The G protein-coupled receptor kinase-2 is a TGFβ-inducible antagonist of TGFβ signal transduction
2005; Springer Nature; Volume: 24; Issue: 18 Linguagem: Inglês
10.1038/sj.emboj.7600794
ISSN1460-2075
AutoresJoanne Ho, Eftihia Cocolakis, Víctor Dumas, Barry I. Posner, Stéphane A. Laporte, Jean‐Jacques Lebrun,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle25 August 2005free access The G protein-coupled receptor kinase-2 is a TGFβ-inducible antagonist of TGFβ signal transduction Joanne Ho Joanne Ho Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Eftihia Cocolakis Eftihia Cocolakis Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Victor M Dumas Victor M Dumas Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Canada Search for more papers by this author Barry I Posner Barry I Posner Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Canada Search for more papers by this author Stéphane A Laporte Stéphane A Laporte Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Jean-Jacques Lebrun Corresponding Author Jean-Jacques Lebrun Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Joanne Ho Joanne Ho Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Eftihia Cocolakis Eftihia Cocolakis Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Victor M Dumas Victor M Dumas Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Canada Search for more papers by this author Barry I Posner Barry I Posner Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Canada Search for more papers by this author Stéphane A Laporte Stéphane A Laporte Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Jean-Jacques Lebrun Corresponding Author Jean-Jacques Lebrun Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada Search for more papers by this author Author Information Joanne Ho1, Eftihia Cocolakis1, Victor M Dumas2, Barry I Posner2, Stéphane A Laporte1 and Jean-Jacques Lebrun 1 1Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada 2Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Canada *Corresponding author. Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Canada H3A 1A1. Tel.: +1 514 934 1934x34846; Fax: +1 514 982 0893; E-mail: [email protected] URL: www.hcru.mcgill.ca The EMBO Journal (2005)24:3247-3258https://doi.org/10.1038/sj.emboj.7600794 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signaling from the activin/transforming growth factor β (TGFβ) family of cytokines is a tightly regulated process. Disregulation of TGFβ signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFβ-signaling cascade. TGFβ-induced expression of GRK2 acts in a negative feedback loop to control TGFβ biological responses. Upon TGFβ stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFβ-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFβ-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction. Introduction The transforming growth factor β (TGFβ) superfamily of growth factors represents a large group of pluripotent polypeptides, comprised of activins, TGFβs and bone morphogenetic proteins (BMPs) among others, that regulate cell growth, differentiation and apoptosis, in nearly all cell types (Shi and Massague, 2003). TGFβ, activin and their receptors are widely expressed in all tissues, and the regulatory role played by these growth factors is of central importance to human diseases. The involvement of these growth factors in human cancer is multifaceted. While they initially contribute to tumor suppression by efficiently inhibiting the proliferation of cells derived from epithelial, endothelial and hemaetopoietic origins, the TGFβ growth-inhibitory responses in cancer cells are often replaced by invasive and prometastatic responses as tumors progress, highlighting the dual role of TGFβ as both a tumor suppressor and a tumor-promoting agent (Derynck et al, 2001; Wakefield and Roberts, 2002). The current paradigm of TGFβ/activin signal transduction begins with ligand binding to a single-transmembrane-spanning, constitutively auto-phosphorylated serine/threonine kinase, type II receptor. Following ligand binding, the type II receptor recruits and transphosphorylates the type I receptor within a juxtamembrane glycine and serine-rich region, thereby activating the kinase activity of the type I receptor. The activated type I receptor then phosphorylates intracellular mediators known as receptor-regulated Smads (R-Smads), Smad2 and 3, on their carboxy-terminal serine residues (SxS motif). This phosphorylation event results in the release of auto-inhibitory intramolecular interactions between the Mad-homology 1 (MH1) and Mad-homology 2 (MH2) domains of Smads 2 and 3, allowing for their subsequent heterodimerization with their common partner, Smad4. Once activated, the Smad complex translocates into the nucleus, where it associates with coactivators or corepressors of transcription to regulate the expression of various target genes (Shi and Massague, 2003). Although mechanistically simple, the activin/TGFβ signaling cascade has numerous auto-regulatory mechanisms that exist to maintain a tightly regulated ligand-induced message. The inhibitory Smad7 functions through a negative feedback loop mechanism to terminate signaling by sterically preventing access of Smad2/3 to the kinase domain of the type I receptor (Hayashi et al, 1997; Nakao et al, 1997) and by recruiting protein phosphatases and ubiquitin ligases to the activated TGFβ receptor (Kavsak et al, 2000; Shi et al, 2004). Receptor internalization and/or downregulation are also now emerging as predominant means of regulating signaling from the TGFβ pathways (Di Guglielmo et al, 2003). These receptors can be constitutively internalized through clathrin-independent or -dependent mechanisms, which may involve the recruitment to the TGFβ receptor of endocytic adaptors like AP-2 and βarrestins (Yao et al, 2002; Chen et al, 2003). At the level of the R-Smads, nuclear translocated Smad2 and 3 can be subject to ubiquitin–proteasome-mediated degradation (Lo and Massague, 1999) or dephosphorylation (Xu et al, 2002), leading to termination of signaling. Crosstalk from other signaling pathways, which interferes with the nuclear translocation of the Smads, can further act to negatively regulate Smad-dependent signal transduction (Kretzschmar et al, 1997, 1999). In fact, nonreceptor kinases have been shown to utilize the R-Smads as substrates to regulate TGFβ signaling (Wicks et al, 2000; Matsuura et al, 2004; Waddell et al, 2004). In the present study, we describe a novel inhibitory mechanism downstream of activin/TGFβ. We have identified GRK2 to act in a negative feedback loop downstream of the activin/TGFβ receptors. GRK2 is known to phosphorylate a large number of G protein-coupled receptors (GPCRs), leading to the uncoupling of the GPCRs from their heterotrimeric G proteins, subsequent receptor desensitization and downregulation to terminate signaling (Pitcher et al, 1998). Although GRK2 is critical to GPCR signaling and has been shown to be involved in receptor tyrosine kinase desensitization (Freedman et al, 2002), to date no implications of GRK2 acting downstream of single-transmembrane serine/threonine kinase growth factor receptors have been established. We show here that GRK2 expression levels are upregulated in response to activin/TGFβ signaling, and that GRK2 physically interacts with the MH1 and MH2 domains of the receptor-regulated Smads and phosphorylates their linker region on a specific single serine/threonine residue. GRK2-induced Smad phosphorylation blocks activin/TGFβ-induced Smad activation, nuclear translocation and target gene expression. The net effect of GRK2 on activin/TGFβ responses leads to an inhibition of their antiproliferative and proapoptotic functions. We further demonstrate that GRK2 antagonizes activin/TGFβ responses ex vivo and potently inhibits activin-mediated cell death in primary hepatocytes from liver perfused animals. Thus, GRK2 appears as a novel TGFβ antagonist that strongly inhibits activin/TGFβ-mediated cell growth arrest and apoptosis in both normal and cancer liver cells. Results and discussion Activin/TGFβ induces cell growth arrest and apoptosis in human hepatocarcinoma cells Human hepatocellular carcinoma (HuH7) and hepatoblastoma (HepG2) cells, treated with activin or TGFβ for 72 h, led to a clear inhibition of cell growth (Figure 1A). Using flow cytometry (FACS) analysis, we found that activin/TGFβ regulates cell growth of these two hepatocarcinoma cell lines by inhibiting cell proliferation (G1 arrest) and inducing apoptosis (Figure 1B). The strong proapoptotic effect of these growth factors was confirmed by Annexin V/propidium iodide (PI) staining (Figure 1C). Collectively, these results indicate that the two human hepatoma cell lines, HepG2 and HuH7, respond in a highly similar manner to activin and TGFβ. These findings are consistent with previous studies demonstrating that activin and TGFβ play a major role in regulating liver function by modulating growth arrest and apoptosis in normal and cancer liver cells (Oberhammer et al, 1992; Yasuda et al, 1993; Ho et al, 2004). Figure 1.Activin/TGFβ's biological effect in human heptocarcinoma cells. (A) Normal HepG2 and HuH7 cells were stimulated or not with either activin or TGFβ for 72 h before cell growth was measured by cell viability colorimetric (MTT) assay. Values are representative of three independent experiments performed in triplicate, and are expressed in arbitrary units. P<0.05 compared with no activin/TGFβ treatment. (B) HepG2 and HuH7 cells were stimulated or not with either activin or TGFβ for 48 h. The distribution of cells in the cell cycle was quantified by analysis of PI-stained cells using flow cytometry, and the data are representative of three independent experiments. (C) HepG2 and HuH7 cells either treated or not with activin for 0, 8 and 24 h were labeled for Annexin V-FITC and PI to ensure that necrotic cells were not included in the analysis (data not shown) and analyzed by confocal microscopy. Download figure Download PowerPoint Activin/TGFβ selectively induces GRK2 expression in human hepatocarcinoma cells To identify novel activin/TGFβ target genes which may be responsible for mediating their growth-inhibitory effects, we performed Affymetrix human Gene Chip U95A microarray experiments using activin or TGFβ-treated human hepatocarcinoma (HuH7) cells. From our microarray experiments, we found the mRNA level of the GPCR kinase-2 (GRK2) to be significantly increased in HuH7 cells treated for 8 h with activin or TGFβ (3.5 and 3, respectively). Our initial microarray findings were verified by Northern blot analysis (Figure 2A). Figure 2.Activin/TGFβ induces upregulation of GRK2 in human hepatocarcinoma cells. (A) HuH7 cells were stimulated with activin for 0, 1, 2, 4, 8, 16 and 24 h, and total RNA was analyzed by Northern blot using specific probes for GRK2 (upper panel). Equal loading was assessed by ethidium bromide staining (lower panel). (B) Reverse transcription reactions were performed using oligo-dT and cDNAs were amplified for 30 cycles using specific oligonucleotides to GRKs 2–6 and GAPDH as a control. The samples were treated or not with the translation inhibitor cycloheximide and stimulated or not with activin for 16 h. (C) HuH7 and HepG2 cells were stimulated with activin for 0, 4, 8, 16 and 24 h and total cell lysates were analyzed by Western blot using a specific monoclonal antibody against GRK2 (upper panels) or tubulin (lower panels). (D) Vascular smooth muscle and MCF7 breast cancer cells were stimulated or not with activin for 24 h and total cell lysates were analyzed by Western blot using a specific monoclonal antibody against GRK2 (upper panels) or tubulin (lower panels). Download figure Download PowerPoint The activin-induced increase in GRK2 mRNA levels was further confirmed by RT–PCR using primers specific for GRK2, and appeared to occur through a direct transcriptional regulatory mechanism, as it was not affected by treatment with the translational inhibitor cycloheximide (Figure 2B). To determine whether activin/TGFβ could also induce expression of other GRK family members, semiquantitative RT–PCR experiments were performed in HuH7 cells treated or not with activin, and, as shown in Figure 2B, only GRK2 levels were affected by activin treatment. Thus, this suggests that activin selectively regulates GRK2 mRNA levels in these cells. Consistent with the increase in the mRNA levels of GRK2, we also observed an increase in GRK2 protein levels in response to activin in the two hepatocarcinoma cell lines, HuH7 and HepG2 (Figure 2C). This effect is not liver specific, as activin was also able to induce GRK2 protein expression levels in breast cancer cells (MCF7) and vascular smooth muscle cells (VSMC), two distinct activin-responsive cell lineages (Figure 2D). Thus, our findings identify activin/TGFβ to be key modulators of the expression levels of GRK2 in both normal and cancer cells. GRK2 inhibits activin/TGFβ-mediated gene expression and cell growth arrest To determine the role of an increase in GRK2 expression on activin/TGFβ-mediated signal transduction, we cotransfected HuH7 liver cells with an activin/TGFβ-responsive gene promoter construct (3TP-luc) fused to the luciferase gene. As shown in Figure 3A, in the presence of GRK2, a 75% decrease in 3TP-luc activity was observed upon activin stimulation, as compared to cell expressing 3TP-luc alone. Moreover, transfection of a GRK2 mutant in which the kinase domain was inactivated by point mutation (K220M) reversed this inhibitory effect, indicating that the kinase domain of GRK2 is required for inhibition of activin-induced target gene expression (Figure 3A). Figure 3.GRK2 inhibits activin-mediated Smad2 phosphorylation, gene transcription, cell growth arrest, apoptosis and target gene expression. (A) HuH7 cells were cotransfected with pcDNA3, GRK2-WT or GRK2-K220M and the 3TP-luc reporter construct, together with a β-galactosidase expression plasmid. Cells were treated with activin for 18 h and luciferase assays were performed. *P<0.05 compared with no activin treatment. (B) HuH7 cells were cotransfected with pcDNA3, GRK2-WT, GRK3-WT, GRK4-WT, GRK5-WT or GRK6-WT and the 3TP-luc reporter construct, together with a β-galactosidase expression plasmid. Cells were treated with activin or TGFβ for 18 h and luciferase assays were performed. *P<0.05 compared with no activin treatment. (C) Magnetically enriched HuH7 GFP-expressing cells and HuH7 GFP-GRK2-expressing cells were stimulated or not with activin for 72 h before cell growth was measured by cell viability MTT assay. Values are expressed in arbitrary units. P<0.05 compared with no activin treatment. (D) WT CHO cells, CHO cells stably overexpressing either GRK2 or GRK2-K220M were stimulated or not with TGFβ for 48 h. The distribution of cells in the cell cycle was quantified by analysis of PI-stained cells using flow cytometry, and the data are representative of three independent experiments. (E) WT CHO cells, CHO cells stably overexpressing either GRK2 or GRK2-K220M, either treated or not with activin for 8 h and labeled with Annexin V-FITC and PI (data not shown), and analyzed by confocal microscopy. (F) HuH7 cells were mock transfected, or transfected with a scrambled siRNA oligonucleotide sequence, or with a specific siRNA sequence to GRK2. Cells were treated with activin for 24 h and total cell lysates were analyzed by Western blot using either specific polyclonal antibodies against Bax (first panel), p15 (second panel) or c-myc (third panel), or using monoclonal antibodies against GRK2 (fourth panel) or tubulin (fifth panel). Download figure Download PowerPoint Given the high homology of the kinase domains of the GRK family members, we examined whether any of the other GRKs could have potential effects upon activin/TGFβ-induced promoter activity. Similar to GRK2, overexpression of any of GRKs 3, 4, 5 or 6 resulted in an inhibition of activin/TGFβ-induced 3TP-luc promoter activity (Figure 3B). This ability of the other GRK family members to block activin/TGFβ-induced 3TP-luc promoter activity emphasizes the importance of the highly homologous kinase domain of the GRKs in mediating their inhibitory effect upon activin/TGFβ signaling. Having established that activin/TGFβ treatment induces an upregulation of GRK2 protein levels in liver cells and that overexpression of GRK2 inhibits activin/TGFβ-mediated gene transcription, we next examined the effect of GRK2 on activin/TGFβ-induced cell growth inhibition. For this, HuH7 hepatocarcinoma cells were cotransfected with a GFP-tagged GRK2 (GFP-GRK2) expression construct and a bicistronic pMACS Kk II vector encoding the truncated mouse H-2Kk surface marker. MACselect Kk microbeads conjugated to a monoclonal antibody directed against the surface marker H-2Kk were then used to magnetically select, enrich and purify GFP-GRK2/H-2Kk-overexpressing cells. Control GFP/pMACS-transfected cells, which went through a similar purification process, and GFP-GRK2-positive cells were subsequently treated or not with activin, and cell viability was assessed by MTT assay. In control HuH7 cells, activin stimulation resulted in a 42% cell growth inhibition, as compared to cells that had been left untreated (Figure 3C). In contrast, in an enriched population of overexpressing GFP-GRK2 HuH7 cells, activin-mediated cell growth inhibition was reversed. Furthermore, we performed FACS analysis on either wild-type (WT) Chinese hamster ovary (CHO) cells, CHO cells stably overexpressing GRK2 or the kinase-inactive (K220M) mutant. As seen in Figure 3D, TGFβ treatment of WT-CHO cells clearly induced an increase in apoptosis, and, to a more modest extent, G1 arrest. However, stable overexpression of GRK2 reversed these TGFβ-mediated effects, while overexpression of the kinase-inactive mutant GRK2 had no effect. GRK2's inhibitory effect upon activin-induced apoptosis was further confirmed by Annexin V staining. As shown in Figure 3E, the activin-induced onset of apoptosis, detectable following activin treatment of WT cells, was clearly inhibited in GRK2 stable cells, while the K220M stable cells behaved in a manner similar to control cells. These findings indicate that elevated levels of GRK2 serve to antagonize activin/TGFβ regulation of cell growth and apoptosis. Activin/TGFβ-induced apoptosis and G1 arrest are mediated through upregulation of proapoptotic factors, such as Bax (Kanzler and Galle, 2000), and cyclin-dependent kinase inhibitors, such as p15 (Beach, 1994; Reynisdottir and Massague, 1997; Ho et al, 2004), and downregulation of proto-oncogenic factors, such as c-myc (Frederick et al, 2004). To examine the effect of GRK2 on the expression levels of these known activin/TGFβ target genes, we used siRNA to specifically block GRK2 expression in liver cancer cells. In both normal HuH7 cells and cells transfected with a scrambled siRNA, activin stimulation led to a clear increase in both endogenous Bax and p15 expression levels, as well as a decrease in c-myc protein levels (Figure 3F). However, decreasing endogenous GRK2 levels using a specific GRK2 siRNA resulted in a potentiated activin-induced upregulation of Bax and p15, coupled with a robust activin-induced downregulation of c-myc (Figure 3F). Thus, removal of GRK2 activity in liver cancer cells leads to increased activin/TGFβ regulation of target genes, which may contribute to GRK2's antagonistic effect upon activin/TGFβ-induced growth inhibition and apoptosis. Collectively, these findings indicate that GRK2 overexpression antagonizes activin/TGFβ-mediated transcriptional activity, target gene expression, G1 arrest and apoptosis in human hepatocarcinoma cells. Taken together, these results also suggest that GRK2 acts in a negative feedback loop manner to block activin/TGFβ-mediated physiological effects on cell growth inhibition, thus defining the kinase GRK2 as a multipotent inhibitor of activin/TGFβ signaling. Norepinephrine (NE) induces GRK2 expression and antagonizes activin-induced cell growth inhibition Previous studies have demonstrated that NE, which signals through the GPCR α1-adrenergic receptor (α1-AR), can increase GRK2 gene promoter activity (Ramos-Ruiz et al, 2000). As such, we decided to examine if NE could increase GRK2 protein expression levels in human hepatocarcinoma cells. As shown in Figure 4A, NE treatment resulted in a clear increase in GRK2 protein expression levels. We then tested whether utilizing NE to upregulate GRK2 levels would have an antagonistic effect upon activin-induced cell growth inhibition. As shown in Figure 4B, activin strongly inhibited cell growth, while NE by itself had no effect. Interestingly, a 24-h pretreatment of HuH7 cells with NE, prior to activin stimulation, led to a partial reversal of the activin-inhibitory effect, indicating that these two growth factors exert antagonistic effects in human liver cells, and further supports a role for GRK2 as an antagonist to activin-mediated signaling. Figure 4.NE induces upregulation of GRK2 and antagonizes activin growth-inhibitory effect. (A) HuH7 cells were stimulated with NE for 0, 4, 8, 16 and 24 h. Total cell lysates were analyzed by Western blot using a specific monoclonal antibody to GRK2 (upper panels) or tubulin (lower panels). (B) HuH7 cells were pretreated or not with NE for 24 h before being stimulated or not with activin for 72 h. Cell growth was assessed by cell viability MTT assay. Values are expressed in arbitrary units. P<0.05 compared with no activin treatment. Download figure Download PowerPoint GRK2 blocks activin/TGFβ-induced Smad phosphorylation and nuclear translocation To further elucidate the mechanism by which GRK2 inhibits activin/TGFβ-induced gene expression, we examined the effect of overexpressing GRK2 on Smad phosphorylation and nuclear accumulation. For this, CHO cells were transfected or not with cDNA encoding Flag-GRK2 or a Flag-K220M kinase-inactive mutant, and stimulated with activin for varying time periods. As shown in Figure 5A, in total cell lysates analyzed by Western blot, using a specific antibody recognizing C-terminal phosphorylated serine residues of Smad2, activin-induced Smad2 phosphorylation was inhibited in the presence of GRK2, but not by the kinase-inactive mutant. Nuclear extracts from CHO cells transfected or not with GRK2 were analyzed using anti-phospho-Smad2 and anti-Smad2/3 antibodies and, as shown in Figure 5B, while activin strongly induced Smad2 phosphorylation and nuclear translocation in control cells, these effects were robustly diminished in cells overexpressing GRK2. Figure 5.GRK2 inhibits Smad2 carboxyl-terminal phosphorylation. (A) Whole-cell lysates from CHO cells transfected with empty vector, Flag-GRK2 or Flag-GRK2-K220M and stimulated with activin were analyzed by Western blot using anti-phospho-Smad2, anti-Smad2/3, anti-Flag and anti-tubulin antibodies. (B) Nuclear extracts from CHO cells transfected or not with Flag-GRK2 and stimulated with activin for the indicated periods of time were analyzed by Western blot using anti-phospho-Smad2 antibody, anti-Smad2/3, anti-Smad4 and anti-GRK2 antibodies. (C) An immunofluorescence field of view of HuH7 cells containing both transfected GFP-GRK2 cells and nontransfected cells, stimulated or not for 30 min with 0.1 nM TGFβ and labeled for endogenous phospho-Smad3 in rhodamine. (D) HuH7 nuclear extracts from cells either mock transfected or transfected with a scrambled siRNA oligonucleotide sequence, or with a specific siRNA sequence to GRK2, that were either treated or not with activin for 30 min and analyzed by Western blot using anti-phospho-Smad2 antibody, anti-Smad2/3, anti-GRK2 and anti-Tbp antibodies. Download figure Download PowerPoint In Figure 5C, using confocal microscopy, we further demonstrate that, while normal HuH7 liver cancer cells (left panels, nonfluorescent cells) exhibited a strong nuclear accumulation of phosphorylated Smad3 (middle panels, control nonstimulated as compared to TGFβ-stimulated cells), cells overexpressing GFP-GRK2 (left panel, green cells) failed to exhibit nuclear accumulation and phosphorylation of Smad3 upon 30 min TGFβ treatment as compared to cells not overexpressing GFP-GRK2. Taken together, our results indicate that the kinase GRK2 prevents Smad phosphorylation and nuclear translocation, thus inhibiting Smad signaling. We next examined the effect of blocking GRK2 expression on activin-mediated Smad activation. As shown in Figure 5D, transfection of HuH7 liver cells with siRNA to GRK2 significantly enhanced Smad2 phosphorylation and nuclear translocation. Together, these results clearly indicate that induced expression of the kinase GRK2 inhibits activin/TGFβ signaling, while knocking down the GRK2 gene potently enhances their signaling effects. GRK2 physically interacts with the Smads in an activin-dependent manner CHO cells were stimulated or not with activin for either 4 or 24 h, and cell lysates were immunoprecipitated using an anti-Smad2/3 antibody and proteins in the complex were revealed by immunoblot analysis with an antibody directed against GRK2. Results indicate that ligand stimulation induces complex formation between endogenous GRK2 and Smad2/3 (Figure 6A), an association that is markedly increased after 24 h, possibly due to the activin-induced increase in GRK2 expression at the later time point. Figure 6.Activin induces complex formation between GRK2 and the Smads. (A) Cell lysates from CHO cells stimulated with activin for 0, 4 or 24 h were immunoprecipitated with an anti-Smad2/3 antibody and analyzed by Western blot with an anti-GRK2 antibody (top panel). (B) Total cell lysates representing 10% of the immunoprecipitating input were analyzed by Western blot using antibodies to Smad2/3 and tubulin for loading controls. (C) Cell lysates from CHO cells overexpressing Flag-GRK2 or Flag-PRLR were incubated with either GST–Smad2 (C), GST–Smad3 (D) or GST–Smad4 (E) fusion constructs, and analyzed by Western blot using anti-Flag antibody (top panels). Ponceau staining of membranes used in the pulldown experiment, to illustrate relative levels of the fusion proteins used (bottom panels). Download figure Download PowerPoint Distinct modular domains within the Smads mediate their interactions with various partner proteins. As such, we set out to map the domains of the Smads required to mediate their interaction with GRK2. GST-fusion constructs encompassing the MH1, linker or MH2 domains of Smad2 were used in GST pulldown experiments using lysates from cells overexpressing either Flag-GRK2 or a nonrelated Flag-cDNA (prolactin receptor) as a negative control. The results indicate that Flag-GRK2 interacts with both MH1 and MH2, but not the linker region of Smad2 and 3 (Figure 6C and D). Interestingly, all domains of Smad4 were found to interact with the Flag-GRK2 (Figure 6E). Taken together, these results indicate that GRK2 can interact with all activin/TGFβ-specific Smads, and that this complex formation is ligand regulated. The linker domains of Smad2/3 are substrates for the kinase GRK2 As GRK2 kinase domain is required to block activin/TGFβ biological effects, we investigated whether Smads were substrates for GRK2. GST-fusion constructs containing the MH1, linker and MH2 domains of Smad2 and 3 were used in an in vitro kinase assay with purified GRK2. A representation of the relative amounts of fusion protein used for the kinase assays is shown in Figure 7A. Our results indicate that the GST-linker domain, but not the MH1 and MH2 domains of Smad2 and Smad3, are highly phosphorylated by GRK2 in vitro, and suggest that the Smads are substrates for the kinase GRK2 (Figure 7B). Figure 7.The linker region of Smad2/3 is phosphorylated by GRK2 in an in vitro kinase assay. (A) Coomassie blue staining demonstrating the relative amounts of GST–Smad2 (top panel) and GST–Smad3 (top panel) fusion protein used for the kinase assays. (B) Purified GRK2
Referência(s)