Differential involvement of H- and K-Ras in Raf-1 activation determines the role of calmodulin in MAPK signaling

Cellular Signalling 21 (2009) 1827–1836

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Differential involvement of H- and K-Ras in Raf-1 activation determines the role of calmodulin in MAPK signaling Jemina Moretó a, Maite Vidal-Quadras a, Albert Pol a,b, Eugenio Santos c, Thomas Grewal d, Carlos Enrich a, Francesc Tebar a,⁎ a Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain b Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010-Barcelona, Spain c Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-University of Salamanca, Campus Unamuno, E-37007 Salamanca, Spain d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia

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Article history: Received 26 May 2009 Received in revised form 27 July 2009 Accepted 29 July 2009 Available online 8 August 2009 Keywords: Calmodulin K-Ras H-Ras Raf-1 MAPK EGFR Signaling

a b s t r a c t We have previously demonstrated that inhibition of calmodulin (CaM) and the concomitant reduction of PI3K interfere with H-Ras-mediated activation of Raf-1 [1]. In the present study, we show that CaM has completely opposite effects on K-Ras-mediated Raf-1 activation. The differential contribution of CaM in the regulation of Raf-1 kinase activity via K- or H-Ras correlates with the stimulatory or inhibitory effect of CaM on MAPK phosphorylation depending on the cell type analyzed. FRET microscopy and biochemical analysis show that inhibition of CaM increases K-Ras-GTP levels and consequently its association with Raf-1. Though inhibition of CaM, using the CaM antagonist W-13, significantly increased Raf-1 activation by K-Ras-GTP, MAPK activation downstream K-Ras/ Raf-1 was strongly reduced in COS-1 and several other cell lines. In contrast, in other cell lines such as NIH3T3-wt8, W-13-mediated inhibition of CaM increased Raf-1 activity, but resulted in an increase in MAPK phosphorylation. These findings suggest that modulation of K-Ras activity via CaM regulates MAPK signaling only in certain cell types. In support of this hypothesis, the comparison of H- and K-Ras expression, GTP loading and Raf-1 interaction in COS-1 and NIH3T3-wt8 suggests that the overall role of CaM in MAPK signal output is determined by the ratio of activated H- and K-Ras and the cell-specific contribution of each isoform in Raf-1 activation. © 2009 Elsevier Inc. All rights reserved.

1. Introduction The Ras/Raf/Mek/ERK pathway regulates fundamental processes such as proliferation, differentiation, migration and apoptosis. Receptor tyrosine kinases induce activation of Ras GTPases which transmit extracellular signals to Raf kinases, a family of serine/threonine kinases that comprises three members, A-Raf, B-Raf and Raf-1. Raf-1 is the most extensively studied Raf kinase and plays a central role in the EGF receptor (EGFR) mediated activation of the MAPK pathway. However, Raf-1 activity is regulated by an interplay of complex and still incompletely understood mechanisms. Raf-1 is recruited to the plasma membrane through binding to activated Ras proteins (Ras-GTP). Once

Abbreviations: CaM, Calmodulin; CD, β-Methyl-cyclodextrin; CFP, Cyan fluorescent protein; EGF, Epidermal growth factor; FRET, Fluorescence Resonance Energy Transfer; MAPK, Mitogen-activated protein kinase; Mek, MAPK/ERK kinase; PI3K, Phosphoinositide 3-kinase; RBD, Ras-binding domain of Raf-1; W-13, N-(4-aminobutyl)-5-chloro-2naphthalenesulfonamide; YFP, Yellow fluorescent protein. ⁎ Corresponding author. Tel.: +34 2275400x3337; fax: +34 934021907. E-mail address: [email protected] (F. Tebar). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.07.018

translocated to the plasma membrane, Raf-1 is activated by sequential dephosphorylation and phosphorylation events. Several phosphatases, kinases and scaffold proteins have now been identified to finely coordinate and guarantee the proper spatial–temporal activation of Raf-1 and subsequently Mek and ERK [2]. Upon membrane recruitment of Raf-1 through binding to Ras-GTP, phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) dephosphorylate phopho-Ser259 residue of Raf-1 and overall favor the release of its autoinhibitory conformation. This step facilitates the subsequent phosphorylation of Tyr341 and the key activating Ser338 residue [3–5]. In particular the phosphorylation of Ser338 strongly correlates with the activated state of Raf-1 and several kinases including, p21-activated kinases (PAKs) and casein kinase 2 (CK2), have been described to phosphorylate Ser338 [5–8]. However there are still some caveats about the physiological significance and involvement of these kinases in Raf-1 regulation in vivo. Besides the phoshorylation events listed above, an additional key phosphorylation occurs at Ser471 to promote Mek binding. Furthermore Thr491 and Ser494, two residues in the kinase domain activation loop [9] and several other Ser residues (Pos. 29, 43, 289, 296, 301 and 642) appear to act as inhibitory ERK feedback phosphorylation sites [10].

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Given that most cells express several Ras and Raf isoforms, Rasmediated activation of Raf kinases provides a platform to create signal diversity and differential signaling. Mammalian cells express four highly related proteins of either 188 or 189 amino acids: H-Ras, N-Ras, K4A-Ras and K4B-Ras (hereafter K-Ras) [11] and numerous studies have addressed the potential of Ras isoforms to differentially regulate downstream effectors. Indeed, despite the high degree of homology among the Ras isoforms, differences at their C-terminal domain result in differential subcellular localization and consequently, differential signaling [12–14]. Along these lines, several studies have addressed the role of Ras isoforms in Raf-1 activation. In fact, numerous studies have shown that all Ras isoforms generate quantitatively different signal outputs through their effectors: Raf, PI3-K, Cdc42 and Rac, which in turn results in qualitative or quantitative distinct biological responses [14–17]. For instance, Roy et al. demonstrated that activation of Raf-1 by H-Ras, but not K-Ras, requires endocytosis and PI3K activity [18]. Lately, inhibition of EGFR endocytosis has been shown to reduce N- and H-Ras activation, whereas K-Ras was unaffected [19]. CaM is a ubiquitiously expressed calcium sensor, and we and others showed that CaM acts on the EGFR-mediated activation of the Ras/MAPK signaling pathway at different levels [20–22]. CaM not only binds and modulates EGFR activation [23,24] but also interacts with K-Ras to control K-Ras localization and its activation via PKC [25–29]. We previously showed that the role of CaM in the regulation of the EGFR/Ras/Raf/MAPK pathway is extremely complex, as inhibition of CaM in COS-1 cells increased EGFR phosphorylation and Ras activity, but simultaneously reduced and interfered with Raf-1-mediated MAPK activation [22,30]. Most relevant to the studies presented here, we demonstrated that inhibition of CaM, in a PI3K- and endocytosisdependent manner, interfered with H-Ras-induced activation of Raf-1 [1]. In the present study, we examined the role of CaM in K-Rasmediated Raf-1 activation. Intriguingly, opposite from the previously described requirement of CaM for H-Ras dependent Raf-1 activation [1], we demonstrate that inhibition of CaM increases GTP loading of K-Ras and Raf-1 activation. We propose that the differential effects of CaM on Raf-1 activity observed in various cell lines determine MAPK signal output and can be explained by the expression levels, ratio as well as cell-specific contribution of active H-Ras and K-Ras in Raf-1 activation.

cells with stably overexpressing EGF receptor (NIH3T3-wt8; kindly provided by Dr. Alexander Sorkin, University of Colorado, Denver, CO, USA) and mouse embryo fibroblasts (MEFs) from knockout mice of K-Ras (K-Ras−/−; kindly provided by Dr. Mariano Barbacid, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain) or doble knockout of H- and N-Ras (H-Ras−/−, N-Ras−/−). 2.3. Plasmid and siRNA transfection H-Ras and K-Ras 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 N-terminus of the full length proteins. GFP-H-RasG12V and GFP-K-RasG12V were transfected with Effectene (QIAGEN, Valencia, CA) in p-100 dishes of COS-1 or NIH3T3wt8 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 were siRNA1 (5′-CCUUCUACAC GUUGGUGCGUGAGAU-3′) and siRNA2 (5′-AUCUCACGCACCAACGUGUAGAAGG-3′). The siRNA sequences for targeting K-Ras were siRNA1 (5′-ACUCCUACAGGAAACAAGUAGUAAU-3′) and siRNA2 (5′AUUACUACUUGUUUCCUGUAGGAG-3′). After resuspending both duplex RNAs (20 µM solution), 6 µl of this was transfected in p-60 plates of COS-1 or NIH3T3-Wt8 cells (40–50% confluence) with 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 lyses were conducted 72 h after transfection. 2.4. Cellular extracts After the various treatments (as indicated in figure legends), 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 [31] were electrophoresed and immunoblotted. 2.5. Fluorescence Resonance Energy Transfer (FRET) microscopy, sensitized emission method

2. Experimental procedures 2.1. Reagents Mouse receptor-grade EGF, AG1478 and W-13 were from Sigma Chemical Co. (Madrid, Spain). Wortmannin (KY 12420) was from Calbiochem (Merck KGaA, Darmstadt, Germany). Monoclonal antibodies to Raf-1 are from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against phosphorylated MAPK, phosphorylated Mek, Mek, or MAPK were from Cell Signaling, New England Biolabs (Beverly, MA). The monoclonal antibody against phospho-Raf-1 (Ser338) was from Upstate (New York, USA). Monoclonal anti-actin antibody was from ICN. Polyclonal anti-GFP antibody was from Abcam (Cambridge, UK). Polyclonal H-Ras (C-20) and 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) were grown in DMEM containing 10% fetal calf serum (FCS), pyruvic acid, antibiotics and glutamine. DMEM and FCS were purchased from Biological Industries (Beit Haemek, Israel). Cells were grown to 90% confluence for immunoprecipitation, pulldown or radioactivity experiments, and 60% confluence for FRET microscopy experiments. We also used murine embryo NIH3T3

FRET analysis was based on the sensitized emission method previously described [32,33] 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 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-bypixel-basis for the entire image by using the equation: c

FRET = FRET
ðA × CFPÞ
ðB × YFPÞ;

ð1Þ

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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: c

FRETN = FRET = YFP

ð2Þ

where FRETc and YFP are the mean intensities of FRETc and YFP fluorescence in the selected subregion of the image. All calculations were performed using the FRET sensitized emission wizard from Leica Confocal Software and Microsoft Excel. 2.6. Immunoprecipitation Starved COS-1 and NIH3T3-wt8 cells expressing GFP-H-RasG12V or GFP-K-RasG12V 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, and 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 [PMSF], 10 mg/ml leupeptin, and 10 mg/ml aprotinin) followed by gentle rotation for 10 min at 4 °C.

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Lysates were then centrifuged at 14,000 ×g for 10 min at 4 °C. Supernatants were incubated 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). SDS-polyacrylamide gels, 8%, were used to separate proteins of the immunoprecipitates, previously solubilized with Laemmli loading buffer. Proteins were then transferred to Immobilon-P (Millipore, Bedford, MA) and immunoblotted using anti-phospho-Raf-1 (Ser338) and anti-Raf-1 followed by the appropriate peroxidaseconjugated secondary antibody and ECL detection (Amersham Pharmacia Biotechnology, Buckinghamshire, UK).

2.7. Raf-1 kinase activity assays To measure Raf-1 activity in COS-1 cells, kinase assays were performed after immunoprecipitation, as previously described [34]. Endogenous Raf-1 was co-immunoprecipitated in cells transiently expressing GFP-H- or GFP-K-RasG12V using anti-GFP polyclonal antibody. Treated cells were harvested on ice in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1% [vol/vol] Triton X-100, 5 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM aprotinin, and 20 µM leupeptin) and clarified by centrifugation at 10,000 ×g. Supernatants (equalized for protein concentration) were immunoprecipitated for 2 h at 4 °C with 2 µg of the antibodies, precoupled with 30 µl Protein A-sepharose. Immunoprecipitates were then washed three times in buffer (30 mM Tris, 0.1 mM EDTA, 0.3% mercaptoethanol, 10% glycerol, 0.1% [vol/vol] Triton X-100, 5 mM NaF, and 0.2 mM Na3VO4) with decreasing amounts of NaCl. Washed immunoprecipitates were incubated for 30 min at 30 °C in 20 µl of MEK

Fig. 1. CaM inhibition increases K-Ras-GTP levels and K-Ras interaction with Raf-1 at the plasma membrane. (A) Starved COS-1 cells were treated with W-13 (15 μg/ml) for 7 min and/or with EGF (50 ng/ml) for the last 5 min at 37 °C. Lysates were subjected to RBD pulldowns and the amounts of activated K-Ras (K-Ras-GTP) and H-Ras (H-Ras-GTP) were determined using specific antibodies. Actin expression in each lysate is shown. Histogram shows the densitometry quantification (n=3) of K-Ras-GTP signals. Statistical significance between controls and corresponding W-13 treatments was determined using the Student's t test, ⁎⁎P b 0.01. (B) CFP-K-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. 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). Bar is 10 μm. (C) FRET emission was quantified in different plasma membrane areas from 10–15 cells per condition. Mean values (± S.D.) from 2 independent experiments normalized to YFP are shown. Statistical significance between different treatments and the control was determined using the Student's t test, ⁎P b 0.05, ⁎⁎P b 0.01.

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buffer (30 mM Tris, 0.1 mM EDTA, 0.3% mercaptoethanol, 10 mM MgCl2, 0.1% TX100, 5 mM NaF, 0.2 mM Na3VO4, 0.8 mM ATP, 6.5 µg/ml GSTMEK, and 100 µg/ml GST-ERK2), and the reaction was terminated by the addition of 20 µl of ice-cold stop buffer (30 mM Tris, 6 mM EDTA, 0.3% mercaptoethanol, 0.1% TX100, 5 mM NaF, and 0.2 mM Na3VO4). After

centrifugation, 6-µl aliquots of supernatants were incubated for 15 min at 30 °C with 24 µl of MBP buffer (50 mM Tris, 0.1 mM EDTA, 0.3% mercaptoethanol, 10 mM MgCl2, 0.1% [vol/vol] Triton X-100, 5 mM NaF, 0.2 mM Na3VO4, 0.1 mM ATP, 2.5 µl 32P-ATP, 0.5 µg/µl MBP, and 0.16 µg/ µl BSA); 24 µl was then loaded on P81 sheets, washed three times

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(20 min each) in 75 mM orthophosphoric acid, and counted for incorporation.

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P

2.8. Measurement of Ras activation The capacity of Ras-GTP to bind to RBD (Ras-binding domain of Raf-1) was used to analyze the amount of active Ras [35]. After treatments, cells were lysed in the culture dish with lysis buffer (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 5 mM MgCl 2, 1% [vol/ vol] Triton X-100, 5 mM NaF, 10% [vol/vol] glycerol, and 0.5% [vol/vol] 2-mercaptoethanol) plus protease and phosphatase inhibitors. Cleared lysates (10,000 ×g) were assayed for protein concentration by the Bradford method and protein-equalized supernatants were incubated for 2 h at 4 °C with glutathione sepharose-4B beads precoupled to GST-RBD. Beads were washed three times in the lysis buffer. Bound proteins were electrophoresed on 12% SDS-PAGE gels. The amount of K-Ras or H-Ras in the bound fraction was analyzed by Western blotting using specific antibodies. 3. Results Previously, we demonstrated that inhibition of CaM leads to tyrosine phosphorylation and activation of EGFR [22], which correlated with increased levels of total Ras-GTP [22,30]. However, we recently identified that CaM, through PI3K, is required for H-Ras-mediated activation of Raf-1 [1]. In this study, using the highly specific inhibitor of CaM, W-13, we analyzed the role of CaM on Raf-1 activation by K-Ras. Firstly, we examined the effect of W-13 on K-Ras-GTP levels in COS-1 cells. Starved COS-1 were treated with W-13 for 7 min and the last 5 min EGF (100 ng/ml) was added. Similar to the previously described stimulation of Total Ras-GTP upon inhibition of CaM [22,30], W-13 with or without EGF increased K-Ras-GTP and H-Ras-GTP levels in COS-1 cells (Fig. 1A). Next, to examine if elevated K-Ras-GTP levels upon W-13 incubation could result in an increased interaction with the downstream effector Raf-1, the capability of K-Ras to bind Raf-1 in the presence of W-13, EGF or both was analyzed by FRET (Fluorescence Resonance Energy Transfer) microscopy. COS-1 cells were transiently co-transfected with CFP-tagged K-Ras (CFP-K-Ras) and YFP-tagged Raf1 (YFP-Raf-1) and treated ± EGF, W-13 or both. CFP, YFP and FRET measurements were based on the sensitized emission method [1]. After background subtraction, correction for spectral bleed through and cross excitation, normalized FRET (FRETN) values were used to compare FRET efficiencies in selected plasma membrane regions. Very low background FRET signals were detected in untreated cells (Fig. 1B, Control) supporting inactive CFP-K-Ras at the plasma membrane not interacting with predominantly cytosolic YFP-Raf-1. In contrast, after treatment with W-13, EGF or both, YFP-Raf-1 was recruited and co-localized with CFP-K-Ras on plasma membrane (Fig. 1B), indicating CFP-K-Ras activation and interaction with YFP-Raf-1. Consequently, the mean FRET efficiency was significantly higher in EGF-stimulated cells compared to control cells (Fig. 1C). Moreover, W-13 treatment also significantly increased CFP-K-Ras/YFP-Raf-1 interaction. Next we addressed if W-13-mediated effects upstream of Ras, for example at the level of EGFR, rather than W-13-induced K-Ras

Fig. 3. Inhibition of CaM with W-13 has opposite effects on MAPK activation in COS-1 and NIH3T3-wt8 cells. Serum starved COS-1 (A, B) and NIH3T3-wt8 (C, D) cells grown in 100-mm dishes were incubated with W-13 (15 µg/ml) for 15 min and/or with EGF (100 ng/ml) for the last 10 min at 37 °C. Lysates with equal amount of protein were electrophoresed and phosphorylated and total levels of Mek (A, C) and MAPK (B, D) were analyzed by Western blotting. In both cell lines, phosphorylation levels of Mek and MAPK were quantified by densitometry from 3 and 5 different experiments respectively, and mean values were expressed as relative phosphorylation levels. Statistical significance between controls and corresponding W-13 treatments was determined using the Student's t test. ⁎P b 0.05, ⁎⁎P b 0.01.

activation could increase K-Ras/Raf-1 interaction. Therefore COS-1 cells were co-transfected with GFP-K-RasG12V and CFP-Raf-1, treated with W-13, EGF or both and FRET efficiency was determined as described above (Fig. 2A). Neither EGF nor inhibition of CaM appeared to modify the association between active CFP-K-RasG12V and YFPRaf-1 as FRET efficiency remained comparable under all conditions tested (Fig. 2A). Previously we demonstrated that W-13 does not interfere with the association of active H-Ras with Raf-1 [1]. However, activity and

Fig. 2. Inhibition of CaM increases EGFR-dependent and K-RasG12V-mediated activation of Raf-1. (A) COS-1 cells were co-transfected with CFP-Raf-1 and YFP-K-RasG12V and treated with EGF, W-13 and both W-13 and EGF as described (Fig. 1B). Cells were fixed and FRET analysis was performed (for details see Experimental procedures and Fig. 1B). Images show the CFP channel (left column), YFP channel (central column) and the quantitative pseudocolor of FRETc (right column). FRET emission was quantified in different plasma membrane areas from 10– 15 cells per condition. The results (Mean ± S.D.) from two independent experiments normalized to YFP are shown. Bar is 10 μm. (B) Starved COS-1 cells expressing GFP-K-RasG12V or GFPH-RasG12V were treated with W-13 or wortmannin (1 μM) for 15 min at 37°C. The kinase activities of endogenous Raf-1 co-immunoprecipitated with anti-GFP polyclonal antibody were determined in radioactive kinase assay (see Experimental procedures). The histogram shows the mean values (± S.D.) of one representative experiment (n = 2) with triplicate samples. Statistical significance between controls and W-13 or wortmannin treated samples was determined using the Student's t test (⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001). (C) COS-1 cells expressing GFP-K-RasG12V were pre-incubated ± the EGFR-Tyr-kinase inhibitor AG1478 (5 µM) for 10 min, and then treated with W-13 (15 μg/ml) for 7 min and/or with EGF (100 ng/ml) for the last 5 min at 37 °C. After immunoprecipitation with monoclonal anti-GFP antibody, the immunoprecipitated GFP-K-RasG12V, the phosphorylation of Raf-1-Ser338 and total amount of coimmunoprecipitated Raf-1 were determined by Western blotting. Results were quantified and the mean values (± S.D.) from 3 independent experiments are shown. Statistical significance between different treatments and the control was determined using the Student's t test, ⁎P b 0.05, ⁎⁎P b 0.01.

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Ser338 phosphorylation of Raf-1 associated with H-RasG12V expressed in COS-1 cells were significantly inhibited in the presence of W-13 [1]. In order to analyze if inhibition of CaM would exert similar effects on Raf-1 interacting with active K-Ras, we determined

Raf-1 kinase activity (Fig. 2B) and Ser338 phosphorylation (Fig. 2C) in GFP-K-RasG12V transfected cells. Interestingly, and completely opposite to the results obtained with GFP-H-RasG12V ([1] and Fig. 2B) W-13 significantly enhanced Raf-1 activity by GFP-K-

Fig.4. CaM interfering MAPK activation in NIH3T3-wt8 cells depends on K-Ras isoform. (A) Starved NIH3T3-wt8 cells were treated with β-methyl-cyclodextrin (CD, 5 mM) for 60 min and subsequently with W-13 for 10 min and EGF for additional 5 min. The phosphorylation of MAPK (P-ERK) was determined by Western blotting from equal amount of protein lysates. Densitometry quantification (n = 3; mean ± S.D.) showed that CD did not affect W-13- or EGF-induced P-ERK activation. (B, C) Small interference RNA was used to specifically inhibit K-Ras expression in COS-1 cells (B) and in NIH3T3-wt8 cells (C). The siRNA for GFP was used as a control. The inhibition of K-Ras expression was confirmed by Western blot with a specific antibody after 96 h of siRNA transfection. One of three representative experiments of each cell line is shown. Statistical significances between controls and corresponding K-Ras siRNA were determined using the Student's t test. ⁎P b 0.05. Cells were treated with W-13 for 10 min and EGF for the last 5 min at 37 °C. Equal amounts of protein were electrophoresed and analyzed by Western blotting with anti-phospho MAPK. Anti-actin antibody was used as a loading control. Histogram shows the effect of W-13 on P-MAPK determined by densitometry quantification (n = 3). Statistical significance was determined using the Student's t test. ⁎P b 0.05. (D) NIH3T3-wt8 cells transiently expressing GFP-H-RasG12V were starved and treated with W-13 and/or EGF. Levels of GFP-H-RasG12V, phosphorylated Ser338-Raf-1 and total Raf-1 from cell lysates were determined by Western blotting. Activation of Raf-1 was assayed by immunoprecipitation with anti-GFP antibody followed by Western blotting with anti-phospho-Ser338. One of three representative experiments is shown. Statistical significances between controls and corresponding W-13 treatments were determined using the Student's t test. ⁎P b 0.05, ⁎⁎P b 0.01. Cells expressing GFP alone was used as a control for specific immunoprecipitation. Raf-1 was not co-immunoprecipitated using anti-GFP antibody.

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RasG12V (Fig. 2B). Furthermore, while PI3K is involved in H-Rasmediated Raf-1 activation [1], pharmacological inhibition of PI3K with wortmannin did not modify GFP-K-RasG12V induced Raf-1 kinase activity (Fig. 2B). To ascertain whether W-13-activation of Raf-1 by GFP-K-Ras involves EGFR regulation by CaM, GFP-K-RasG12V expressing cells were pre-incubated with the specific EGFR-tyrosine kinase inhibitor AG1478 and Ser338 phosphorylation of Raf-1 was analyzed (Fig. 2C). In contrast to the non-treated controls, W-13 and/or EGF did not further increase Ser338 phosphorylation of Raf-1, suggesting a potential CaM-dependent involvement of EGFR in K-RasG12V mediated Raf-1 activation. Hence these results suggest that CaM exerts opposite effects on the downstream signaling potential of constitutively active H- and K-Ras isoforms. In contrast to results obtained for H-RasG12V, the K-RasG12V-dependent stimulatory effect of W-13 on Raf-1 kinase activity and Ser338 phosphorylation does not involve PI3K, but is possibly linked to the interference of EGFR activation by CaM [22]. In summary, whereas CaM appears necessary to guarantee Raf-1 activation through H-Ras, the results shown above strongly implicate that CaM is dispensable for K-Rasmediated Raf-1 activation. The opposite effects of W-13 on H-Ras and K-Ras-mediated Raf-1 activation, prompted us to analyze whether this could be connected to the well-known but different roles of CaM on MAPK signal output depending on the cell type analyzed. Therefore we treated a variety of cell lines with W-13 and first analyzed the activation of the MAPK signaling pathway (Fig. 3). Consistent with previous findings in COS-1 cells [22], W-13-induced stimulation of EGFR (data not shown) did not correlate with the phosphorylation of Mek and ERK. The amount of basal or EGF-stimulated P-Mek and P-MAPK was significantly reduced in the presence of W-13 (Fig. 3A and B, respectively). Interestingly, the inhibitory effect of W-13 on Mek and ERK activation was not observed in NIH3T3-wt8 cells (EGFR overexpressing mouse NIH3T3 cell line; ~4 × 105 EGFR/cell). In these cells, the inhibition of CaM rather increased the level of activated Mek and MAPK (Fig. 3C and D, respectively). As observed by others, this differential effect of W-13 was also observed in other cell lines, W-13 exerted an inhibitory effect on MAPK activation in COS-1, CHO, NR6, HEK293 cells while stimulatory in NIH3T3, NIH3T3-wt8, A431 and NRK cells (data not shown). It was tempting to speculate that the opposite effect of W-13 on MAPK activation may be related to the differential involvement and contribution of H- or K-Ras isoforms in overall MAPK signal output in the various cell lines. To further compare the opposed outcome of CaM inhibition on MAPK activation in the representative COS-1 and NIH3T3-wt8 cell models, two different experimental approaches were undertaken. First, and as shown previously [1,36,37], β-methylcyclodextrin (CD) was used to inactivate H-Ras in both COS-1 and NIH3T3-wt8 cells. While CD treatment reduced EGF-mediated MAPK stimulation and abolished the inhibitory effect of W-13 on ERK phosphorylation in COS-1 cells [1], CD treatment did not modify EGF and/or W-13-induced activation of MAPK in NIH3T3-wt8 cells (Fig. 4A). Thus, H-Ras appears to be involved in the inhibitory effect on MAPK activation after W-13 treatment in COS-1, but not in the stimulatory effect of W-13 on ERK phosphorylation in NIH3T3-wt8 cells. Besides, we examined the involvement of K-Ras in W-13 effects on MAPK activation in RNAi knockdown experiments. Fig. 4B and C shows that siRNA oligonucleotides specifically targeting K-Ras effectively reduced K-Ras protein expression without modifying H-Ras levels in COS-1 and NIH3T3-wt8 cells. K-Ras depleted COS-1 and NIH3T3-wt8 cells and GFP-siRNA transfected controls were treated with W-13 and the lysates were analyzed for P-ERK levels. Similar to Fig. 3B and previous data [1], W-13 inhibited MAPK activation not only in GFPsiRNA transfected controls, but also upon knockdown of K-Ras in COS-1 cells (Fig. 4B). In contrast in NIH3T3-wt8 cells depleted of K-Ras, the stimulatory effect of W-13 on EGF-mediated MAPK stimulation was

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diminished and rather an inhibitory effect was observed (Fig. 4C). This last result may indicate that in the absence of K-Ras, H-Ras becomes more relevant to activate MAPK and consequently CaM would then also be required to ensure H-Ras-mediated activation of the MAPK pathway in NIH3T3-wt8 cells. To examine whether CaM is able to regulate Raf-1 activation by H-Ras in NIH3T3-wt8 as in a similar fashion to COS-1 cells, GFP-H-RasG12V was transiently expressed in NIH3T3-wt8 cells and Ser338 phosphorylation of Raf-1 was analyzed in H-RasG12V immunoprecipitates. Indeed, in NIH3T3-wt8 W-13 significantly inhibited HRasG12V-induced Ser338 phosphorylation of Raf-1 in both basal and

Fig. 5. The amounts of total and activated H- and K-Ras are different in COS-1 and NIH3T3-wt8 cells. (A) Equal amounts of protein from COS-1 and NIH3T3-wt8 cells were electrophoresed and analyzed by Western blotting using anti-H-Ras, anti K-Ras and anti-pan-Ras. The levels of H- and K-Ras were determined by densitometry and were expressed in relation to Pan-Ras levels. The bar diagrams show the relative amount of H- and K-Ras in every cell line. (B) Lysates of EGF-stimulated COS-1 and NIH3T3-wt8 cells were subjected to RBD pulldown and the amounts of activated H- and K-Ras (HRas-GTP and K-Ras-GTP) were determined using H- and K-Ras specific antibodies. Histogram shows the ratio of activated H-Ras versus activated K-Ras determined by densitometry quantification (n = 3). (C) CFP-K-Ras or CFP-H-Ras and YFP-Raf-1 were transiently expressed in COS-1 and NIH3T3-wt8 cells grown in glass coverslips. After serum starvation cells were treated with EGF for 2 min at 37 °C. FRET emission was quantified in different plasma membrane areas from 10–15 cells per condition. Mean values (± S.D.) from 2 independent experiments normalized to YFP are shown. Statistical significance was determined using the Student's t test. ⁎⁎P b 0.01.

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EGF-stimulated conditions (Fig. 4D). In further support of these findings, W-13 did not impact on EGF-stimulated-MAPK phosphorylation upon H-Ras knockdown in NIH3T3-wt8 cells (not shown). Given the results presented above we hypothesized that the expression levels of H- and K-Ras and the ratio of GTP-loaded H-Ras and K-Ras are major determinants for the overall inhibitory or stimulatory effect of W-13 on MAPK signal output in COS-1 cells and NIH3T3-wt8 cells, respectively. Therefore, we compared the expression levels and the activation of H- or K-Ras in COS-1 and NIH3T3-wt8 cells (Fig. 5). Supporting a dominant role for H-Ras in MAPK regulation in COS-1 cells, densitometry quantification analysis of different Western blots shows that the amount of K-Ras is comparable in COS-1 and NIH3T3-wt8 lysates, whereas expression of H-Ras was increased by 25–30% in COS-1 compared to NIH3T3-wt8 cells (Fig. 5A). GST-RBD pulldown experiments revealed that the ratio of H-Ras-GTP versus K-Ras-GTP after EGF stimulation was 25–30%

higher in COS-1 compared to NIH3T3-wt8 cells (Fig. 5B). Moreover, the comparison of EGF-induced interaction of ectopically expressed CFP-H-Ras with YFP-Raf-1 compared to CFP-K-Ras and YFP-Raf-1, as judged by FRET efficiency at the plasma membrane, shows that H-Ras is 1.82 fold more efficient to interact with Raf-1 in COS-1 than in NIH3T3-wt8 cells. In contrast K-Ras is 1.4 times more potent to recruit Raf-1 in NIH3T3-wt8 than in COS-1 cells (Fig. 5C). Finally, to further strengthen a model of CaM being differentially involved in H- and K-Ras-mediated Raf-1 activation and MAPK signal output, the effect of W-13 was examined in mouse embryonic fibroblasts (MEFs) lacking K-Ras (K-Ras−/−) or both H- and N-Ras (H-Ras−/−, N-Ras−/−). Similar to the results obtained from COS-1 cells ([1]; Fig. 3A), W-13 inhibited basal or EGF-stimulated Raf-1 and MAPK in wild type MEFs (Fig. 6A). In further agreement with data obtained from COS-1 [1], CD treatment also completely abolished the inhibitory effect of W-13 on phosphorylation of P-Ser338-Raf-1 and

Fig. 6. The different roles of CaM in MAPK activation depend on the activated Ras isoform. (A) Starved wild type MEFs cells were treated with β-methyl-cyclodextrin (CD, 5 mM) for 60 min and then with W-13 for 10 min and/or EGF for 5 min thereafter as indicated. The phosphorylations of Ser338-Raf-1 (P-Ser338) and MAPK (P-ERK) were detected by Western blotting from equal amount of protein lysates. Actin levels served as a loading control. Phosphorylation levels of Ser338-Raf-1 were quantified by densitometry from 3 independent experiments and mean values were expressed as relative phosphorylation levels. (B) Wild type MEFs (WT) and MEFs lacking K-Ras (K-Ras −/−) or H- and N-Ras (H-Ras−/−, N-Ras−/−) cells were starved and treated with W-13 for 10 min and/or EGF for the last 5 min. The amounts of phosphorylated Ser338-Raf-1 (P-Ser338), MAPK (PERK) and actin in the lysates were determined by Western blotting as indicated. Histogram shows the densitometry quantification (n = 3; mean ± S.D.) of relative P-Ser338-Raf-1 levels. Statistical significance between controls and corresponding W-13 treatments was determined using the Student's t test. ⁎P b 0.05, ⁎⁎P b 0.01.

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MAPK (Fig. 6A). The results suggest that signaling from Ras proteins, which localize on cholesterol rich domains at the plasma membrane (H- and N-Ras), is regulated by CaM. The fact that W-13 inhibits Raf-1 and MAPK activation only in MEFs lacking K-Ras but not in the double H-Ras−/−, N-Ras−/− knock-out MEFs further supports this hypothesis (Fig. 6B). 4. Discussion This study has examined the role of CaM in the regulation of Raf-1 activity and MAPK signaling and our data strongly suggests that the inhibitory effect of W-13 on MAPK activation is Ras isoform specific and cell-type dependent. Our data therefore illustrate a novel signaling mechanism mediated through CaM that differentially regulates the activity of H-Ras and K-Ras isoforms and the Raf-1/ MAPK signaling pathway (Fig. 7). 4.1. Dual regulation of Ras signaling by CaM It is well-known that inhibition of CaM can have opposing effects on MAPK activity depending on cell type analyzed. W-13 and another CaM antagonist, W-7, both increase MAPK activity in NIH3T3, SWISS3T3 or MYC 83 [38–40] but decrease MAPK activity in PC12, MTN or COS-1 cells [1,22,41,42]. The cause for the differential involvement of CaM in MAPK activation has yet not been identified, but it appears that opposite effects of CaM inhibition on the MAPK pathway can be linked to increased levels of Total Ras-GTP and to inhibition of Raf-1 activity, respectively. Raf-1 is regulated at different levels. A first step towards Raf-1 activation is the

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binding to Ras-GTP and the subsequent recruitment of Raf-1 to plasma membrane. This process releases its autoinhibitory conformation and allows access of the kinases and phosphatases involved in its activation. FRET analysis and determination of H- and K-Ras-GTP levels presented here and previously show that CaM prevents interaction of Raf-1 with H-Ras [1] as well as K-Ras (Figs. 1 and 2) by reducing the amount of H-Ras-GTP and K-Ras-GTP, respectively. However, CaM does not interfere with the binding of activated H- or K-Ras to Raf-1. We also demonstrated that CaM modulates Raf-1 activity at the level of its phosphorylation. We have described previously that CaM, through PI3K, regulates phosphorylation of Ser-338 residue of Raf-1 [1], one of the most important key phosphorylation sites that control Raf-1 activity [43]. Ser338 phosphorylation of endogenous Raf-1 is increased by extracellular ligands/signals and leads to Raf-1 activation. Mutation of S338 to alanine significantly impaired the response to growth factors indicating that ligand-mediated activation of Raf-1 requires Ser338 phosphorylation [4]. Previously we have shown that inhibition of CaM/ PI3K with W-13/wortmannin impairs the Ser338 phosphorylation of Raf-1 activated by H-Ras, which was 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 [5,7]. Interestingly, in the present study, we observed that W-13, as a consequence of EGFR activation, slightly increases the kinase activity of Raf-1 in K-RasG12V expressing cells. In contrast, W-13 inhibits H-Ras-mediated Raf-1 activation. We conclude from these findings that CaM differentially regulates Ras isoforms and is necessary for H-Ras but not K-Ras-mediated activation of Raf-1. 4.2. Differential regulation of Raf-1 by specific Ras isoforms determines the role of CaM in MAPK signal output

Fig. 7. Model for the regulation of CaM-mediated effects on the Ras/MAPK signaling pathway. (1) CaM binds and modulates EGFR activation [23,24] and through CaMKII, phosphorylates EGFR and inhibits its intrinsic tyrosine kinase activity [44]. In addition, CaM interferes the shedding of autocrine/paracrine growth factors, such as HB-EGF, though PKC/matrix-metalloproteinases stimulation [22,30]. (2) CaM binds to K-Ras and controls its cellular localization and activation through PKC-stimulation [25–27,30]. Therefore, inhibition of CaM results in EGFR and Ras activation. Consequently this leads to Raf-1 recruitment to the plasma membrane. Finally K-Ras facilitates Raf-1 activation at the plasma membrane. On the other hand, internalization and endosomal localization are required to ensure a completely H-Ras-mediated Raf-1 activation. (3) CaM, through PI3K, is necessary to stimulate Raf-1 via activated H-Ras in the endosomal compartment [1]. In this study, using COS-1 and NIH3T3-wt8 as model systems, we demonstrated that CaM-mediated regulation of Raf-1 activation via H- or K-Ras results in cell-type specific and different signal outputs through the MAPK signaling cascade. While H-Ras is more relevant than K-Ras to activate Raf-1 in COS-1 cells (continuous arrows), K-Ras is the main pathway to stimulate Raf-1 in NIH3T3-wt8 cells (discontinuous arrows). CaM, calmodulin; EC, endocytic compartment; PM, plasma membrane.

The differential involvement of H- and K-Ras in Raf-1 activation could explain the different MAPK signal outcomes observed in NIH3T3wt8 and COS-1 cells upon treatment with CaM inhibitor. Several findings support this hypothesis. First, inhibition of CaM promotes Raf-1 and MAPK activation in NIH3T3-wt8 but impairs Raf-1/MAPK signaling in COS-1 cells. Previous results from cells expressing a dominant negative mutant of dynamin, suggested that CaM, in a PI3K-dependent manner, regulates H-Ras-mediated phosphorylation and activation of Raf-1 in the endosomal compartment. In contrast, CaM appears to regulate Raf-1 via K-Ras almost exclusively at the plasma membrane as K-Ras-mediated Raf-1 activation in the presence of W-13 was not affected by overexpression of dominant negative dynamin (not shown). These results are consistent with results from Roy et al. showing that HRas and K-Ras signaling differentially depend on endocytosis [18]. Similarly, Omevoric et al. recently demonstrated that inhibition of receptor internalization reduces H-, N-, but not K-Ras-mediated Raf-1activation [19]. Second, in COS-1 cells inhibition of H-Ras signaling using membrane cholesterol depletion or siRNA knockdown experiments, CaM is unable to stimulate MAPK [1]. In the other hand, cholesterol depletion in NIH3T3-wt8 cells did neither substantially modify MAPK activation nor alter the effect of MAPK signaling upon CaM inhibition. Moreover, when K-Ras was depleted in NIH3T3-wt8 cells using specific siRNAs CaM appears necessary to facilitate MAPK activation. In addition, W-13 also inhibits H-RasG12V-activated Raf-1 in NIH3T3-wt8 cells, suggesting that this cell line has the molecular machinery necessary for CaM to regulate H-Ras-mediated Raf-1 activation. Third, the results obtained by means of FRET microscopy and GST-RBD pulldown assays; point to the activation of H-Ras being higher in COS-1 as compared to NIH3T3-wt8 cells. In further support of our model, K-Ras activates Raf-1 more efficiently than H-Ras [17]. Although only 20–30% differences on K-Ras versus H-Ras activity between both cell lines were observed, this may be sufficient to account for the difference observed at the level of MAPK regulation by CaM. Overall the results indicate that K-Ras exerts the major role on Raf-1 and MAPK activation in NIH3T3-wt8,

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while H-Ras is the most important upstream regulator of the MAPK pathway in COS-1 cells. Indeed, the results obtained from MEFs lacking either K-Ras or both H- and N-Ras further confirm a differential contribution of CaM in H- and K-Ras dependent signaling pathways. In summary, H- and K-Ras isoform expression and the ratio of HRas-GTP/K-Ras-GTP seem to control Raf activity. This tightly balanced repertoire of Ras/Raf signaling proteins is specific for each particular cell type and will modify and determine the overall contribution of CaM in MAPK signal output. Thus, CaM might have an important role orchestrating fine-tuning of different signaling pathways and their related functions in different cell and tissues. Acknowledgements We thank Drs. Richard Marais, Antonio Chiloeches, and Mariano Barbacid for donating plasmids and cells, respectively. This study was supported by grants BFU2007-67652 from Ministerio de Educación y Ciencia (MEC) of Spain to F.T.; BFU2006-01151 (MEC), RTICC program (Instituto de Salud Carlos III, Spain) and PI040236 from Maratò TV3 to C.E.; BFU2008-00345 (MEC) to A. P.; and grants 510293, 510294 from the National Health and Medical Research Council of Australia) to T. G. M.V-Q. is a recipient of a pre-doctoral RTICC program fellowship. We thank Dra. Maria Calvo, Dra. Anna Lladó and Anna Bosch for assistance in the confocal imaging (Unitat Microscopia Confocal, Serveis Cientificotècnics, Universitat de Barcelona-IDIBAPS) and Maria Molinos for technical assistance. References [1] J. Moretó, A. Lladó, M. Vidal-Quadras, M. Calvo, A. Pol, C. Enrich, F. Tebar, Cell. Signal. 20 (6) (2008) 1092–1103. [2] M.M. McKay, D.K. Morrison, Oncogene 26 (22) (2007) 3113–3121. [3] A.S. Dhillon, S. Meikle, Z. Yazici, M. Eulitz, W. Kolch, Embo J. 21 (1–2) (2002) 64–71. [4] C.S. Mason, C.J. Springer, R.G. Cooper, G. Superti-Furga, C.J. Marshall, R. Marais, Embo J. 18 (8) (1999) 2137–2148. [5] A.J. King, H. Sun, B. Diaz, D. Barnard, W. Miao, S. Bagrodia, M.S. Marshall, Nature 396 (6707) (1998) 180–183. [6] M. Zang, C. Hayne, Z. Luo, J. Biol. Chem. 277 (6) (2002) 4395–4405. [7] A. Chaudhary, W.G. King, M.D. Mattaliano, J.A. Frost, B. Diaz, D.K. Morrison, M.H. Cobb, M.S. Marshall, J.S. Brugge, Curr. Biol. 10 (9) (2000) 551–554. [8] D.A. Ritt, M. Zhou, T.P. Conrads, T.D. Veenstra, T.D. Copeland, D.K. Morrison, Curr. Biol. 17 (2) (2007) 179–184. [9] H. Chong, J. Lee, K.L. Guan, Embo J. 20 (14) (2001) 3716–3727. [10] J.M. Kyriakis, Biochim. Biophys. Acta 1773 (8) (2007) 1238–1247. [11] M. Barbacid, Annu Rev Biochem 56 (1987) 779–827. [12] J.F. Hancock, Nat. Rev., Mol. Cell Biol. 4 (5) (2003) 373–384.

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