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Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis
Cellular Signalling 22 (2010) 955–960
Contents lists available at ScienceDirect
Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis Johan Lennartsson ⁎, Fatima Burovic, Barbara Witek, Aleksandra Jurek, Carl-Henrik Heldin Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE-751 24, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 13 January 2010 Accepted 30 January 2010 Available online 6 February 2010 Keywords: PDGF Erk5 Akt Caspase Apoptosis
a b s t r a c t Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Platelet-derived growth factor (PDGF) is a potent mitogen for connective tissue cells and plays an important role during embryonic development and wound healing. PDGF is a family of disulfide-bonded dimers of four homologous polypeptide chains, PDGF-AA, AB, BB, CC and DD [1]. Binding of PDGF isoforms to the related α or β tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively) results in dimerization and subsequent autophosphorylation, leading to initiation of downstream signaling cascades. Well established signaling proteins recruited to phosphorylated PDGF receptors include PI3-kinase, PLCγ, Src, Grb2 and Shp2, leading to, for example, subsequent activation of Akt, PKC and MAP kinases. These pathways transduce signals ultimately resulting in increased cell proliferation, migration and survival. The MAP kinase cascade consists of a conserved three-layered kinase module that serves as an important link between cell surface receptors and intracellular effects such as gene expression. In mammalian cells there are several MAP kinase pathways, i.e. Erk1/2, p38, Jnk and Erk5, as well as the atypical Erk3, Erk4 and Erk7 [2]. Erk5 is also denoted big MAP kinase 1 (Bmk1) because its molecular weight is approximately twice that of the other MAP kinases, and differs
Abbreviations: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; Erk, extracellular regulated kinase; MAPK, mitogen activated protein kinase; PAE, porcine aortic endothelial cell; PI3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; FBS, fetal bovine serum. ⁎ Corresponding author. Ludwig Institute for Cancer Research, Box 595, Biomedical Center, SE-751 24 Uppsala, Sweden. Tel.: +46 18 160406; fax: +46 18 160420. E-mail address: [email protected] (J. Lennartsson). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.01.020
structurally from Erk1/2 by the presence of a C-terminal transactivation domain that can be activated by phosphorylation [3]. Initially, Erk5 was thought to be activated by cell stress [4], but subsequent studies have shown that also growth factors induce its activation [5–7]. The biological function of Erk5 has been associated with cell survival, proliferation and migration [8]. The pro-survival function of Erk5 has been associated to Erk5-dependent phosphorylation of Bad and Akt [9,10]. Diminished Akt phosphorylation may sensitize cells toward apoptosis through reduced Bad phosphorylation thereby promoting release of cytochrome c from the mitochondria, as well as enhanced Foxo3adependent transcription of death promoting genes. Deletion of the Erk5 gene in the mouse results in embryonic lethality due to vascular defects [11–13]. Furthermore, conditional deletion of Erk5 in endothelial cells displayed a similar phenotype as the conventional knock-out suggesting an important role for Erk5 in this cell type, and deletion of Erk5 in the adult mouse showed an important function for Erk5 in maintaining vessel homeostasis [14]. In this study, we have investigated the involvement of Erk5 in PDGF-BB-induced signaling. Our results suggest that Erk5 has a central role in maintaining an extended PDGF-BB-induced Akt phosphorylation, suppression of caspases 3 cleavage and inhibition of nuclear apoptotic alterations. 2. Materials and methods 2.1. Reagents Recombinant human PDGF-BB was generously provided by Amgen (Thousand Oaks, CA). The inhibitors U0126, SU6656 and LY294002
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were from Calbiochem (San Diego, CA). Antibodies against phosphorylated Erk5 (#3371), total Erk5 (#3372), phosphorylated Erk1/2 (#9106), phosphorylated Akt (#9271) and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology (Beverly, MA). Antisera against phosphotyrosine (PY99, sc-7029), total Akt (sc8312) and PDGFRβ (sc-339) were from Santa Cruz Biotechnology (Santa Cruz, CA). α-Tubulin antibody was purchased from Sigma (St. Louis, MI). A rabbit antiserum recognizing Erk2 was raised against a peptide corresponding to the carboxy-terminal sequence EETARFQPGYRS conjugated to KLH. The rabbit antiserum cst-1, recognizing Src, Yes and Fyn, was prepared as described [15]. γ[32P]ATP (BLU502A) were purchased from Perkin Elmer (Shelton, CT).
2.6. Nuclear morphology analysis Cells were transfected with siRNA and treated as indicated and then fixed for 30 min in paraformaldehyde. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, followed by washing in PBS and blocking with 10 mM glycine for 1 h at room temperature. After washing with PBS, coverslips were incubated for 10 min with DAPI (Sigma) diluted 1:1000 in PBS, followed by further washings in PBS and once in water. Coverslips were mounted using Fluoromount-G (Southern Biotech) anti-fade, followed by fluorescence microscopy using Zeiss axioplan2. 3. Results
2.2. Cell culture
3.1. PDGF-BB induces a rapid and transient Erk5 activation
Porcine aortic endothelial (PAE) cells stably transfected with wild-type or mutated PDGFRβ were cultured in Ham's F-12 with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. For serum starvation, cells were washed once and incubated in Ham's F-12 medium containing 1% FBS.
To assess the involvement of Erk5 downstream of PDGFRβ, we used porcine aortic endothelial (PAE) cells stably expressing PDGFRβ. We stimulated cells for up to 6 h with PDGF-BB and analyzed the activation of PDGFRβ and Erk5. As expected, the PDGFRβ was rapidly activated with peak phosphorylation occurring at 5–15 min of stimulation followed by a decreased receptor phosphorylation, although the remaining above base line throughout the time course of the experiment (Fig. 1A). Erk5 was fully phosphorylated already at 5 min of stimulation and returned to an undetectable level by 30 min of PDGF-BB treatment (Fig. 1B). On closer inspection of the onset of Erk5 activation, we observed phosphorylation already after 2 min of stimulation, but peak phosphorylation occurred after 5 min of PDGFBB treatment (Fig. 1C). In addition to PDGFRβ, also the PDGFRα isoform was able to mediate induction of Erk5 phosphorylation with similar kinetics (data not shown).
2.3. siRNA knockdown Downregulation of Erk5 was performed by using 30 nM of specific siRNA (RNA sequence: GGUGUUGGCUUUGACCUGGAGGAAUdTdT or CCCUAUGGAGAAUUCAGAUCUGUCUdTdT) purchased from Invitrogen. For every experiment performed, luciferase-targeting siRNA (RNA sequence CGUACGCGGAAUACUUCGAdTdT) was used as a control. Transfection of siRNA was done for 24 h with SilentFect from BioRad. Levels of knockdown were tested after 40 h by measuring protein levels by immunoblotting.
2.4. Western blotting Subconfluent cells were starved and incubated with vehicle or inhibitors at the indicated concentrations and thereafter stimulated with PDGF-BB (20 ng/ml) for the indicated periods of time. Cells were washed with ice-cold phosphate-buffered saline and lysed in 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 1 mM Pefa Bloc and 1 mM sodium orthovanadate. Extracts were clarified by centrifugation, and protein concentration was determined by the BCA protein assay (Pierce). Equal amounts of lysates were boiled with SDS sample buffer containing dithiothreitol. Proteins were separated by SDS-PAGE and then electro-transferred to polyvinylidene difluoride membranes (Immobilon P), which were blocked in 5% bovine serum albumin in Tris-buffered saline solution containing 0.1% Tween-20. Primary antibodies were diluted according to the manufacturer's instructions and membranes incubated overnight at 4 °C. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (both from Amersham Biosciences) and proteins were visualized using ECL Western blotting detection systems from Roche Applied Science on a cooled charge-coupled device camera (Fuji, Minami-Ashigata, Japan).
3.2. Src kinases and Shp2 are required for PDGF-BB-induced Erk5 activation In an attempt to identify upstream requirements for Erk5 activation, we exploited low molecular weight inhibitors against selected pathways known to be robustly activated downstream of PDGFRβ, i.e. SU6656, U0126 and LY294002 inhibiting Src kinases, Mek1/2 and PI3-kinase, respectively. Inhibition of Src kinases effectively blocked PDGF-BB-induced Erk5 activation, whereas inhibition of Mek1/2 or PI3-kinase did not have any
2.5. Src kinase assay Subconfluent cells were starved overnight in serum-free medium and stimulated with PDGF-BB for 5 min, followed by lysis and immunoprecipitation with cst-1 antibodies against Src and in vitro kinase assay using acid-denatured enolase as a substrate, as described [15]. Quantitation of radioactivity incorporated into enolase was done using a Fuji FLA3000 Bioimager.
Fig. 1. Stimulation of PDGFRβ results in activation of the Erk5 pathway. PAE/PDGFRβ cells were serum-starved and then stimulated with PDGF-BB for the indicated periods of time. PDGFRβ (A), Erk5 (B and C) levels and activation were assayed by immunoblotting (Ib) of total cell lysates (TCL) for Erk5 and immunoprecipitated (Ip) material for PDGFRβ with antibodies against total or phosphorylated proteins, as indicated.
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significant effect (Fig. 2A and B). Consistently, a mutated receptor unable to interact with Src family kinases (PDGFRβY579F) was also incapable of inducing Erk5 phosphorylation (Fig. 2C). A previous report had implicated the tyrosine phosphatase Shp2 in the activation of Erk5 in response to PDGF stimulation [16]. We therefore analyzed the importance of Shp2 for Erk5 activation in PAE cells using a mutated receptor lacking the docking sites for Shp2 (PDGFRβY763/1009F). As shown in Fig. 3A and B, the mutant receptor had a severely reduced ability to mediate Erk5 phosphorylation, whereas the capacity to induce Erk1/2 phosphorylation was not affected. It has been suggested that Shp2 plays a role in growth factormediated Src activation [17]. To address the involvement of Shp2 in PDGF-BB-induced Src activation, we performed in vitro kinase assays with Src after stimulation of the wild-type receptor or the mutant receptor deficient in Shp2 binding (PDGFRβY763/1009F). We found that the PDGFRβY763/1009F mutant receptor was unable to effectively mediate Src activation (Fig. 3C).
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In summary, PDGF-BB stimulation transiently activates Erk5 in a manner requiring Src kinase activity, consistent with a previous report [18]. We also demonstrate that Src activation to a large extent is dependent on Shp2 docking to the PDGFRβ. Our results are consistent with the previous findings that Shp2 [16] and Src [18] are required for PDGF-BB-induced Erk5 phosphorylation and show that Shp2 is a critical component in the pathway leading to Src activation downstream of PDGFRβ. 3.3. Erk5 is necessary for sustained Akt phosphorylation in response to PDGF-BB, protection from serum-starvation-induced caspase 3 cleavage and suppression of apoptotic chromatin alterations Erk5 has been implicated in the process of Akt phosphorylation in neuronal cells [9]. Therefore, we downregulated Erk5 by siRNA and analyzed the effect on PDGF-BB-induced Akt phosphorylation. Our results showed that Erk5 was not critical for the initial onset of Akt
Fig. 2. Activation of Erk5 in response to PDGF-BB treatment requires Src kinase activity. Serum-starved PAE/PDGFRβ cells pretreated for 1 h with inhibitors (10 μM LY294002, 10 μM U0126 and 2 μM SU6656), as indicated, and then stimulated with PDGF-BB for 5 min (A and B). Erk5, Erk1/2, Akt and PDGFRβ levels and activation were assayed by immunoblotting (Ib) of total cell lysates (TCL) for Erk5, Erk1/2 and Akt, and immunoprecipitated (Ip) material for PDGFRβ with antibodies against total or phosphorylated proteins, as indicated. Serum-starved PAE/PDGFRβY579F mutant cells were stimulated with PDGF-BB for 5 min and activation of Erk5 measured with antibodies against total or phosphorylated protein (C). Quantifications shown in panels B and C are the averages of three independent experiments ± standard deviation.
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Fig. 3. Shp2 docking to PDGFRβ is necessary for Erk5 and Src activation. Serum-starved PAE/PDGFRβ and PAE/PDGFRβY763/1009F mutant cells were stimulated with PDGF-BB for 5 min and activation of Erk5, Erk1/2 and PDGFRβ was measured with antibodies against total or phosphorylated protein (A) and quantified in (B). PAE/PDGFRβ or PAE/PDGFRβY763/1009F expressing cells were stimulated with PDGF-BB, lysed and subjected to immunoprecipitation using the cst-1 antiserum, recognizing Src, Yes and Fyn (C). The washed immunoprecipitates were incubated with γ[32P]ATP and acid-denatured enolase for 10 min. Proteins were separated by SDS-PAGE and exposed on Fuji FLA3000 Image Analyzer. The amount of radioactivity incorporated into enolase was quantified. The average of three independent experiments ± standard deviation is shown.
phosphorylation seen after 5 min of PDGF-BB stimulation, but was required to sustain this phosphorylation up to 60 min of stimulation (Fig. 4). Sustained Akt activation has a well established role in promoting cell survival. Therefore, we wanted to address the possibility that Erk5 has a function in PDGF-BB-promoted suppression of apoptosis. We found that under conditions of reduced Erk5 expression, PDGF-BB was no longer able to suppress cleavage of caspase 3 in response to serumwithdrawal (Fig. 5A). Notably, in cells with reduced Erk5 expression and in the absence of serum or PDGF-BB, we were unable to recover sufficient number of cells for caspase 3 cleavage analysis because of extensive cell death. Using a second independent siRNA targeting Erk5, we observed similar effects on cell survival (not shown). Consistent with the increased level of caspase 3 cleavage when Erk5 expression was downregulated, we could visualize morphological
Fig. 4. Erk5 is essential for sustained PDGF-BB-induced Akt phosphorylation. PAE/ PDGFRβ cells transfected with siRNA against Erk5 or control siRNA were serum-starved and stimulated with PDGF-BB. Akt activation and levels, as well as Erk5 levels, were assayed by immunoblotting (Ib) of total cell lysates with antibodies against total Erk5, Akt and phosphorylated Akt. α-Tubulin immunoblotting served as a loading control. Quantification of a representative experiment is shown as the ratio between p-Akt and total Akt.
apoptotic features such as nuclear condensation and fragmentation by DAPI staining of fixed cells under these conditions (Fig. 5B and quantifications in Fig. 5C). 4. Discussion In the present report we have investigated the molecular mechanisms for PDGF-BB-induced Erk5 activation and its functional role in PDGF-BB stimulated cells. We found that activation of PDGFRβ rapidly and transiently promoted Erk5 phosphorylation, in concurrence with recent reports [16,18]. In a study by Rovida et al., the PDGFinduced activation of Erk5 was shown, using low molecular weight inhibitors, to depend on the activity of Src kinases [18]. This observation was confirmed and extended in our study, in which we show that in addition to the low molecular weight inhibitor SU6656, also a point-mutated PDGF receptor (PDGFRβY579F) unable to bind Src kinases prevents activation of Erk5. Notably, in cells expressing the PDGFRβY579F mutant receptor, Src retains basal activity and can potentially be activated indirectly, which explains the weak Src activation observed in the kinase assay by the mutant receptor; thus, our finding indicates that Src kinases have to be recruited to the receptor in order to mediate Erk5 activation. In the study by Izawa et al., the catalytic activity of the tyrosine phosphatase Shp2 was demonstrated to be essential for PDGF-induced Erk5 phosphorylation [16]. To explore this finding further, we used a mutant receptor unable to interact with Shp2 (PDGFRβY763/1009F); using this approach we could implicate Shp2 in Erk5 activation, confirming the previous finding. Interestingly, Shp2 has been connected to Src activation through its ability to dephosphorylate PAG/Cbp thereby restricting the ability of Csk to access and negatively phosphorylate Src kinases [17]. Indeed, we found that the Y763/1009F mutant PDGFRβ unable to
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Fig. 5. Erk5 is important for PDGF-BB suppressed caspase 3 activation and chromatin condensation in response to serum-withdrawal. PAE/PDGFRβ cells transfected with siRNA against Erk5 or control siRNA were serum-starved for 24 h and then treated with PDGF-BB or FCS for 24 h as indicated, and activation of caspase 3 was measured by immunoblotting against cleaved caspase 3 (A, left panel). Erk5 downregulation and equal loading was verified by immunoblotting against total Erk5 and α-tubulin, respectively. Quantifications shown are the averages of three independent experiments ± standard deviation (A, right panel). PAE/PDGFRβ was stained with DAPI to visualize morphological apoptotic features such as nuclear condensation and fragmentation (B) and quantifications and results are plotted as mean % of at least three experiments ± standard deviation (C).
interact with Shp2, was also defective in its ability to activate Src kinases. Furthermore, the rapid kinetics of PDGF-BB-induced Erk5 phosphorylation was mirrored by equally rapid activation of Src kinases (data not shown). Moreover, it has been seen that Erk5, but not Erk1/2, is constitutively active in v-Src transformed NIH3T3 fibroblasts [19]. In conclusion, our data support the notion that the requirement of Shp2 in Erk5 activations stems from its involvement in Src activation. Erk5 has been shown to be important for cell survival of for example endothelial and neuronal cells [9,14]. In the neuronal cells it was demonstrated that loss of Erk5 expression resulted in reduced Akt and RSK phosphorylation and thereby increased expression of proapoptotic proteins Bad and Bim [9]. It is well established that Akt provides important survival signals downstream of growth factor receptors; therefore we investigated whether Erk5 is important also for PDGF-BB-induced Akt phosphorylation. Under normal conditions, PDGF-BB stimulation induced a sustained Akt phosphorylation, however, under conditions where Erk5 expression was reduced by siRNA, PDGF-BB was only able to promote a transient activation of
Akt. Thus, Erk5 appears not to be essential for the initial PI3-kinasedependent Akt activation, but is in contrast critical for prolonged Akt phosphorylation. It is possible that Erk5 phosphorylates and thereby inactivates or mislocates a phosphatase that otherwise would promote Akt dephosphorylation, alternatively, Erk5 may control the level of PIP3. An obvious Erk5 target candidate is PTEN that can restrict Akt signaling by dephosphorylating the lipid PIP3, but we have been unable to obtain data supporting an involvement of PTEN in the mechanism whereby Erk5 promotes Akt activation (data not shown). Consistent with the inability of PDGF-BB to promote sustained Akt phosphorylation when Erk5 was silenced, we observed that when Erk5 expression was reduced, PDGF-BB was not able to protect cells from activation of caspase 3 under serum-free conditions; however, addition of 10% FCS was able to efficiently block caspase 3 cleavage. Visualization and quantification of apoptotic morphological nuclear changes showed that under conditions with reduced Erk5 expression there was a significant increase in apoptotic nuclei both under serumfree conditions (10.7% vs. 1.2% in control cells) and in the presence of PDGF-BB (4.9% vs. 0.7%). In concurrence, it has been shown that the
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Acknowledgements This work was supported by the Ludwig Institute for Cancer Research, the Swedish Research Council and the Swedish Cancer Foundation.
References
Fig. 6. Schematic representation of Erk5 involvement in PDGF-BB-induced signal transduction. Stimulation of the PDGFRβ results in recruitment of Shp2 to the receptor and subsequently to Src activation. Activated Src then promotes activation of Erk5. Akt activation occurs in a PI3-kinase-dependent manner, but the sustained Akt phosphorylation requires Erk5. Akt is a key molecule in promoting cell survival through several mechanisms, including suppression of the caspases cascade.
loss of the Erk5 pathway makes fibroblasts more sensitive to sorbitolinduced caspase 3 activation and apoptosis [10,20]. Mechanistically, Akt can phosphorylate and thereby inactivate the initiator caspases 9 which lies upstream of caspase 3, thus establishing a link between Akt activation and inhibition of the caspase cascade [21]. In summary, our data suggest that Erk5 has a role in survival signaling by ensuring sustained Akt phosphorylation and suppression of caspase 3 cleavage. In conclusion, our data suggest the model depicted in Fig. 6. Upon PDGF-BB stimulation, Shp2 and Src family kinases are recruited to the receptor and Shp2 plays an important role in activation of Src family kinases. Src kinase activity is critical for the activation of Erk5 through a still poorly characterized mechanism. Erk5 is necessary for sustained activation of the pro-survival kinase Akt. In the absence of significant Erk5 expression PDGF-BB cannot suppress serum-starvation-induced caspase 3 cleavage in contrast to 10% FCS which efficiently inhibit caspase 3 activation. In concurrence, under conditions with reduced Erk5 expression PDGF-BB can only partially inhibit chromatin condensation and fragmentation in response to serum starvation, whereas 10% FCS can do so completely.
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Contents lists available at ScienceDirect
Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis Johan Lennartsson ⁎, Fatima Burovic, Barbara Witek, Aleksandra Jurek, Carl-Henrik Heldin Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE-751 24, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 13 January 2010 Accepted 30 January 2010 Available online 6 February 2010 Keywords: PDGF Erk5 Akt Caspase Apoptosis
a b s t r a c t Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Platelet-derived growth factor (PDGF) is a potent mitogen for connective tissue cells and plays an important role during embryonic development and wound healing. PDGF is a family of disulfide-bonded dimers of four homologous polypeptide chains, PDGF-AA, AB, BB, CC and DD [1]. Binding of PDGF isoforms to the related α or β tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively) results in dimerization and subsequent autophosphorylation, leading to initiation of downstream signaling cascades. Well established signaling proteins recruited to phosphorylated PDGF receptors include PI3-kinase, PLCγ, Src, Grb2 and Shp2, leading to, for example, subsequent activation of Akt, PKC and MAP kinases. These pathways transduce signals ultimately resulting in increased cell proliferation, migration and survival. The MAP kinase cascade consists of a conserved three-layered kinase module that serves as an important link between cell surface receptors and intracellular effects such as gene expression. In mammalian cells there are several MAP kinase pathways, i.e. Erk1/2, p38, Jnk and Erk5, as well as the atypical Erk3, Erk4 and Erk7 [2]. Erk5 is also denoted big MAP kinase 1 (Bmk1) because its molecular weight is approximately twice that of the other MAP kinases, and differs
Abbreviations: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; Erk, extracellular regulated kinase; MAPK, mitogen activated protein kinase; PAE, porcine aortic endothelial cell; PI3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; FBS, fetal bovine serum. ⁎ Corresponding author. Ludwig Institute for Cancer Research, Box 595, Biomedical Center, SE-751 24 Uppsala, Sweden. Tel.: +46 18 160406; fax: +46 18 160420. E-mail address: [email protected] (J. Lennartsson). 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.01.020
structurally from Erk1/2 by the presence of a C-terminal transactivation domain that can be activated by phosphorylation [3]. Initially, Erk5 was thought to be activated by cell stress [4], but subsequent studies have shown that also growth factors induce its activation [5–7]. The biological function of Erk5 has been associated with cell survival, proliferation and migration [8]. The pro-survival function of Erk5 has been associated to Erk5-dependent phosphorylation of Bad and Akt [9,10]. Diminished Akt phosphorylation may sensitize cells toward apoptosis through reduced Bad phosphorylation thereby promoting release of cytochrome c from the mitochondria, as well as enhanced Foxo3adependent transcription of death promoting genes. Deletion of the Erk5 gene in the mouse results in embryonic lethality due to vascular defects [11–13]. Furthermore, conditional deletion of Erk5 in endothelial cells displayed a similar phenotype as the conventional knock-out suggesting an important role for Erk5 in this cell type, and deletion of Erk5 in the adult mouse showed an important function for Erk5 in maintaining vessel homeostasis [14]. In this study, we have investigated the involvement of Erk5 in PDGF-BB-induced signaling. Our results suggest that Erk5 has a central role in maintaining an extended PDGF-BB-induced Akt phosphorylation, suppression of caspases 3 cleavage and inhibition of nuclear apoptotic alterations. 2. Materials and methods 2.1. Reagents Recombinant human PDGF-BB was generously provided by Amgen (Thousand Oaks, CA). The inhibitors U0126, SU6656 and LY294002
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were from Calbiochem (San Diego, CA). Antibodies against phosphorylated Erk5 (#3371), total Erk5 (#3372), phosphorylated Erk1/2 (#9106), phosphorylated Akt (#9271) and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology (Beverly, MA). Antisera against phosphotyrosine (PY99, sc-7029), total Akt (sc8312) and PDGFRβ (sc-339) were from Santa Cruz Biotechnology (Santa Cruz, CA). α-Tubulin antibody was purchased from Sigma (St. Louis, MI). A rabbit antiserum recognizing Erk2 was raised against a peptide corresponding to the carboxy-terminal sequence EETARFQPGYRS conjugated to KLH. The rabbit antiserum cst-1, recognizing Src, Yes and Fyn, was prepared as described [15]. γ[32P]ATP (BLU502A) were purchased from Perkin Elmer (Shelton, CT).
2.6. Nuclear morphology analysis Cells were transfected with siRNA and treated as indicated and then fixed for 30 min in paraformaldehyde. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, followed by washing in PBS and blocking with 10 mM glycine for 1 h at room temperature. After washing with PBS, coverslips were incubated for 10 min with DAPI (Sigma) diluted 1:1000 in PBS, followed by further washings in PBS and once in water. Coverslips were mounted using Fluoromount-G (Southern Biotech) anti-fade, followed by fluorescence microscopy using Zeiss axioplan2. 3. Results
2.2. Cell culture
3.1. PDGF-BB induces a rapid and transient Erk5 activation
Porcine aortic endothelial (PAE) cells stably transfected with wild-type or mutated PDGFRβ were cultured in Ham's F-12 with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. For serum starvation, cells were washed once and incubated in Ham's F-12 medium containing 1% FBS.
To assess the involvement of Erk5 downstream of PDGFRβ, we used porcine aortic endothelial (PAE) cells stably expressing PDGFRβ. We stimulated cells for up to 6 h with PDGF-BB and analyzed the activation of PDGFRβ and Erk5. As expected, the PDGFRβ was rapidly activated with peak phosphorylation occurring at 5–15 min of stimulation followed by a decreased receptor phosphorylation, although the remaining above base line throughout the time course of the experiment (Fig. 1A). Erk5 was fully phosphorylated already at 5 min of stimulation and returned to an undetectable level by 30 min of PDGF-BB treatment (Fig. 1B). On closer inspection of the onset of Erk5 activation, we observed phosphorylation already after 2 min of stimulation, but peak phosphorylation occurred after 5 min of PDGFBB treatment (Fig. 1C). In addition to PDGFRβ, also the PDGFRα isoform was able to mediate induction of Erk5 phosphorylation with similar kinetics (data not shown).
2.3. siRNA knockdown Downregulation of Erk5 was performed by using 30 nM of specific siRNA (RNA sequence: GGUGUUGGCUUUGACCUGGAGGAAUdTdT or CCCUAUGGAGAAUUCAGAUCUGUCUdTdT) purchased from Invitrogen. For every experiment performed, luciferase-targeting siRNA (RNA sequence CGUACGCGGAAUACUUCGAdTdT) was used as a control. Transfection of siRNA was done for 24 h with SilentFect from BioRad. Levels of knockdown were tested after 40 h by measuring protein levels by immunoblotting.
2.4. Western blotting Subconfluent cells were starved and incubated with vehicle or inhibitors at the indicated concentrations and thereafter stimulated with PDGF-BB (20 ng/ml) for the indicated periods of time. Cells were washed with ice-cold phosphate-buffered saline and lysed in 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 1 mM Pefa Bloc and 1 mM sodium orthovanadate. Extracts were clarified by centrifugation, and protein concentration was determined by the BCA protein assay (Pierce). Equal amounts of lysates were boiled with SDS sample buffer containing dithiothreitol. Proteins were separated by SDS-PAGE and then electro-transferred to polyvinylidene difluoride membranes (Immobilon P), which were blocked in 5% bovine serum albumin in Tris-buffered saline solution containing 0.1% Tween-20. Primary antibodies were diluted according to the manufacturer's instructions and membranes incubated overnight at 4 °C. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (both from Amersham Biosciences) and proteins were visualized using ECL Western blotting detection systems from Roche Applied Science on a cooled charge-coupled device camera (Fuji, Minami-Ashigata, Japan).
3.2. Src kinases and Shp2 are required for PDGF-BB-induced Erk5 activation In an attempt to identify upstream requirements for Erk5 activation, we exploited low molecular weight inhibitors against selected pathways known to be robustly activated downstream of PDGFRβ, i.e. SU6656, U0126 and LY294002 inhibiting Src kinases, Mek1/2 and PI3-kinase, respectively. Inhibition of Src kinases effectively blocked PDGF-BB-induced Erk5 activation, whereas inhibition of Mek1/2 or PI3-kinase did not have any
2.5. Src kinase assay Subconfluent cells were starved overnight in serum-free medium and stimulated with PDGF-BB for 5 min, followed by lysis and immunoprecipitation with cst-1 antibodies against Src and in vitro kinase assay using acid-denatured enolase as a substrate, as described [15]. Quantitation of radioactivity incorporated into enolase was done using a Fuji FLA3000 Bioimager.
Fig. 1. Stimulation of PDGFRβ results in activation of the Erk5 pathway. PAE/PDGFRβ cells were serum-starved and then stimulated with PDGF-BB for the indicated periods of time. PDGFRβ (A), Erk5 (B and C) levels and activation were assayed by immunoblotting (Ib) of total cell lysates (TCL) for Erk5 and immunoprecipitated (Ip) material for PDGFRβ with antibodies against total or phosphorylated proteins, as indicated.
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significant effect (Fig. 2A and B). Consistently, a mutated receptor unable to interact with Src family kinases (PDGFRβY579F) was also incapable of inducing Erk5 phosphorylation (Fig. 2C). A previous report had implicated the tyrosine phosphatase Shp2 in the activation of Erk5 in response to PDGF stimulation [16]. We therefore analyzed the importance of Shp2 for Erk5 activation in PAE cells using a mutated receptor lacking the docking sites for Shp2 (PDGFRβY763/1009F). As shown in Fig. 3A and B, the mutant receptor had a severely reduced ability to mediate Erk5 phosphorylation, whereas the capacity to induce Erk1/2 phosphorylation was not affected. It has been suggested that Shp2 plays a role in growth factormediated Src activation [17]. To address the involvement of Shp2 in PDGF-BB-induced Src activation, we performed in vitro kinase assays with Src after stimulation of the wild-type receptor or the mutant receptor deficient in Shp2 binding (PDGFRβY763/1009F). We found that the PDGFRβY763/1009F mutant receptor was unable to effectively mediate Src activation (Fig. 3C).
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In summary, PDGF-BB stimulation transiently activates Erk5 in a manner requiring Src kinase activity, consistent with a previous report [18]. We also demonstrate that Src activation to a large extent is dependent on Shp2 docking to the PDGFRβ. Our results are consistent with the previous findings that Shp2 [16] and Src [18] are required for PDGF-BB-induced Erk5 phosphorylation and show that Shp2 is a critical component in the pathway leading to Src activation downstream of PDGFRβ. 3.3. Erk5 is necessary for sustained Akt phosphorylation in response to PDGF-BB, protection from serum-starvation-induced caspase 3 cleavage and suppression of apoptotic chromatin alterations Erk5 has been implicated in the process of Akt phosphorylation in neuronal cells [9]. Therefore, we downregulated Erk5 by siRNA and analyzed the effect on PDGF-BB-induced Akt phosphorylation. Our results showed that Erk5 was not critical for the initial onset of Akt
Fig. 2. Activation of Erk5 in response to PDGF-BB treatment requires Src kinase activity. Serum-starved PAE/PDGFRβ cells pretreated for 1 h with inhibitors (10 μM LY294002, 10 μM U0126 and 2 μM SU6656), as indicated, and then stimulated with PDGF-BB for 5 min (A and B). Erk5, Erk1/2, Akt and PDGFRβ levels and activation were assayed by immunoblotting (Ib) of total cell lysates (TCL) for Erk5, Erk1/2 and Akt, and immunoprecipitated (Ip) material for PDGFRβ with antibodies against total or phosphorylated proteins, as indicated. Serum-starved PAE/PDGFRβY579F mutant cells were stimulated with PDGF-BB for 5 min and activation of Erk5 measured with antibodies against total or phosphorylated protein (C). Quantifications shown in panels B and C are the averages of three independent experiments ± standard deviation.
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Fig. 3. Shp2 docking to PDGFRβ is necessary for Erk5 and Src activation. Serum-starved PAE/PDGFRβ and PAE/PDGFRβY763/1009F mutant cells were stimulated with PDGF-BB for 5 min and activation of Erk5, Erk1/2 and PDGFRβ was measured with antibodies against total or phosphorylated protein (A) and quantified in (B). PAE/PDGFRβ or PAE/PDGFRβY763/1009F expressing cells were stimulated with PDGF-BB, lysed and subjected to immunoprecipitation using the cst-1 antiserum, recognizing Src, Yes and Fyn (C). The washed immunoprecipitates were incubated with γ[32P]ATP and acid-denatured enolase for 10 min. Proteins were separated by SDS-PAGE and exposed on Fuji FLA3000 Image Analyzer. The amount of radioactivity incorporated into enolase was quantified. The average of three independent experiments ± standard deviation is shown.
phosphorylation seen after 5 min of PDGF-BB stimulation, but was required to sustain this phosphorylation up to 60 min of stimulation (Fig. 4). Sustained Akt activation has a well established role in promoting cell survival. Therefore, we wanted to address the possibility that Erk5 has a function in PDGF-BB-promoted suppression of apoptosis. We found that under conditions of reduced Erk5 expression, PDGF-BB was no longer able to suppress cleavage of caspase 3 in response to serumwithdrawal (Fig. 5A). Notably, in cells with reduced Erk5 expression and in the absence of serum or PDGF-BB, we were unable to recover sufficient number of cells for caspase 3 cleavage analysis because of extensive cell death. Using a second independent siRNA targeting Erk5, we observed similar effects on cell survival (not shown). Consistent with the increased level of caspase 3 cleavage when Erk5 expression was downregulated, we could visualize morphological
Fig. 4. Erk5 is essential for sustained PDGF-BB-induced Akt phosphorylation. PAE/ PDGFRβ cells transfected with siRNA against Erk5 or control siRNA were serum-starved and stimulated with PDGF-BB. Akt activation and levels, as well as Erk5 levels, were assayed by immunoblotting (Ib) of total cell lysates with antibodies against total Erk5, Akt and phosphorylated Akt. α-Tubulin immunoblotting served as a loading control. Quantification of a representative experiment is shown as the ratio between p-Akt and total Akt.
apoptotic features such as nuclear condensation and fragmentation by DAPI staining of fixed cells under these conditions (Fig. 5B and quantifications in Fig. 5C). 4. Discussion In the present report we have investigated the molecular mechanisms for PDGF-BB-induced Erk5 activation and its functional role in PDGF-BB stimulated cells. We found that activation of PDGFRβ rapidly and transiently promoted Erk5 phosphorylation, in concurrence with recent reports [16,18]. In a study by Rovida et al., the PDGFinduced activation of Erk5 was shown, using low molecular weight inhibitors, to depend on the activity of Src kinases [18]. This observation was confirmed and extended in our study, in which we show that in addition to the low molecular weight inhibitor SU6656, also a point-mutated PDGF receptor (PDGFRβY579F) unable to bind Src kinases prevents activation of Erk5. Notably, in cells expressing the PDGFRβY579F mutant receptor, Src retains basal activity and can potentially be activated indirectly, which explains the weak Src activation observed in the kinase assay by the mutant receptor; thus, our finding indicates that Src kinases have to be recruited to the receptor in order to mediate Erk5 activation. In the study by Izawa et al., the catalytic activity of the tyrosine phosphatase Shp2 was demonstrated to be essential for PDGF-induced Erk5 phosphorylation [16]. To explore this finding further, we used a mutant receptor unable to interact with Shp2 (PDGFRβY763/1009F); using this approach we could implicate Shp2 in Erk5 activation, confirming the previous finding. Interestingly, Shp2 has been connected to Src activation through its ability to dephosphorylate PAG/Cbp thereby restricting the ability of Csk to access and negatively phosphorylate Src kinases [17]. Indeed, we found that the Y763/1009F mutant PDGFRβ unable to
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Fig. 5. Erk5 is important for PDGF-BB suppressed caspase 3 activation and chromatin condensation in response to serum-withdrawal. PAE/PDGFRβ cells transfected with siRNA against Erk5 or control siRNA were serum-starved for 24 h and then treated with PDGF-BB or FCS for 24 h as indicated, and activation of caspase 3 was measured by immunoblotting against cleaved caspase 3 (A, left panel). Erk5 downregulation and equal loading was verified by immunoblotting against total Erk5 and α-tubulin, respectively. Quantifications shown are the averages of three independent experiments ± standard deviation (A, right panel). PAE/PDGFRβ was stained with DAPI to visualize morphological apoptotic features such as nuclear condensation and fragmentation (B) and quantifications and results are plotted as mean % of at least three experiments ± standard deviation (C).
interact with Shp2, was also defective in its ability to activate Src kinases. Furthermore, the rapid kinetics of PDGF-BB-induced Erk5 phosphorylation was mirrored by equally rapid activation of Src kinases (data not shown). Moreover, it has been seen that Erk5, but not Erk1/2, is constitutively active in v-Src transformed NIH3T3 fibroblasts [19]. In conclusion, our data support the notion that the requirement of Shp2 in Erk5 activations stems from its involvement in Src activation. Erk5 has been shown to be important for cell survival of for example endothelial and neuronal cells [9,14]. In the neuronal cells it was demonstrated that loss of Erk5 expression resulted in reduced Akt and RSK phosphorylation and thereby increased expression of proapoptotic proteins Bad and Bim [9]. It is well established that Akt provides important survival signals downstream of growth factor receptors; therefore we investigated whether Erk5 is important also for PDGF-BB-induced Akt phosphorylation. Under normal conditions, PDGF-BB stimulation induced a sustained Akt phosphorylation, however, under conditions where Erk5 expression was reduced by siRNA, PDGF-BB was only able to promote a transient activation of
Akt. Thus, Erk5 appears not to be essential for the initial PI3-kinasedependent Akt activation, but is in contrast critical for prolonged Akt phosphorylation. It is possible that Erk5 phosphorylates and thereby inactivates or mislocates a phosphatase that otherwise would promote Akt dephosphorylation, alternatively, Erk5 may control the level of PIP3. An obvious Erk5 target candidate is PTEN that can restrict Akt signaling by dephosphorylating the lipid PIP3, but we have been unable to obtain data supporting an involvement of PTEN in the mechanism whereby Erk5 promotes Akt activation (data not shown). Consistent with the inability of PDGF-BB to promote sustained Akt phosphorylation when Erk5 was silenced, we observed that when Erk5 expression was reduced, PDGF-BB was not able to protect cells from activation of caspase 3 under serum-free conditions; however, addition of 10% FCS was able to efficiently block caspase 3 cleavage. Visualization and quantification of apoptotic morphological nuclear changes showed that under conditions with reduced Erk5 expression there was a significant increase in apoptotic nuclei both under serumfree conditions (10.7% vs. 1.2% in control cells) and in the presence of PDGF-BB (4.9% vs. 0.7%). In concurrence, it has been shown that the
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Acknowledgements This work was supported by the Ludwig Institute for Cancer Research, the Swedish Research Council and the Swedish Cancer Foundation.
References
Fig. 6. Schematic representation of Erk5 involvement in PDGF-BB-induced signal transduction. Stimulation of the PDGFRβ results in recruitment of Shp2 to the receptor and subsequently to Src activation. Activated Src then promotes activation of Erk5. Akt activation occurs in a PI3-kinase-dependent manner, but the sustained Akt phosphorylation requires Erk5. Akt is a key molecule in promoting cell survival through several mechanisms, including suppression of the caspases cascade.
loss of the Erk5 pathway makes fibroblasts more sensitive to sorbitolinduced caspase 3 activation and apoptosis [10,20]. Mechanistically, Akt can phosphorylate and thereby inactivate the initiator caspases 9 which lies upstream of caspase 3, thus establishing a link between Akt activation and inhibition of the caspase cascade [21]. In summary, our data suggest that Erk5 has a role in survival signaling by ensuring sustained Akt phosphorylation and suppression of caspase 3 cleavage. In conclusion, our data suggest the model depicted in Fig. 6. Upon PDGF-BB stimulation, Shp2 and Src family kinases are recruited to the receptor and Shp2 plays an important role in activation of Src family kinases. Src kinase activity is critical for the activation of Erk5 through a still poorly characterized mechanism. Erk5 is necessary for sustained activation of the pro-survival kinase Akt. In the absence of significant Erk5 expression PDGF-BB cannot suppress serum-starvation-induced caspase 3 cleavage in contrast to 10% FCS which efficiently inhibit caspase 3 activation. In concurrence, under conditions with reduced Erk5 expression PDGF-BB can only partially inhibit chromatin condensation and fragmentation in response to serum starvation, whereas 10% FCS can do so completely.
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