PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR
2006; Springer Nature; Volume: 25; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601059
ISSN1460-2075
AutoresKohji Nagano, Beat Bornhäuser, Gayathri D. Warnasuriya, Alan Entwistle, Rainer Cramer, Dan Lindholm, Søren Naaby‐Hansen,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoArticle13 April 2006free access PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR Kohji Nagano Kohji Nagano Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UKPresent address: Department of Proteomics Research, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Beat C Bornhauser Beat C Bornhauser Department of Neuroscience, University of Uppsala, Uppsala, SwedenPresent address: Department of Oncology, Childrens Hospital, University of Zyrich, Zyrich, Switzerland Search for more papers by this author Gayathri Warnasuriya Gayathri Warnasuriya Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Search for more papers by this author Alan Entwistle Alan Entwistle Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Search for more papers by this author Rainer Cramer Rainer Cramer Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Department of Biochemistry and Molecular Biology, University College London, London, UKPresent address: The BioCentre and School of Chemistry, University of Reading, Reading, UK Search for more papers by this author Dan Lindholm Dan Lindholm Department of Neuroscience, University of Uppsala, Uppsala, Sweden Minerva Research Institute, Biomedicum Helsinki, Helsinki, Finland Search for more papers by this author Soren Naaby-Hansen Corresponding Author Soren Naaby-Hansen Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Department of Biochemistry and Molecular Biology, University College London, London, UK Search for more papers by this author Kohji Nagano Kohji Nagano Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UKPresent address: Department of Proteomics Research, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Beat C Bornhauser Beat C Bornhauser Department of Neuroscience, University of Uppsala, Uppsala, SwedenPresent address: Department of Oncology, Childrens Hospital, University of Zyrich, Zyrich, Switzerland Search for more papers by this author Gayathri Warnasuriya Gayathri Warnasuriya Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Search for more papers by this author Alan Entwistle Alan Entwistle Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Search for more papers by this author Rainer Cramer Rainer Cramer Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Department of Biochemistry and Molecular Biology, University College London, London, UKPresent address: The BioCentre and School of Chemistry, University of Reading, Reading, UK Search for more papers by this author Dan Lindholm Dan Lindholm Department of Neuroscience, University of Uppsala, Uppsala, Sweden Minerva Research Institute, Biomedicum Helsinki, Helsinki, Finland Search for more papers by this author Soren Naaby-Hansen Corresponding Author Soren Naaby-Hansen Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK Department of Biochemistry and Molecular Biology, University College London, London, UK Search for more papers by this author Author Information Kohji Nagano1,‡, Beat C Bornhauser2,‡, Gayathri Warnasuriya1, Alan Entwistle1, Rainer Cramer1,3, Dan Lindholm2,4 and Soren Naaby-Hansen 1,3 1Ludwig Institute for Cancer Research, Royal Free and University College London, Medical School, London, UK 2Department of Neuroscience, University of Uppsala, Uppsala, Sweden 3Department of Biochemistry and Molecular Biology, University College London, London, UK 4Minerva Research Institute, Biomedicum Helsinki, Helsinki, Finland ‡These authors contributed equally to this work *Corresponding author. Ludwig Institute for Cancer Research, Royal Free and University College London Medical School, Courtauld Building, 91 Riding House Street, London W1W 7BS, UK. Tel.: +44 208 346 4948; Fax: +44 207 878 4040; E-mail: [email protected] The EMBO Journal (2006)25:1871-1882https://doi.org/10.1038/sj.emboj.7601059 Present address: Department of Proteomics Research, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan Present address: Department of Oncology, Childrens Hospital, University of Zyrich, Zyrich, Switzerland PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin–myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin -ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators. Introduction Changes in cellular morphology and the induction of locomotion in response to extracellular stimuli are mainly determined by concerted reorganization of the actin cytoskeleton, which forms a dynamic structural framework around which cell shape, polarity, motility and mitosis are defined. The organization of the actin filaments is coordinated by a multitude of accessory proteins whose functions are, in turn, regulated by many signals, including the activity of small GTPases, reversible phosphorylation, phosphoinositides, intracellular pH, oxygen radicals and free Ca2+ levels. Controlled spatiotemporal activation of Rho-family GTPases and their regulative interactions control the organization of F-actins and alter motility in many cell types, including fibroblasts. Several studies have identified myosin regulatory light chain (MRLC) and its regulatory molecules as a functional link between Rho and the formation of stress fibers (SFs) (Sellers et al, 1981; Ikebe et al, 1987; Satterwhite et al, 1992; Kimura et al, 1996; Kureishi et al, 1997; Hall, 1998), and phosphoactivation of MRLC is essential for the formation of actin SF and focal adhesion (FA) complexes (Totsukawa et al, 2000). SFs are bundles of actin filaments with associated myosin filaments and other key proteins of the actin–myosin-based contractile system found in nonmuscle cells. The ends of most SF are anchored to FA complexes, which are established by clustering of integrins and enable the forces generated by actomyosin contractions to be transferred to extracellular structures. While highly organized spatial orientation of the SF provides the cell with solid anchorage within tissue structures, the ability of actin polymers to assemble and disassemble rapidly within the core of retractile surface extensions enables migration. PDGF is a potent mitogen and a physiological chemotactic factor for fibroblasts. Short-term exposure to PDGF triggers dynamic cytoskeletal changes, including a reduction of SF and FA complexes and the formation of lamellipodia, whereas long-term exposure leads to the induction of motility (Bockus and Stiles, 1984; Ridley and Hall, 1994; Wennstrom et al, 1994). PDGF also suppresses SF and FA promotion induced by other stimuli, such as lysophosphatidic acid and bombesin, in a dose-dependent manner (Rankin and Rozengurt, 1994; Seufferlein and Rozengurt, 1994; Jimenez et al, 2000). Indeed, the ability of PDGF to cause disruption of SF and FA complexes in a variety of cell types is well established, but the regulatory mechanisms have, however, remained elusive (Jimenez et al, 2000). In the present study, we have investigated the molecular mechanisms activated by PDGF to change actin organization in 3T3 fibroblasts, in concurrence with altered adhesive characteristics and subsequent induction of motility. Specifically, the regulatory pattern and rate of post-translational modifications and turn-over of proteins, induced during both short- and long-term treatment with growth factors individually or in combinations, have been examined. Results Treatment with PDGF induces dynamic morphological changes in Swiss 3T3 cells, and dominates over EGF- and IGF-1-induced phenotypes The sequence and timing of actin cytoskeletal changes induced in Swiss 3T3 fibroblasts during sustained stimulation with PDGF, IGF-1, EGF and combinations thereof were investigated. PDGF induced a unique morphological phenotype, which was characterized by an elongated cell shape, dendritic protrusions and an attenuation of central SF and FA (Figure 1). While PDGF reduced SF and FA within the first 30 min, 12 h of exposure were required for the complete development of the dynamic surface protrusions. The appearance of mature protrusions coincided with a significant enhancement in the motility of PDGF-treated cells (see Supplementary data 1A). Analysis by time-lapse microscopy revealed that the increased motility, characterized by continuous extension and retraction of the dendritic protrusions and changes in cell shape, was sustained for the rest of the observation period (12–22 h). Although stimulation with EGF also increased membrane movements over the same time period, the fibroblasts remained stationary and no forward movement of their nuclei was observed (see Supplementary data 1B). Conversely, long-term stimulation with EGF, IGF-1 or both resulted in moderate increases in SF and FA formation, compared to nonstimulated cells; FA were found in 20% of nonstimulated cells, in 26% of IGF-1 and in 28% of EGF stimulated cells (Figure 1, top graph). EGF and IGF-1 induced a large, flat immotile phenotype and almost doubled the cell surface area compared to that of untreated or PDGF-stimulated cells (Figure 1, bottom graph). The EGF- or IGF-1-induced morphological changes were greatly influenced by cell confluence, whereas the PDGF-induced changes were less affected. This suggested that PDGF receptor signalling may over-ride that induced by cell–cell contacts in Swiss 3T3 cells. Furthermore, costimulation experiments demonstrated that PDGF had a dominant influence on morphology over EGF and IGF-1, resulting in the formation of dendritic protrusions and suppression of SF and FA, even in cells simultaneously exposed to all three GFs (Figure 1). In contrast, costimulation with EGF plus IGF-1 increased the formation of SF and FA in about 25% of the murine fibroblasts. Figure 1.Cytoskeletal changes induced by long-term stimulation with PDGF, IGF-1, EGF and their combinations. Swiss 3T3 cells were stimulated with growth factors for 18 h and stained with α-vinculin antibody and rhodamine–phalloidin. Arrowheads and arrows show FAs and dendritic protrusions, respectively. Note the dominance of PDGF in costimulation experiments, resulting in an elongated cell shape with dendritic protrusions and disruption of SFs and FAs. Download figure Download PowerPoint PDGF-specific reduction of MRLC expression A functional proteomic analysis was undertaken to identify changes in the 3T3 fibroblast proteome, which defined the resulting morphological phenotypes. The rate of protein synthesis was monitored by biosynthetic labelling with [35S]-methionine and -cysteine, and GF-induced changes in the expression of proteins were determined by quantitation and comparison of the protein spots' volumes and intensities on fluorescence-stained gel images. The three growth factors induced de novo synthesis of several hundred protein isoforms within the first 6 h of treatment, in addition to the 631, which were radiolabelled in untreated cells (Supplementary Figure 1). While the vast majority of the detected proteins showed similar regulation over time, 21% of the matched spots were differentially regulated by the three GFs. In all, 117 unique gene products were identified by mass spectrometry (ms) analysis of 115 differentially regulated protein features. PDGF stimulation induced the highest overall rate of protein synthesis at any given time, followed by IGF-1 and then EGF, as judged by the total quantity of radioisotope incorporation measured in the 3T3 cells' proteome by autoradiography, following separation on broad pH-range two-dimensional (2D) gels (Supplementary Figure 1). To facilitate identification of proteins whose specific regulation contribute to the establishment of a motile cytoskeleton in 3T3 fibroblasts, we focused on proteins that were dominantly induced or suppressed by PDGF in costimulation experiments. A newly synthesized 22 kDa acidic protein, which was highly abundant in untreated, and EGF- and IGF-1-treated cells, was strongly and dominantly suppressed by PDGF (Figure 2A, B and D). Mass spectrometry analysis identified the protein as MRLC 2A, a key regulator of the actin–myosin-based contractile system (Fukata et al, 2001). In all, 14 peptides were identified by matrix-assisted laser desorption/ionization analysis, corresponding to 61% of the primary structure of MRLC 2A. De novo sequencing by electrospray ionization–ms/ms analysis confirmed the amino-acid sequence in two of these peptides (data not shown). Of the 23 cytoskeletal regulators, we identified based on GF-specific radiolabelling, only MRLC was dominantly suppressed by both short- and long-term PDGF-stimulation. Figure 2.PDGF-specific suppression of MRLC. (A) Abundance of radiolabelled MRLC in growth factor-treated Swiss 3T3 fibroblasts at 6, 12 and 18 h (encircled in black). Protein synthesis was studied by labelling with [35S]-methionine and -cysteine during the last 3 h of stimulation. (B) Changes in the abundance of nascent acidic MRLC induced by PDGF, IGF-1 and EGF (left side of graphs), and the effects of costimulation for 18 h (right). Quantitative data represent average of three independent analyses of cells at different times. (C) Semiquantitative RT–PCR analysis of MRLC 2A gene expression in response to growth factor stimulation. (D) PDGF treatment dominantly suppressed the levels of nascent MRLC in costimulation experiments at 18 h. The acidic isoform is encircled in white, and indicated by horizontal arrow pointing left. (E) Effect of various inhibitors on the acidic MRLC isoform in cells exposed to PDGF for 18 h. DMSO (1 μl/ml), LY294002, rapamycin, PD98059 or PS-341 in DMSO was added 1 h before and the 3 h labelling period. (F) Immunoblots of MRLC after GF stimulation. Staining was performed with anti-MLC and β-actin antibodies from Sigma. (G) MRLC levels in controls and in cells stimulated for 4 h with PDGF in the presence of cycloheximide (CHX) (10 μg/ml) or CHX plus lactacystin (50 μM). (H) Changes in MRLC expression after CHX-chase. Cells were stimulated with GF for 1 h before CHX. Stained with anti-MLC and anti β-actin antibodies as above. (I) Localization of MRLC. Growth factor-treated 3T3 cells were stained with anti-MLC antibody (1:25; Sigma) and rhodamine–phalloidin for visualization of actin. Colocalization with actin SFs is indicated by arrowheads in left images and local concentrations at the termination of the PDGF-specific surface extensions are indicated by arrowheads to the right. Download figure Download PowerPoint Semiquantitative RT–PCR analysis demonstrated slightly increased MRLC gene expression in PDGF-treated cells compared to the levels detected in EGF- and IGF-1-treated cells (Figure 2C), indicating that the PDGF-specific reduction occurred after transcription. The abundance of newly synthesized, 35S-labelled MRLC copies in PDGF-stimulated or -costimulated cells was reduced to less than 10% of the levels detected in untreated, and EGF- and IGF-1-treated cells at the 18 h time point (Figure 2A and B). Treatment of cells with both EGF and IGF-1 induced a number of newly synthesized MRLC copies, similar to that detected in cells stimulated with IGF-1 alone (Figure 2B). Cells treated with PDGF in the presence of 10 μM LY294002 or 50 μM PD98059, inhibitors of phosphatidylinositol-3 kinase (PI3 K) and MAPK kinase (MEK), respectively, showed no increase in isotope labelling of the 22 kDa MRLC form, indicating that the suppressive effects caused by PDGF occurred independently of the pathways regulated downstream of these kinases (Figure 2E). Inhibition of mTOR kinase by treatment with 50 nM rapamycin reduced the abundance of newly synthesized MRLC in PDGF-treated cells further (Figure 2E), suggesting that its translation was under the control of the p70s6 kinase. PDGF-induced reduction of newly synthesized MRLC in 3T3 cells was, in contrast, reversed completely by treatment with PS-341 (1 μM), a specific inhibitor of the 26S proteasome (Adams et al, 1999) (Figure 2E). While inhibition of the 26S proteasome had no effect on the abundance of radiolabelled MRLC copies in untreated cells (Figure 2E, lower panel), the number of newly synthesized species found in cells treated with PDGF in the presence of a proteasome inhibitor was significantly higher than those detected in untreated, and EGF- and IGF-1-stimulated cells (Figure 2A and E). Immunoblotting of whole-cell extracts showed that the abundance of MRLC in cells stimulated with PDGF was only marginally reduced compared to the levels found in IGF-1- and EGF-treated cells after 18 h (Figure 2F). Taken together, these findings suggest that PDGF augment the turn-over rate of MRLC in Swiss 3T3 fibroblasts by increasing its transcription and translation rates, and promoting its removal via the 26S proteasome. To investigate this further, the abundance of MRLC at various time points after the cells de novo protein synthesis was blocked by addition of cycloheximide (10 μg/ml) was examined (Figure 2G and H). While the half-life of MRLC was more than 18 h in untreated and IGF-1-stimulated cells, it was significantly shortened by PDGF treatment (Figure 2G). The addition of lactacystin (50 μM), a proteasome-specific inhibitor structurally distinct from PS-341, to the cycloheximide-chase experiments abrogated the enhanced removal of MRLC from PDGF-treated cells, but had no effect on MRLC expression in untreated or IGF-1-stimulated cells (Figure 2G and H). These findings confirmed that the turn-over rate of MRLC is significantly increased in PDGF-treated cells and further support the notion that PDGF targets the actin–myosin regulator for proteasome-mediated degradation. Immunofluorescent staining showed colocalization of MRLC and actin SFs in nonstimulated, and EGF- and IGF-1-stimulated fibroblasts, and that the concentrations of both proteins were elevated at the base of the adhesion plaques situated at the end of the bundled fibers (yellow arrowheads in Figure 2I). MRLC was also localized in the nuclei of nonstimulated (35%), EGF (31%), IGF-1 (30%) and costimulated (EGF and IGF-1: 32%) cells, whereas in PDGF-stimulated cells, MRLC was found in the cytoplasm and dendritic protrusions, but rarely in the nucleus (<10%) (Figure 2H). Staining for MRLC was additionally found to decorate the arched actin filaments seen in the periphery and at the termination of PDGF-specific surface protrusions (orange arrowheads in Figure 2H). Finally, costimulation with PDGF together with EGF or IGF-1 abolished nuclear presence of MRLC, showing a distribution that could not be distinguished from that generated by PDGF treatment alone (Figure 2H). These findings imply that in addition to shortening the half-life of MRLC, PDGF also dominantly regulates its intracellular distribution in Swiss 3T3 cells. PDGF-induced degradation of MRLC via the ubiquitin–proteasome system. Inhibition of 26S proteasome activity, by the addition of 1 μM PS-341 during the last 4 h of a 18 h treatment period, multiplied the abundance of the 22 kDa MRLC form in PDGF-stimulated cells (encircled and indicated by horizontal arrow in Figure 3A). This also caused several high molecular weight (HMW) protein species that were immunologically crossreactive with MRLC, but not found in extracts from cells treated with PDGF alone, to be detected (framed by black rectangles in Figure 3A). The vertical appearance and abundance of these 140–160 kDa, PS-341-sensitive species (indicated by oblique arrow) suggested that the MRLC was polyubiquitinated in response to PDGF. Figure 3.PDGF-specific ubiquitination of MRLC in 3T3 fibroblasts. (A) Cells were treated with PDGF for 18 h and 150 μg of proteins separated by 2DE. MRLC was detected by immunoblotting with a murine IgM mAb (1:200; NEB). The 22 kDa isoform migrating at a pI of 4.55 is encircled in black, and indicated by horizontal arrow at the right. (B) Controls- (C) and PDGF- (P) treated cells were lysed and MRLC isolated by immunoprecipitation (1:25; sc-9448). Immunostaining with anti-ubiquitin antibody (1:1000; Affiniti Research Products, PW8810) demonstrated increased ubiquitination of MRLC in treated cells. The poly-ubiquitin-MRLC form migrated at a molecular weight of approximately 150 kDa, similar to the size of the HMW immunoreactive species indicated by oblique upwards arrows in (A). (C) Polyubiquitinated MRLC was increased by PDGF stimulation for 1 h in the presence of 1 μM PS-341, compared with that in EGF- or IGF-1-treated cells. Costimulation with PDGF also enhanced MRLC ubiquitination. The anti-ubiquitin-stained membrane was subsequently stripped and reprobed with antibody against MRLC (1:200; NEB) (bottom row). (D) The membrane was restained to show clearly the presence of ubiquitinylated MRLC at 150 kDa. (E) The 150 kDa polyubiquitinated MRLC was less abundant in IGF-1- than in PDGF-stimulated cells. Download figure Download PowerPoint Immunostaining of immunoprecipitated (IP) MRLC with antibody against ubiquitin confirmed that PDGF induced ubiquitination of MRLC (Figure 3B and C). Proteasome inhibition significantly increased the abundance of a 150 kDa, PDGF-induced polyubiquitinylated MRLC form, in agreement with the HMW antigens detected by 2D IB under similar conditions. Ubiquitination of MRLC occurred at a several-fold higher rate in the PDGF-treated cells than in nontreated, EGF- or IGF-1-treated cells (Figure 3C, top row). Stripping and reprobing of the membrane with an antibody against MRLC and staining of IP samples from IGF-1- and PDGF-treated cells with antibody against MRLC confirmed that the 150 kDa PDGF-specific protein band represented polyubiquitinylated MRLC (Figure 3C–E). Consistent with the dominant suppressive effect of PDGF, enhanced rates of MRLC ubiquitination were also found in cells costimulated with this growth factor together with EGF and/or IGF-1. The levels of polyubiquitinated MRLC detected in cells, costimulated with all three growth factors for 1 h, was more than fivefold higher than those found in nontreated, EGF- or IGF-1-stimulated cells (Figure 3C). Together, these results demonstrated that PDGF controlled MRLC activity in 3T3 fibroblasts by regulating its expression through targeted degradation. PDGF-specific induction of the E3-ligase MIR in 3T3 fibroblasts To identify the mechanism mediating the effects of PDGF on MRLC ubiquitination, the expression of MRLC-interacting protein (MIR), an E3-ligase (Olsson et al, 1999) recently shown to promote ubiquitination of MRLC in N2-A neuroblastoma cells (Bornhauser et al, 2003), was examined. The expression of MIR in 3T3 fibroblasts was strongly upregulated within minutes of exposure to PDGF (Figure 4A). Maximum levels of MIR were observed after 1–2 h stimulation, and its expression remained elevated during sustained exposure (Figure 4A). MIR synthesis was several fold higher in PDGF-stimulated cells than in nontreated fibroblasts and in cells stimulated with IGF-1 or EGF, at all time points (Figure 4A and B). The induction of MIR by PDGF was prevented by pretreating the cells with 2 μM SU6656, a Src-kinase family selective inhibitor (Figure 4B). Intriguingly, MRLC phosphorylated at threonine 18 and serine 19 copurified with MIR in immunoprecipitates from PDGF-stimulated, but not from -untreated cells (Figure 4C). While PDGF induces ubiquitination of diphosphorylated MRLC, it simultaneously increases the abundance of Ser19-phosphorylated MRLC compared to the levels found in untreated and EGF-stimulated 3T3 fibroblasts (Supplementary Figure 2). Figure 4.Induction of the MIR E3-ligase by PDGF is abrogated by Src-kinase inhibition. (A) Increase in the 50 kDa MIR ubiquitin-ligase after GF stimulation. Rabbit anti-MIR antiserum was used at 1:3000. The levels were increased at 15 min, with a maximum attained at 1 h after PDGF stimulation. (B) Effects of DMSO, EGF, PDGF or PDGF plus 2 μM SU6656 (all in DMSO) on MIR. (C) Copurification of MIR and phosphorylated MRLC. Cells were labelled with 33P-ortho-phosphate for 6 h before PDGF stimulation and MIR isolated by immunoprecipitation. A 22 kDa phosphoprotein specifically copurified with MIR is indicated by oblique downward arrow in the autoradiogram (left). Subsequent staining of the membrane with the antibody specific for MRLC 2 phosphorylated at Thr18 and Ser19 (no. 3674; Cell Signaling) is shown to the right. (D) Inhibition of Src-kinase activity by SU6656 abolished the PDGF-specific increase in MRLC degradation. (E) Morphology of Swiss 3T3 cells after 18 h GF stimulation in the presence and absence of 2 μM SU6656. (F) 2D autoradiograms of phosphoisotope-labelled proteins immunoprecipitated with a monospecific antibody against hnRNP-K protein (enlarged gel area is shown). The direction of IEF and SDS–PAGE is indicated in the upper right corner, and in all images, the acidic side is to the left. A hnRNP-K phosphoform induced in response to all three GFs, but absent in untreated cells, is indicated by white downward arrows. The position of hnRNP-K was confirmed by immunoblotting (G) and is indicated in the image from EGF-treated cells. Note the increased hnRNP-K phosphorylation in PDGF-stimulated and -costimulated cells, and the appearance of new phosphoforms (oblique downward arrows in PDGF images). A copurified protein (indicated by a horizontal black arrow) migrating slightly lower than hnRNP-K and demonstrating increased phosphorylation in IGF-1- and PDGF-stimulated cells was identified as p85α using a mixture of the mAbs U9 and U14 (H). IGF-1- and PDGF- but not EGF-induced PI3 K activity in the 3T3 cells (see Supplementary Figure 2), and phospho-activation of hnRNP-K-associated p85 coincites with increased PI3 K activity in IGF-1- and PDGF-treated cells, further verifying the specificity of the phosphoisotope labeling procedure. (I) RT–PCR amplification of hnRNP-K associated MIR mRNA isolated by immuno precipitation from samples from nonstimulated and cells exposed to GF for 60 min using the monospecific antibody 54. Download figure Download PowerPoint Consistent with Src-dependent MIR induction being responsible for the enhanced removal of MRLC from PDGF-treated cells, the presence of 1 μM SU6656 prevented PDGF-specific MRLC degradation in cycloheximide-chase experiments, but had no effect upon its expression in IGF-1-treated cells (Figure 4D). Inhibition of Src activity also blocked the establishment of the elongated motile morphology normally induced by long-term PDGF stimulation, resulting in formation of a flat, immotile cell shape with an enlarged surface area and an abundance of FA typically found in EGF- and IGF-1-stimulated 3T3 cells (Figures 1 and 4E). In contrast, inhibition of Src activity did not affect the flat, immotile morphology induced by EGF and IGF-1. These findings link PDGF-induced, Src-dependent MIR activity to the fate of MRLC and morphological differentiation of fibroblasts, and suggest that the association between the E3-ligase and MRLC is regulated by the phosphorylation status of the substrate. A recent report suggested that RNA-binding proteins may be involved in the regulation of cell spreading (de Hoog et al, 2004). This analysis of GF-induced phosphosignalling in 3T3 cells showed a strong increase in the phosphorylation of hnRNP-K protein soon after stimulation with PDGF (Figure 4F). Phosphorylation of hnRNP-K by a c-Src-regulated mechanism has previously been shown to regulate translation of hnRNP-K-associated mRNAs (Ostareck-Lederer et al, 2002), and here PDGF treatment dominantly increased hnRNP-K phosphorylation with induction of new specific acidic phosphoforms, one of which was also detected in cells costimulated with all three GFs (oblique downward
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