The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B
2004; Springer Nature; Volume: 23; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7600477
ISSN1460-2075
AutoresMartin Schmidlin, Min Lu, Sabrina Angélique Leuenberger, Georg Stoecklin, Michel Mallaun, Brigitte Gross, Roberto Gherzi, Daniel Heß, Brian A. Hemmings, Christoph Moroni,
Tópico(s)RNA modifications and cancer
ResumoArticle11 November 2004free access The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B Martin Schmidlin Martin Schmidlin Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Min Lu Min Lu Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Sabrina A Leuenberger Sabrina A Leuenberger Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Georg Stoecklin Georg Stoecklin Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michel Mallaun Michel Mallaun Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Brigitte Gross Brigitte Gross Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Roberto Gherzi Roberto Gherzi Gene Transfer Laboratory, Instituto Nazionale per la Ricerca sul Cancro, Genova, Italy Search for more papers by this author Daniel Hess Daniel Hess Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Brian A Hemmings Brian A Hemmings Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Christoph Moroni Corresponding Author Christoph Moroni Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Martin Schmidlin Martin Schmidlin Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Min Lu Min Lu Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Sabrina A Leuenberger Sabrina A Leuenberger Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Georg Stoecklin Georg Stoecklin Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michel Mallaun Michel Mallaun Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Brigitte Gross Brigitte Gross Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Roberto Gherzi Roberto Gherzi Gene Transfer Laboratory, Instituto Nazionale per la Ricerca sul Cancro, Genova, Italy Search for more papers by this author Daniel Hess Daniel Hess Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Brian A Hemmings Brian A Hemmings Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Christoph Moroni Corresponding Author Christoph Moroni Institute for Medical Microbiology, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Martin Schmidlin1, Min Lu1, Sabrina A Leuenberger1, Georg Stoecklin2, Michel Mallaun1, Brigitte Gross1, Roberto Gherzi3, Daniel Hess4, Brian A Hemmings4 and Christoph Moroni 1 1Institute for Medical Microbiology, University of Basel, Basel, Switzerland 2Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 3Gene Transfer Laboratory, Instituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4Friedrich Miescher Institute, Basel, Switzerland *Corresponding author. Institute für Medizinische Mikrobiologie, Universität Basel, Petersplatz 10, Basel 4003, Switzerland. Tel.: +41 61 267 3264; Fax: +41 61 267 3283; E-mail: [email protected] The EMBO Journal (2004)23:4760-4769https://doi.org/10.1038/sj.emboj.7600477 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3′ untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3. Introduction The stability of mRNA varies considerably among different species of transcripts and is in many cases regulated in a complex fashion in response to external stimuli. Short-lived transcripts carry cis elements that appear to regulate the access to the mRNA decay machinery. A major element is the so-called AU-rich element (ARE), which is located in the 3′ untranslated region (3′UTR) of many short-lived transcripts from cytokines, proto-oncogenes, growth factors or cell cycle regulators (Shaw and Kamen, 1986; Chen and Shyu, 1995; Bakheet et al, 2001). AREs promote deadenylation and decapping (Gao et al, 2001), followed by degradation of the mRNA body (Shyu et al, 1991; Xu et al, 1997). Mammalian ARE-containing mRNA (ARE-mRNA) is thought to be degraded mainly by the exosome, a multiprotein complex containing 3′–5′ exonucleases and helicases (Chen et al, 2001; Mukherjee et al, 2002). Stabilization of short-lived ARE-containing transcripts by exogenous signals leads to a rapid accumulation of mRNA, with a consequent increase of protein levels. Physiological examples are found in T-cells activated by immune stimulation (Lindsten et al, 1989), mast cells responding to IgE-linked allergens (Wodnar-Filipowicz et al, 1989; Wodnar-Filipowicz and Moroni, 1990), or macrophages stimulated by IL-1/TNFα (Huang et al, 2000), where increased cytokine production occurs. Deregulated ARE-dependent mRNA turnover can contribute to oncogenic transformation (Schuler and Cole, 1988; Nair et al, 1994; Stoecklin et al, 2003), inflammation (Carballo et al, 1998) and immunopathology (Kontoyiannis et al, 1999), underlying the physiological relevance of this process. Also, stress stimuli, including UV exposure (Gorospe et al, 1998; Wang et al, 2000), heat shock (Laroia et al, 1999) and hypoxia (Paulding and Czyzyk-Krzeska, 2000), can lead to stabilization of ARE-mRNA. In addition to mRNA turnover, the ARE has been reported to regulate translation (Kontoyiannis et al, 1999; Piecyk et al, 2000). Several signal transduction pathways have been implicated in mRNA decay control. Activation of stress-induced c-jun N-terminal kinase (JNK) (Chen et al, 1998; Ming et al, 1998), p38 mitogen-activated protein kinase (MAPK) (Dean et al, 1999; Winzen et al, 1999; Brook et al, 2000; Clark et al, 2003; Frevel et al, 2003), MAPKAP kinase 2 (MK2) (Neininger et al, 2002), phosphatidylinositol 3-kinase (PI3-K) (Ming et al, 2001) and wnt-β-catenin pathway (Briata et al, 2003) has been shown to trigger stabilization of various transcripts. Several ARE-binding proteins (AUBPs) affecting mRNA turnover have been identified. HuR, a member of the embryonic-lethal abnormal vision (ELAV in Drosophila melanogaster) family of RNA-binding proteins (Ma et al, 1996), stabilizes ARE-containing transcripts (Fan and Steitz, 1998). AUF1 (hnRNPD) exerts a stabilizing as well as a destabilizing function depending on cell type (Zhang et al, 1993; Chen et al, 2002) and isoform (Raineri et al, 2004). The Tis11 protein family members tristetraprolin (TTP) and butyrate response factors (BRF1 and BRF2) share a highly conserved CCCH tandem zinc-finger (Zn-finger) motif that binds RNA (Lai et al, 1999). TTP was initially identified by the Blackshear group as a positive regulator of TNFα mRNA decay (Carballo et al, 1998) by studying the autoimmune-like phenotype of TTP knockout (k.o.) mice. Subsequently, it was found to act by targeting ARE-mRNA to the exosome (Chen et al, 2001). BRF1 was recently identified by a functional genetic screen aimed at finding genes responsible for ARE-dependent decay (Stoecklin et al, 2002). Interestingly, BRF1 has been found to be a circadian gene (Storch et al, 2002) and its k.o. is lethal at embryonic day 11 (Stumpo et al, 2004). Both TTP and BRF1 can stimulate deadenylation (Lai et al, 2003). TIA/TIAR are RNA recognition motif (RRM)-containing proteins that regulate translational silencing of TNFα mRNA and recruitment into stress granules (Gueydan et al, 1999; Piecyk et al, 2000). Lastly, KSRP, a K homology (KH)-motif RNA-binding protein originally described as a neuronal specific splicing enhancer of c-src (Min et al, 1997), has been recently demonstrated to be an ARE-mRNA-specific destabilizing factor both in vitro and in vivo (Gherzi et al, 2004). An ARE-binding helicase has been described by Tran et al (2004). AUBPs are obvious candidate targets for signaling pathways regulating mRNA turnover. HuR has been shown to act in concert with p38 in stabilizing IL-3 mRNA (Ming et al, 2001), although direct phosphorylation of HuR was not detected. TTP was found to be phosphorylated by p38, which inhibited its ARE-binding activity (Carballo et al, 2001; Zhu et al, 2001). In addition, the extracellular signal-related kinase 2 (ERK2) (Taylor et al, 1995) and MK2 (Mahtani et al, 2001) are able to phosphorylate TTP in vitro. Phosphorylation of TTP by MK2 leads to 14-3-3 binding, inhibition of TTP activity and exclusion of TTP from stress granules (Johnson et al, 2002; Chrestensen et al, 2003; Stoecklin et al, 2004). 14-3-3 may also interact with BRF1, as suggested by results from a yeast two-hybrid screening (Bustin and McKay, 1999) and from GST-pulldown experiments (Johnson et al, 2002). Considerable progress in the analysis of mRNA turnover has been made by the development of in vitro decay systems. As control can be exercised over the choice of reagents to be added/omitted, different aspects of post-transcriptional regulation, including deadenylation, decapping and degradation of ARE-containing RNA, can be studied (Ford et al, 1999; Chen et al, 2000; Gao et al, 2001). In vitro AREs destabilize RNA through binding of AUBPs such as KSRP and TTP, which in turn recruit both the exosome and the deadenylase, thus targeting the RNA for exosomal decay (Chen et al, 2001; Lai et al, 2003; Gherzi et al, 2004). In vitro systems therefore provide a powerful tool to study the regulation of ARE-dependent decay. Here we show that BRF1 is able to promote ARE-dependent decay in an in vitro system, and that this activity is strongly reduced by phosphorylation of BRF1 by protein kinase B (PKB) at serine 92 (S92). Using a phospho-specific antipeptide antibody, we present evidence that BRF1 phosphorylation at S92 mediates insulin-induced mRNA stabilization in vivo. Furthermore, BRF1 phosphorylation was found to provide a docking site to 14-3-3, suggesting that this protein may sequester BRF1 from the cellular decay-promoting machinery. Results Activated PKB stabilizes ARE-containing reporter mRNA Previously, we have shown that activated PI3-K is able to stabilize ARE-containing reporter mRNA upon transfection into NIH 3T3 cells (Ming et al, 2001). We tested whether PKB, a downstream effector of PI3-K, mediates this effect. An activated form of PKB (m/pPKB) (Andjelkovic et al, 1997) was transfected together with a tetracycline-sensitive β-globin reporter construct containing the ARE of IL-3 (Tet-β-globin-IL3UTR) (Stoecklin et al, 2002) into NIH 3T3 B2A2-23 mouse fibroblasts (Xu et al, 1998). These cells stably express the Tet-responsive transcriptional activator tTA. When transcription of the reporter was inhibited by the addition of tetracycline, rapid degradation of the message with a half-life of less than 1 h was observed, while the reporter remained stable in the presence of activated PKB (Figure 1A and B). Activation of PKB was therefore sufficient to stabilize the ARE reporter transcript. Figure 1.ARE-mRNA stabilization by PKB. (A) The Tet-β-globin-IL3UTR reporter gene was transfected alone (lanes 1–3) or in combination with constitutively activated m/pPKB (lanes 4–6). After 24 h, transcription was stopped by addition of doxycycline. Cytoplasmic RNA was isolated at the indicated time points and processed for northern blotting. (B) The graph shows the quantifications of five independent decay assays normalized to the actin signal. Standard errors are shown unless too small to be represented. Download figure Download PowerPoint BRF1 is phosphorylated at S92 in vitro Inspection of the coding sequence of BRF1 (SwissProt database; accession number Q07352) revealed the presence of two overlapping RXRXXS motifs, including S90 and S92, corresponding to PKB consensus phosphorylation sites (Alessi et al, 1996). Interestingly, this PKB recognition motif is highly conserved in human, mouse and Xenopus leavis BRF1 (Figure 2A), but it is not present in TTP, although these closely related proteins show considerable homology throughout other regions. Figure 2.PKB phosphorylates BRF1 on S92. (A) Alignment of BRF1 sequences from human, mouse, rat and X. leavis. S90 and S92 are indicated. (B) In vitro phosphorylation reactions were performed for the times indicated using 40 ng of activated recombinant PKB and 20 ng of either full length rBRF1-wt, a N-terminal fragment (aa 3–110), the Zn-finger domain (aa 111–179) or a C-terminal fragment (aa 180–338). The asterisk denotes the full-length BRF1 protein, lower bands are degradation products. (C) In vitro phosphorylation reactions were performed for the times indicated, using 40 ng of activated recombinant PKB and 20 ng of either rBRF1-wt, rBRF1-S90A or rBRF1-S92A. Download figure Download PowerPoint To address the question whether BRF1 is indeed a substrate of PKB, we first performed in vitro phosphorylation experiments. Either full-length recombinant BRF1 or three fragments consisting of the N-terminus (aa 3–110), the central Zn-finger domain (aa 111–179) or the C-terminus of BRF1 (aa 180–338) were tested as substrates for PKBβ. Only the full-length rBRF1 and the N-terminal fragment containing S90 and S92 proved to be efficiently phosphorylated (Figure 2B, lanes 5–12). We used the N-terminal fragment (aa 3–110) of BRF1 to identify the site phosphorylated by PKB via mass spectrometry (see Materials and methods). This revealed that BRF1 is preferentially phosphorylated at S92. Accordingly, a peptide containing the S92 residue (DSRFRDRSFSEG) was phosphorylated in vitro by purified activated PKBα and β at 60% of the rate of the GSK3 peptide (RPRTSSFAEG), which is the best-characterized substrate of this kinase (data not shown). To investigate whether S92 is indeed the major PKB phosphorylation site, we mutated S90 or S92 to alanine (S90A, S92A), and tested the recombinant proteins for substrate activity. In line with the mass spectrometry data, BRF1-wt and BRF1-S90A were equally phosphorylated by PKB (Figure 2C, lanes 5–12), whereas phosphorylation was considerably reduced by the S92A mutation (Figure 2C, lanes 13–16). It appears that in the absence of the S92 phosphorylation site PKB can phosphorylate S90, which represents an alternative consensus site. BRF1 S92 phosphorylation regulates ARE-dependent RNA decay in vitro We were curious to see whether BRF1 may show similar in vitro decay activity as reported for its close homolog TTP (Chen et al, 2001), as this would allow us to study the nature of the S92 phosphorylation under controlled in vitro conditions. We co-incubated S100 extracts from slowC cells, a line lacking BRF1 expression as the result of frameshift mutations in both alleles (Stoecklin et al, 2002), with a radio-labeled RNA containing the 59 nt long ARE from IL-3 (AREIL3). While AREIL3 was stable in the presence of slowC extracts (Figure 3A, lanes 1–4), it decayed rapidly when 5 ng or more of rBRF1 was added (Figure 3A, lanes 5–20). As a control for both ARE specificity of the observed decay and equal loading, a 250 nt RNA containing the IL-3 3′UTR lacking the ARE (ΔARE) was added at the onset of the reaction. No degradation of the ΔARE control could be observed under all conditions tested. We concluded that rBRF1 is able to promote rapid RNA decay in this ARE-dependent in vitro system. Figure 3.BRF1 S92 phosphorylation regulates ARE-dependent RNA decay in vitro. (A) Radioactively labeled AREIL3 and ΔARE transcripts were incubated together in S100 extract from slowC cells. Decay reactions were performed at 37°C in the presence of increasing amounts of rBRF1-wt, stopped at the indicated time points and resolved on a 10% urea-polyacrylamide gel. ΔARE transcripts served as specificity and loading control. (B) Decay reactions were carried out in the absence (lanes 1–4) or in the presence of 20 ng of rBRF1-wt (lanes 5–12) or rBRF1-S92A (lanes 13–20) after pretreatment with buffer (lanes 5–8/13–16) or activated PKB (lanes 9–12/17–20). The graph below shows quantification of at least four independent experiments after normalizing to the ΔARE signal. Standard error bars were too small to be represented. Download figure Download PowerPoint We next investigated whether phosphorylation of rBRF1 by PKB would affect the in vitro decay rate. Unphosphorylated rBRF1-wt was compared to rBRF1-wt pretreated with activated PKB for 30 min (Figure 3B, lanes 5–12). A striking reduction of AREIL3 decay was observed with phospho-rBRF1-wt (p-rBRF1-wt) as compared to unphosphorylated rBRF1-wt. To check whether this effect was due to phosphorylation of BRF1 at S92, we performed the same experiment using the rBRF1-S92A mutant. Again, rBRF1-S92A promoted degradation of AREIL3 (Figure 3B, lanes 13–16), yet the mutant appeared refractory to inhibition by PKB (Figure 3B, lanes 17–20). Quantification (Figure 3B, lower panel) revealed an intermediate decay pattern, which may result from alternative phosphorylation at S90 (see also Figure 2C). From these data, we concluded that, in vitro, the decay-promoting activity of BRF1 is inhibited by phosphorylation at S92. BRF1 is phosphorylated at S92 in vivo To extend this study to endogenous BRF1, we first raised an antibody directed to a trisdekapeptide containing phosphorylated S92 (FRDRSFpSEGGERL). Affinity-purified antibody recognized rBRF1 phosphorylated by PKB with a high specificity (Figure 4A). As an additional control, we preincubated either the antibody described above or an antibody recognizing the C-terminus of BRF1 (Raineri et al, 2004) with the S92-containing phosphopeptide (PP; see above). In the case of the phospho-antibody, the western signal could specifically be competed away, while detection of unphosphorylated BRF1 was not impaired (Figure 4B, compare lanes 3 and 6). Figure 4.BRF1 is phosphorylated in vivo at S92. (A) Indicated amounts of rBRF1-wt were incubated with buffer alone or activated PKB for 30′ at 30°C and processed for western blotting. Phosphorylated (upper panel) or unphosphorylated BRF1 (lower panel) were detected by corresponding antibodies directed against a p-S92 containing or a C-terminal peptide (see Materials and methods). (B) rBRF1 phosphorylated as in (A) was processed for western blotting. Antibodies recognizing phosphorylated BRF1 (upper panel), BRF1 (middle panel) or PKB (lower panel) were pre-incubated on ice for 30′ with or without 50 μg of the PP used for immunization before adding to the blot. (C) HIRc-B cells were serum starved overnight, treated with WM (200 nM) for 30′ where indicated before stimulation with insulin (20 μg/ml) for 15′ (lanes 1–3). Whole-cell extracts were analyzed by western blot, detecting p-BRF1, phospho-PKB and α-tubulin as a loading control. (D) NIH3T3 B2A2-23 cells were transfected with m/pPKB or kinase-dead PKB (PKBkd) and BRF1-wt or BRF1S90A/S92A (BRF1AA). Whole-cell extracts were analyzed by western blot, detecting p-BRF1, phospho-PKB and α-tubulin as a loading control. Download figure Download PowerPoint We then examined whether BRF1 becomes phosphorylated at S92 in vivo. Insulin, a known activator of PKB (Burgering and Coffer, 1995) was used to stimulate HIRc-B rat fibroblasts, which express the human insulin receptor and are widely used to investigate insulin signaling (McClain et al, 1987). To reduce PKB background activity, we starved the cells overnight in serum-free medium. After stimulation with insulin for 15 min, PKB was strongly activated (Figure 4C, lane 2, middle panel) and we monitored BRF1 phosphorylation by western blotting using the p-BRF1 antibody. Phosphorylation of BRF1 at S92 could be detected under conditions of PKB activation (Figure 4C, lane 2, upper panel). BRF1 levels did not change in response to insulin stimulation (data not shown). This phosphorylation was partially inhibited by pretreating the cells with the PI3-K inhibitor wortmannin (WM), which completely abolishes PKB activation (Figure 4C, lanes 2 and 3). As WM treatment was not able to block BRF1 phosphorylation completely, we hypothesize that, in addition to PKB, another unidentified kinase may also phosphorylate BRF1 at S92. Phosphorylation of a single serine by more than one kinase is known to occur (Johannessen et al, 2004). To obtain in vivo evidence that indeed PKB is phosphorylating BRF1, we transfected BRF1-wt or the BRF1-S90A/S92A double mutant construct together with m/pPKB or a kinase-dead variant of PKB (PKBkd) into 3T3 cells. Strong phosphorylation of BRF1-wt, but not of the mutant, was observed in m/pPKB-transfected cells (Figure 4D). Together, these data indicate that PKB does phosphorylate BRF1 in vivo at S92. ARE-mRNA is stabilized under conditions that phosphorylate BRF1 We then wanted to know whether phosphorylation of BRF1 impairs its decay-promoting activity in vivo. We co-transfected the Tet-β-globin-IL3UTR reporter together with the tTA transactivator (pTetOff, Clontech) into HIRc-B cells. When transcription of the reporter was turned off by adding doxycycline, we observed rapid decay of the reporter mRNA with a half-life of approximately 1 h (Figure 5A, lanes 1–3). After pretreatment of cells with insulin for 15 min, the mRNA was more stable (lanes 4–6), and this effect was sensitive to WM (lanes 7–9). We concluded that in vivo ARE-mRNAs become stabilized under conditions where phosphorylation of BRF1 at S92 occurs. Quantification from three parallel experiments indicated that the observed changes are statistically significant (Figure 5B). Figure 5.Effect of insulin on reporter mRNA decay. (A) At 24 h after co-transfection of the Tet-β-globin-IL3UTR reporter gene and the Tet-responsive transcriptional activator tTA (pTET-on), HIRc-B cells were split into fresh medium for 16 h. Where indicated cells were pretreated with WM (200 nM) for 30′ before addition of insulin (20 μg/ml) for another 15′. Then doxycycline was added (2 μg/ml) to stop transcription, cytoplasmic RNA was isolated at the indicated time points and processed for northern blotting. (B) Quantification of three independent experiments is shown normalized to the 18S rRNA signal. (C) MEF-PKBα−/− and +/+ cells were pretreated with WM for 30′ prior to insulin (Ins) stimulation for 15′ as indicated. p-BRF1, phospho-PKB, PKB and tubulin protein levels were analyzed by western blot. (D) MEF-PKBα−/− and +/+ cells were treated as described for HIRc-B in (A). Reporter RNA was analyzed by northern blot. One representative experiment is shown. Download figure Download PowerPoint To further evaluate the involvement of PKB in stabilizing ARE-mRNA, we made use of mouse embryonic fibroblasts with k.o. of PKBα (MEF-PKBα−/−) (Yang et al, 2003). When MEF-PKBα−/− and +/+ cells were compared by western blotting, we noticed strong PKB phosphorylation upon insulin stimulation even in −/− cells, apparently via the PKBβ and γ isoforms. In unstimulated MEF-PKBα+/+ cells, there was background phosphorylation of BRF1 (Figure 5C), presumably by a kinase other than PKB (see also Figure 4C). Insulin stimulation increased this signal and stabilized the reporter mRNA in a WM-sensitive fashion. In MEF-PKBα cells, the p-BRF1 signal was low with no response to insulin. When mRNA stability of the reporter was examined, decay was generally more rapid in −/− cells and insulin failed to induce stabilization (5D). These data support an in vivo role for PKBα in stabilization of ARE-mRNA. BRF1 phosphorylation leads to 14-3-3 binding To elucidate the mechanism how PKB inhibits BRF1, we first asked whether BRF1 phosphorylation affects ARE binding, as it was found for TTP (Carballo et al, 2001). By electrophoretic mobility shift assay, we compared increasing amounts of unphosphorylated rBRF1 to rBRF1 pretreated with activated PKB for its ability to bind a radio-labeled AREIL3 probe. Phosphorylation did not impair ARE binding (Figure 6A). The inhibitory effect of S92 phosphorylation on BRF1-destabilizing activity could therefore not be explained by a change in the RNA-binding capacity of BRF1. An alternative mechanism responsible could be binding of phosphorylated BRF1 to an inhibitory factor. A candidate binding partner was 14-3-3, as various isoforms of this protein bind to targets phosphorylated by PKB (Tzivion and Avruch, 2002) and, in addition, 14-3-3 has been found to interact with BRF1 (Bustin and McKay, 1999; Johnson et al, 2002). To test this possibility, His-tagged rBRF1, either untreated or phosphorylated by PKB, was added to slowC cell extracts, purified by Ni-NTA beads and processed for western blotting. Phosphorylated but not unphosphorylated BRF1 pulled down 14-3-3 (Figure 6B, lanes 1 and 2). Furthermore, addition of the PP containing phosphorylated S92 antagonized 14-3-3 binding in a dose-dependent fashion (lanes 3–5), whereas the analogous peptide containing the S92A substitution (AP) was unable to interfere with 14-3-3 binding (lanes 6–8). Figure 6.S92 phosphorylation of BRF1 induces complex formation with 14-3-3. (A) Electrophoretic mobility shift assay. Indicated amounts of rBRF1 were pre-incubated with or without activated PKB, mixed with radio-labeled AREIL3 RNA and resolved on 4% nondenaturing PAGE. (B) His-tagged rBRF1 pretreated with buffer (lane 1) or activated PKB (lanes 2–11) was mixed with S100 extract from slowC cells. Increasing concentrations of the phosphorylated peptide surrounding S92 (aa 86–98) of BRF1 (PP) or the corresponding S92A mutant peptide (AP) were added. rBRF1 was purified using Ni-NTA beads, and 14-3-3 and BRF1 were detected with the corresponding antibodies. The lower band represents 14-3-3, the upper His-tagged rBRF1. (C) In vitro decay reactions were carried out as described in Figure 3 in the presence of 0.4 mM of PP (lanes 9–12) or AP (lanes 13–16). The graph on the right shows quantification of four independent experiments after normalizing to the ΔARE signal. Standard error bars were too small to be represented. (D) COS7 cells were transfected with vector, bsdHisBRF1 wt, bsdHisBRF1 aa, either alone (lanes 1–3) or in combination with m/pPKB (lanes 5–8), as indicated. In the experiment shown on the right (lanes 4–8), FCS was withdrawn for 40 h. Expression of His-tagged BRF1, phospho-PKB and 14-3-3 in the cell extracts was determined by western blotting (load). After purification of BRF1 by Ni-NTA beads, co-precipitation of 14-3-3 was tested by western blotting (Co-P). Download figure Download PowerPoint If interaction with 14-3-3 was responsible for BRF1 inhibition, we would expect the PP to affect binding and hence BRF1 activity in the in vitro decay assay. Indeed, addition of PP could restore decay activity of phosphorylated BRF1 (Figure 6C, lanes 9–12), whereas the AP peptide was ineffective (Figure 6C, lanes 13–16). Taken together, these data indicate that PKB-induced inhibition of BRF1 occurs through binding of 14-3-3. Finally, in vivo experiments were performed to verify that PKB regulates the interaction between BRF1 and 14-3-3. For this experiment we chose COS7 cells, as complex formation between phosphorylated TTP and 14-3-3 was recently demonstrated in this line (Stoecklin et al, 2004). Cells were transiently transfected with wt or mutant BRF1 constructs carrying a His-tag. BRF1 was purified from the cell extracts using Ni-NTA beads, and western blotting revealed co-precipitation of 14-3-3 with BRF1-wt (Figure 6D, lane 2). Mutation of BRF1 at S90/92 strongly prevented the interaction with 14-3-3 (lane 3). In order to see whether activation of the PKB pathway would stimulate 14-3-3 binding, cells were co-transfected with m/pPKB and serum-starved to reduce endogenous PKB activity. m/pPKB strongly induced binding of 14-3-3 to BRF1-wt (lane 6). Notably, induced binding of 14-3-3 was not observed with the S90A/S92A double mutant (BRF1AA, lane 8). Together, these data suggest that S92 phosphorylation by PKB leads to interaction of 14-3-3 with BRF1. Whereas this interaction does not impair RNA binding, it may inhibit recruitment of the BRF1–RNA complex to the exosomal decay machinery and thereby lead to stabilization of ARE-mRNA. Discussion In this report, we provide evidence that phosphorylation of BRF1 by PKB stabilizes ARE-mRNA. As initially reported for PI3-K (Ming et al, 2001), its downstream target PKB efficiently stabilized ARE-containing reporter transcripts in
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