kinase. Indeed, it has also been reported that PI3K through activation of PAK proteins is involved in Ser338 phosphorylation and stimulation of Raf-1 [33,40]. The results reinforce the role of calmodulin, through PI3K, in such phosphorylation/ activation of Raf-1 from activated H-Ras.

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30 min. Recycling is highly sensitive to temperature and this characteristic has been extensively used in different studies. Fig. 5C shows that at 18 °C incubation, recycling of 125I-EGF is severely impaired and for this reason it cannot be further inhibited with wortmannin or W-13. Under these conditions the phosphorylation of Ser338-Raf-1 is still inhibited by W-13 and wortmannin (Fig. 5B). Thus, endosomal recycling does not appear to be a critical step in the H-Ras-mediated activation of Raf-1 modulated by calmodulin. Moreover, since PKCδ is responsible for recycling inhibition produced by W-13 [54], we treated COS-1 cells with the specific PKCδ inhibitor, rottlerin. Under inhibition of PKCδ, W-13 or wortmannin still inhibited Raf-1-Ser338 phosphorylation (data not shown). Therefore, the most plausible explanation from the above results, given that the interaction between H-Ras and Raf-1 is maintained in early endosome membranes in W-13 treated cells (Fig. 1B and C), is that Raf-1 activation by H-Ras occurs in the early endosomal compartment. Interestingly, we observed that neither W-13 [18] nor wortmannin interfere with internalization of the EGFR, one of the most well characterized molecules endocytosed via clathrin.

3.3. H-Ras-mediated Raf-1 activation is dependent on endocytosis but not on recycling Since it has been postulated that H-Ras signaling through the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling [35] and given that calmodulin and PI3K regulate endocytic trafficking, next we analyze these correlation on the Raf-1 inhibition by W-13 or wortmannin. When dynamindependent endocytosis was impaired in COS-1 cells by overexpression of the dominant negative mutant DynK44A, as shown by the inhibition of EGF–TRITC internalization (Fig. 5A), Raf-1 was not properly phosphorylated at Ser-338 (Fig. 5A), and the inhibitory effect of W-13 and wortmannin was thus clearly reduced. The internalization of H-Ras/Raf-1 is necessary to complete Raf-1 activation, most probably in endosomes. In fact, Pol et al. identified Ras, Raf-1, Mek and MAPK in endosomes purified from EGF-stimulated rat liver [53] and, in this compartment, Raf-1 showed a shift in its electrophoretic mobility as an indication of its phosphorylated and activated state. Furthermore, in COS-1 cells, we observed that W-13 or wortmannin do not modify 125I-EGF internalization (Fig. 5C), and also that both are necessary for the endocytic recycling process (Fig. 5C). Indeed, W-13 inhibited recycling from early endosomes but did not affect the internalization of 125I-EGF, producing an accumulation of EGF–EGFR in morphologically enlarged endosomes [18]. To elucidate whether inhibition of recycling is involved in W-13 and wortmannin decreased H-Ras-dependent Raf-1 activation, COS-1 cells transiently expressing H-RasG12V were incubated at 18 °C and treated with W-13 or wortmannin for

Fig. 7. Model for the regulation of calmodulin-mediated effects on the Ras/ MAPK signaling pathway. At the plasma membrane: (1) Calmodulin modulates the shedding of autocrine/paracrine growth factors, as HB-EGF, elicited though PKC/matrix-metalloproteinases stimulation [17,18]. (2) Calmodulin also binds and modulates EGFR activation [11,12] and through CaMKII, phosphorylates EGFR and inhibits its intrinsic tyrosine-kinase activity [56]. Therefore, inhibition of calmodulin produces activation of EGFR and downstream Ras activation and consequently Raf-1 translocation/recruitment to plasma membrane. At the endocytic compartment: Functional internalization is required to ensure Raf-1 activation by H-Ras. (3) In this study, we demonstrated that calmodulin, through PI3K, is necessary to stimulate Raf-1 from activated H-Ras. The regulation by calmodulin of Raf-1 activation via H-Ras has a signal output through the MAPK cascade. CaM, calmodulin; Raf-1⁎, activated Raf-1.

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However, both drugs affected EGFR recycling from endosomes to plasma membrane. Although inhibition of recycling with a Rab5-Q79L mutant expression has been reported to impair recruitment to membranes and activation of Raf-1 [35], we found that W-13 and wortmannin still inhibit Raf-1 when recycling process is inhibited at 18 °C, and that Raf-1 recruitment to the membrane fraction is not reduced, but rather increased with W-13. Our results suggest that calmodulin, through PI3K, regulates the phosphorylation and activity of Raf-1 stimulated by H-Ras in the endosome compartment. 3.4. Calmodulin modulation of H-Ras activated Raf-1 affects the final MAPK activation in COS-1 cells To examine the role of calmodulin/PI3K on H-Ras in the final MAPK activation two experimental approaches were taken. First, we used β-methyl-cyclodextrin (CD) to inactivate H-Ras in COS-1 cell; it has been shown that cholesterol depletion caused by CD affects H-Ras signaling [55]. Cells were incubated with CD (5 mM) for 60 min and then stimulated for 10 min with EGF in the presence of W-13. MAPK activation was analyzed from an equal protein amount of lysates by Western blotting with anti-phospho-MAPK specific antibody. After CD treatment, EGF had a reduced stimulation and W-13 lost its inhibitory effect on MAPK in COS-1 cells (Fig. 6A). Thus, H-Ras might be involved in the inhibitory effect on MAPK observed after W-13 treatment in COS-1 cells. Secondly and more precisely, we used specific siRNA to knock-down H-Ras protein expression in COS-1 cells, and examined the W-13 effect on MAPK. Fig. 6B shows that siRNA effectively knock-down H-Ras protein expression without modifying their counterpart K-Ras. COS-1 cells with H-Ras knock-down, or transfected with GFP-siRNA as a control, were treated with W-13 and the lysates analyzed for P-MAPK. As before, W-13 produced an inhibitory effect on MAPK in GFPsiRNA transfected cells. However, this effect could not be reproduced when H-Ras was knock-down with H-Ras-siRNA (Fig. 6B). Fig. 6C shows also that under these conditions EGF activates Akt phosphorylation while this is impaired with W-13, indicating that PI3K pathway was almost unaffected when HRas is knocked-down. In summary, when the signaling through H-Ras is reduced by membrane depletion of cholesterol with CD or by knocking down the H-Ras expression with siRNA, calmodulin is unable to stimulate MAPK in COS-1 cells. Finally, as depicted in Fig. 7, calmodulin can modulate the Ras/MAPK signalling at different levels. We propose that calmodulin regulation of the Raf-1/Mek/MAPK cascade depends on PI3K activity and endocytosis of the H-Ras involved in Raf-1 activation. Acknowledgements We thank Drs. Richard Marais, Antonio Chiloeches and Julian Downward for donating plasmids. This study was supported by grants (BFU2006-01151 and GEN2003-20662C07-01) to C.E., (BMC2003-09496 and BFU2006-15474) to F.T., (BFU2005-01716 and GEN2003-20662) to A. P. and by

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