Effects of protein phosphorylation on ubiquitination and ... - Nature

Oncogene (2008) 27, 811–822

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ORIGINAL ARTICLE

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1 A Elia, C Constantinou1 and MJ Clemens Translational Control Group, Division of Basic Medical Sciences, Centre for Molecular and Metabolic Signalling, St George’s, University of London, London, UK

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecularweight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecularweight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m7GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms. Oncogene (2008) 27, 811–822; doi:10.1038/sj.onc.1210678; published online 23 July 2007 Keywords: calyculin A; eIF4E; protein degradation; protein phosphatases; protein synthesis

Introduction The translation of most mRNAs in eukaryotic cells requires recognition of the 50 m7GTP-containing cap Correspondence: Professor MJ Clemens, Translational Control Group, Division of Basic Medical Sciences, Centre for Molecular and Metabolic Signalling, St George’s, University of London, Cranmer Terrace, London SW17 0RE, UK. E-mail: [email protected] 1 Current address: Yasoo Health Ltd, 1 Poseidon Street, PO Box 25193, 1307 Nicosia, Cyprus. Received 22 February 2007; revised 23 May 2007; accepted 13 June 2007; published online 23 July 2007

structure by polypeptide chain initiation factor eIF4E (Mamane et al., 2004; von der Haar et al., 2004). The availability of the latter for formation of the active eIF4F complex is regulated by the 4E-binding proteins (4E-BP1 and 4E-BP2) (Clemens, 2001; Mamane et al., 2006). The most thoroughly studied of these is 4E-BP1, which in its hypophosphorylated state, binds and sequesters eIF4E. Phosphorylation of 4E-BP1, at multiple sites, decreases the affinity of this protein for eIF4E and thus releases the cap-binding protein for eIF4F complex formation. The phosphorylation of 4E-BP1 is influenced by a wide range of extracellular stimuli. In general, conditions that promote cell growth enhance the phosphorylation of 4E-BP1 and are associated with increased rates of overall protein synthesis; conversely, growth inhibitory conditions and physiological stresses result in dephosphorylation of the protein and cause downregulation of protein synthesis (Mamane et al., 2006). The protein kinase mammalian target of rapamycin (mTOR) plays a crucial role in the phosphorylation of 4E-BP1, although other protein kinases have also been implicated (Herbert et al., 2002; Gingras et al., 2004; Tee and Blenis, 2005). There is evidence that 4E-BP1 associates with protein phosphatase 2A (Peterson et al., 1999), and it is likely that the dephosphorylation of 4E-BP1 is catalysed by this enzyme. The availability of eIF4E has differential effects on the translation of different mRNAs, with those containing complex, structured 50 -untranslated regions being particularly sensitive to changes in the level of the factor (Graff and Zimmer, 2003). The initiation factor also exerts important effects on the nucleo-cytoplasmic transport of a number of mRNAs (Strudwick and Borden, 2002). Overexpression of eIF4E is associated with malignant transformation (De Benedetti and Graff, 2004), probably because the factor promotes the synthesis of antiapoptotic proteins (Clemens, 2004; Mamane et al., 2004), and many naturally occurring tumours contain elevated levels of eIF4E. Conversely, enhanced expression of 4E-BP1 can reverse the transformed phenotype and increase the susceptibility of cells to apoptosis (Li et al., 2002; Proud, 2005). In naturally occurring tumours, the level of phosphorylation of 4E-BP1 correlates with tumour stage and prognosis (Castellvi et al., 2006). Whereas a large body of work has been devoted to the phosphorylation of 4E-BP1, very few studies have

Phosphorylation, ubiquitination and stability of 4E-BP1 A Elia et al

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examined regulation of the stability of the protein. Following fertilization of sea urchin eggs, there is a rapid loss of 4E-BP which occurs in parallel with dramatically enhanced rates of protein synthesis (Cormier et al., 2001; Salau¨n et al., 2003). This loss is inhibited by rapamycin, suggesting a role for protein phosphorylation in the degradation of 4E-BP. In a mammalian cell system dephosphorylation of 4E-BP1 induced by the tumour suppressor protein p53 is accompanied by an increase in the level of the 4E-BP1, in parallel with inhibition of protein synthesis (Tilleray et al., 2006). However, the mechanisms regulating 4E-BP1 levels have not so far been extensively investigated. Many proteins of regulatory significance are targeted for degradation by the 26S proteasome as a result of their polyubiquitination (Fang and Weissman, 2004). Moreover, the susceptibility of such proteins to be processed by this pathway is often controlled by protein phosphorylation. In addition to the now classical cases of the inhibitor of nuclear factor kB (IkB) family (Karin and Ben-Neriah, 2000; Chen, 2005; Krappmann and Scheidereit, 2005) and cyclins (Lin et al., 2006a), other examples of proteins whose (poly)ubiquitination is stimulated by their phosphorylation include the type I interferon receptor IFNAR1 (Kumar et al., 2004), the transcription factor STAT1 (Kim and Maniatis, 1996), the antiapoptotic protein Bcl-2 (Lin et al., 2006b) and bcatenin (Aberle et al., 1997; Orford et al., 1997). On the other hand, phosphorylation can inhibit the ubiquitination of other proteins such as the proto-oncogenes c-jun (Musti et al., 1997; Fuchs et al., 1998), c-fos (Okazaki and Sagata, 1995) and c-mos (Nishizawa et al., 1993). Recent studies have shown that initiation factor eIF4E can undergo ubiquitination and proteasomemediated turnover (Othumpangat et al., 2005a; Murata and Shimotohno, 2006). In contrast, although there have been indications that 4E-BP1 is also degraded by the proteasome (Walsh and Mohr, 2004; Walsh et al., 2005; Wan et al., 2005), no information has been published on the possible ubiquitination of this protein. In this paper, we present evidence from a variety of cell lines indicating that human and mouse 4E-BP1 can be polyubiquitinated, giving rise to high-molecular-weight forms. Our data suggest that this process is controlled by phosphorylation of the protein. Moreover, we show that enhancing the phosphorylation of 4E-BP1 both decreases the level of the normal-sized protein and converts the latter into high-molecular-weight forms that are unable to associate with eIF4E. The implications for the regulation of protein synthesis and cell transformation by eIF4E and 4E-BP1 are discussed.

Results Identification of high-molecular-weight forms of 4E-BP1 We have used immunoblotting with several antibodies against total 4E-BP1 to characterize the size distribution of the protein. In agreement with numerous previous studies (see for example, Lin et al., 1994; Graves et al., Oncogene

1995; Gingras et al., 1996; Mendez et al., 1996), mouse and human 4E-BP1 were detected as multiple bands on gels in the vicinity of 20 kDa. These correspond to differentially phosphorylated forms, with hyperphosphorylated (g) 4E-BP1 migrating more slowly than the less phosphorylated (b and a) forms. In addition, analysis of extracts from mouse embryonic fibroblasts showed that regions of the blots corresponding to higher-molecular-weight proteins contain several other bands that are recognized by antibodies against total 4E-BP1 (Figure 1a). At least some of these forms of the protein are ubiquitinated, as revealed by immunoprecipitation with anti-4E-BP1 followed by immunoblotting with anti-ubiquitin. As shown in Figure 1b (left panel), a band that migrates at around 50 kDa was identifiable by this approach. When the converse experiment was performed, immunoprecipitating a cell extract with antiubiquitin-coated agarose beads and blotting with anti4E-BP1, a similar sized band was seen (Figure 1b, right panel). The mobility of this protein suggests that it contains three or four ubiquitin moieties. The co-precipitation reaction was specific since no anti-4E-BP1 reactive material was observed when unmodified agarose beads were used (Figure 1b, right panel, lane 2) and no unmodified (20 kDa) 4E-BP1 was detected after anti-ubiquitin precipitation.

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Figure 1 Evidence for polyubiquitination of 4E-BP1. (a) A cytoplasmic extract from murine embryonic fibroblasts was subjected to gel electrophoresis followed by immunoblotting for total 4E-BP1. Molecular weight markers were separated in a parallel lane (M) and their sizes (in kDa) are indicated. The regions of the blot corresponding to the a, b and g forms of 4E-BP1 and to higher-molecular-weight cross-reacting bands are shown. The experiment was repeated several times and a typical blot is shown. (b) A cytoplasmic extract from fibroblasts was subjected to immunoprecipitation with anti-4E-BP1, anti-ubiquitin agarose beads or unmodified agarose beads. Left panel: the anti-4E-BP1 immunoprecipitate was analysed by gel electrophoresis followed by immunoblotting with anti-ubiquitin (lane 1). Right panel: the unmodified agarose beads (lane 2) and anti-ubiquitin agarose beads (lane 3) were incubated in 2 SDS sample buffer and the eluted proteins analysed by gel electrophoresis followed by immunoblotting with anti-4E-BP1. The sizes of molecular weight markers (in kDa) are indicated. The regions of the blots corresponding to highmolecular-weight (polyubiquitinated) forms of 4E-BP1 are shown. 4E-BP1, 4E-binding protein 1; SDS, sodium dodecyl sulphate.


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Relationship between phosphorylation and ubiquitination of 4E-BP1 Since 4E-BP1 is modified by phosphorylation and can also become polyubiquitinated, we have investigated whether there is any interaction between the two events, and whether the same protein molecules can undergo both types of modification. Cells with relatively low basal levels of 4E-BP1 phosphorylation (murine erythroleukaemia (MEL) cells in which a temperaturesensitive form of p53 had been activated (Constantinou and Clemens, 2005)) were treated with the protein phosphatase inhibitor calyculin A. Extracts were then examined for the appearance of phosphorylated forms of the protein. Immunoblotting with phospho-specific antibodies showed that calyculin A had a small stimulatory effect on the level of phosphorylation of normal-sized mouse 4E-BP1, particularly on Ser64 (Figure 2a, compare lanes 1 and 3). More dramatically, the phosphatase inhibitor markedly enhanced the appearance of phosphorylated high-molecular-weight forms of the protein. In this case, increased phosphorylation was observed at all sites examined, with a particularly strong effect on Thr36/45. Several bands, with approximate molecular masses in the range of 50– 200 kDa, were observed. Similar results were observed using human Jurkat T-lymphoma cells (Figure 2b). These data suggest that 4E-BP1 may be both phosphorylated and polyubiquitinated simultaneously. In contrast to the results with phospho-specific antibodies, calyculin A had very little effect on the appearance of additional forms of 4E-BP1 when an antibody that recognizes the total protein was used, suggesting that only a relatively small fraction of 4E-BP1 becomes polyubiquitinated in the presence of the phosphatase inhibitor. However the 4E-BP1 that was modified in this manner was heavily phosphorylated, particularly on Thr36/45 in MEL cells (Thr37/46 in Jurkat cells). Because ubiquitination can tag proteins for degradation via the proteasome pathway, we investigated whether inhibition of proteasome activity had any influence on the levels of the normal-sized and highmolecular-weight forms of 4E-BP1. Exposure of both MEL cells and Jurkat cells to the proteasome inhibitor MG132 had two notable effects. First MG132 caused substantial dephosphorylation of the normal-sized protein, especially on Ser64 (Ser65 in Jurkat cells) and enhanced the level of the hypophosphorylated (a) form, particularly in Jurkat cells (Figure 2b). Secondly, MG132 modestly stimulated the appearance of highmolecular-weight forms of 4E-BP1 that were phosphorylated on Thr36/45 (Thr37/46) (Figures 2a–c, compare lanes 1 and 2). The latter effect may reflect inhibition of proteasome-mediated degradation of phosphorylated, ubiquitinated forms of 4E-BP1. However, our data suggest that the phosphorylation of 4E-BP1 drives the ubiquitination process; thus the limited ability of MG132 to cause accumulation of ubiquitinated 4E-BP1, relative to the effect of the inhibitor on total ubiquitinated proteins (Figure 2c, middle panel) may be due to the separate effect of MG132 in inhibiting the phosphorylation of normal-sized 4E-BP1.

To test directly whether the high-molecular-weight forms of 4E-BP1 that accumulate in the presence of calyculin A (7MG132) are both phosphorylated and ubiquitinated, extracts from Jurkat cells were immunoprecipitated with anti-ubiquitin and then immunoblotted for phospho-Thr37/46. Figure 3 shows that the immunoprecipitation brought down high-molecularweight proteins of ca. 50 and 80 kDa that cross-reacted calyculin with anti-phospho-Thr37/46. Moreover, A þ MG132 strongly enhanced the phospho-Thr37/46 signal, in both total cytoplasmic extracts and the immunoprecipitated material, and an even larger form (ca. 200 kDa) was observed under these conditions. These results confirm that at least some forms of highmolecular-weight 4E-BP1 are ubiquitinated and phosphorylated on the same molecules. Relationship between ubiquitination and association of 4E-BP1 with eIF4E We have shown that the high-molecular-weight forms of 4E-BP1 that appear in response to calyculin A in Jurkat cells are highly phosphorylated on Thr37/46. There is also phosphorylation on Ser65 but relatively little on Thr70 (Figure 2b). Since the latter two sites have important roles in regulating the association of 4E-BP1 with eIF4E it was unclear whether ubiquitinated forms of 4E-BP1 would be present in complexes with the initiation factor. To determine this, eIF4E and its associated proteins were purified by m7GTP-Sepharose affinity chromatography from extracts of cells incubated with or without calyculin A for different periods of time. Figure 4a shows that, after 1 h of calyculin A treatment, almost none of the high-molecular-weight 4E-BP1 that was phosphorylated on Thr37/46 could be found in association with eIF4E. This was in contrast to the behaviour of normal-sized 4E-BP1 where the very small amount still associated with eIF4E in calyculin A-treated cells did include material phosphorylated on Thr37/46 (but not on Ser64 or Thr69—data not shown). Similarly, after a longer period of incubation with calyculin A only a trace amount of higher-molecular-weight 4E-BP1 was bound to eIF4E (Figure 4b). Analysis of eIF4E-associated proteins also failed to detect any ubiquitinated species (data not shown). These results suggest that the ubiquitination of 4E-BP1 (but not the phosphorylation of Thr37/46 per se) reduces the affinity of the protein for eIF4E. Phosphorylation and the stability of 4E-BP1 Although calyculin A increased the phosphorylation of the high-molecular-weight bands (especially on Thr37/46), it reduced the total level of normal-sized 4E-BP1 in cytoplasmic extracts, especially in the case of Jurkat cells (Figure 2b, top panel and Figure 4). Analysis of cytoplasmic and nuclear fractions from the cells did not indicate any change in the subcellular distribution of the protein in response to calyculin A (data not shown). To test the effect of calyculin A on the half-life of normal-sized 4E-BP1 levels of the protein were measured after different times in the presence of the protein Oncogene


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synthesis inhibitor cycloheximide. The top panel in Figure 5a shows that under the control conditions, 4EBP1 was a relatively stable protein, with a half-life well in excess of 16 h. A previous study in adipocytes has also indicated that 4E-BP1 is stable (Lin et al., 1995). In contrast, in the presence of calyculin A the normal-sized

protein disappeared with an apparent half-life of about 9 h (Figure 5a, middle panel). This suggests that the protein phosphatase inhibitor either enhances the conversion of 4E-BP1 to larger forms or promotes the complete destruction of the protein. The latter effect would be consistent with enhanced ubiquitination and

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subsequent degradation of phosphorylated 4E-BP1. The extracts were also probed with the antibody against phospho-Thr37/46. As can be seen in Figure 5b, this analysis revealed that the phosphorylated high-molecular-weight forms of 4E-BP1 appeared within 1–2 h of exposure of cells to the phosphatase inhibitor and were maintained throughout the time course with cycloheximide, declining slightly by 16 h. However, these data do not provide accurate information about the half-lives of these phosphorylated forms of the protein since a reservoir of phosphorylated normal-sized 4E-BP1 remained, from which further high-molecular-weight molecules could have been derived. To investigate a possible role for the proteasome in the degradation of ubiquitinated 4E-BP1 the effects of longterm treatment with MG132 (7calyculin A) on the normal-sized and higher-molecular-weight forms of 4E-BP1 were also examined in the presence of cycloheximide. Since extended exposure to MG132 can induce apoptosis (Lin et al., 1998) in this experiment the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z.VAD-FMK) was added to the cells to eliminate any possible role of caspase activation in the degradation of 4E-BP1. Figure 5c shows that, after 16 h, calyculin treatment strongly reduced the level of normal-sized

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4E-BP1, and MG132 did not prevent this. The proteasome inhibitor did slightly increase the calyculin-induced accumulation of the larger form(s), suggesting a possible role for the proteasome in the degradation of high-molecularweight, polyubiquitinated 4E-BP1. However, the latter forms were clearly relatively stable, indicating that ubiquitination per se does not lead to rapid turnover. It is possible that other components of the cell’s proteolytic machinery are involved in the degradation of polyubiquitinated 4E-BP1. For example, lysosomes have been demonstrated to degrade a variety of polyubiquitinated proteins (Hicke, 2001; Kumar et al., 2004). However, treatment of Jurkat cells with the lysosomal inhibitor chloroquine did not prevent the calyculin A-induced loss of normal-sized 4E-BP1 or cause any further accumulation of the larger forms (data not shown). Calyculin A inhibits the dephosphorylation of a wide range of protein phosphatase substrates (Ishihara et al., 1989) and it was possible that it could non-specifically alter the activity of a ubiquitin ligase and/or affect components of the protein degradation machinery itself. To determine whether the effects of calyculin A on 4E-BP1 require the mTOR-mediated phosphorylation of the protein, we therefore examined the fate of 4E-BP1 in the additional presence of rapamycin. This agent is a

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Figure 3 Evidence that 4E-BP1 can be simultaneously phosphorylated and ubiquitinated. Jurkat cells were incubated with or without MG132 for 16 h. Where indicated, calyculin A was then added for the last hour. Cytoplasmic extracts were prepared and samples were immunoprecipitated with anti-ubiquitin agarose beads. The extracts (left panel) and immunoprecipitates (right panel) were then analysed by gel electrophoresis and immunoblotting for phosphorylated 4E-BP1 (Thr37/46). The sizes of molecular weight markers (in kDa) are indicated. The regions of the blots corresponding to the a, b and g forms of 4E-BP1 and to higher-molecular-weight forms of the protein are shown. (Note that the lowest band in the left panel is a smaller fragment of 4E-BP1 that is often observed in Jurkat cell extracts.) 4E-BP1, 4E-binding protein 1.

Figure 2 Effects of protein phosphatase and proteasome inhibitors on 4E-BP1. (a) MEL cells expressing a temperature-sensitive form of p53 were incubated at 321C for 16 h to invoke p53-dependent dephosphorylation of 4E-BP1 (Constantinou and Clemens, 2005) and treated with calyculin A and/or MG132 where indicated. Cytoplasmic extracts were subjected to gel electrophoresis followed by immunoblotting for total 4E-BP1, phosphorylated 4E-BP1 (Ser64, Thr69 and Thr36/45) and a-tubulin as indicated. (b) Jurkat cells were incubated with or without MG132 for 16 h. Where indicated, calyculin A was then added for the last hour. Cytoplasmic extracts were prepared and analysed as in (a). (c) Jurkat cells were incubated with or without MG132 and/or calyculin A for 2 h. Cytoplasmic extracts were prepared and analysed by immunoblotting for phosphorylated 4E-BP1 (Thr37/46), total ubiquitinated proteins and a-tubulin as indicated. In all experiments, molecular weight markers were separated in parallel lanes and their sizes (in kDa) are indicated. The regions of the blots corresponding to the a, b and g forms of normal-sized 4E-BP1 and to higher-molecular-weight forms of the protein are shown. The experiments were repeated several times and typical blots are shown. For the phospho-Thr37/46 blots in (b) and (c) different exposures have been used for lanes 1 and 2, and 3 and 4, respectively to optimize visualization of the bands. Note that the bands at ca. 30 and 50 kDa in the top panel of (b) are non-specific Jurkat cell proteins that cross-react with the antibody used and are not ubiquitinated forms of 4E-BP1. 4E-BP1, 4E-binding protein 1. Oncogene


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Figure 4 Analysis of the association of modified forms of 4E-BP1 with eIF4E. Jurkat cells were incubated with or without MG132 for 16 h. Where indicated, calyculin A was added for the last hour (a) or was present throughout the incubation (b). Cytoplasmic extracts were prepared and eIF4E and its associated proteins were isolated by m7GTP-Sepharose affinity purification. Samples of the affinity purified fractions were analysed for total and phosphorylated 4E-BP1 (Thr37/46) by gel electrophoresis and immunoblotting. The affinity purified fractions were also blotted for eIF4E as a control for equal protein recovery. 4E-BP1, 4E-binding protein 1.

direct inhibitor of mTOR and partially lowers the level of phosphorylation of 4E-BP1 (Gingras et al., 2004). Figure 6 (top panel) shows that rapamycin partially prevented the calyculin A-induced decrease in the level of normal-sized 4E-BP1 in Jurkat cells. Although the mTOR inhibitor was not fully able to reverse this effect of calyculin A, it also had only a modest effect on the phosphorylation of 4E-BP1 (mostly on Ser65 and Thr70), as well as on the calyculin-induced appearance of the phosphorylated high-molecular-weight forms. Nevertheless, our findings suggest that the disappearance of normal-sized 4E-BP1 following calyculin A treatment is likely to be a direct consequence of increased phosphorylation, rather than a non-specific change in the activity of the protein ubiquitination or degradation machinery.

Discussion Our data suggest a model in which the phosphorylation of 4E-BP1 regulates the ubiquitination of this protein. As shown in Figure 7, this may promote the turnover of a fraction of 4E-BP1, mediated by the proteasome and/ or other proteolytic mechanisms, but may have other consequences as well. A previous study (Walsh and Mohr, 2004) proposed a role for the proteasome in the Oncogene

degradation of 4E-BP1 in cells infected by herpes simplex virus-1. Furthermore, their data suggested that degradation was sensitive to the state of phosphorylation of 4E-BP1. However, it is not yet clear whether 4EBP1 is degraded exclusively by the proteasome since interpretation of results from experiments using MG132 to inhibit proteasomal activity is complicated by the fact that this agent also causes the dephosphorylation of one or more sites on 4E-BP1 (Figures 2a and b). This could be an indirect effect reflecting inhibition of a protein kinase or stabilization of a protein phosphatase by MG132 (Torres et al., 2003) and could potentially inhibit the ubiquitination process that is driven by phosphorylation. In addition, as indicated in Figure 7, there may be a role for (poly)ubiquitination of 4E-BP1 in controlling properties of this factor besides stability, as is the case for several other proteins of regulatory significance (Aguilar and Wendland, 2003; Chen, 2005). In this connection it is of interest that the highmolecular-weight forms of 4E-BP1 bind very poorly to eIF4E (Figure 4). Since inhibition of protein phosphatase activity with calyculin A leads to the accumulation of phosphorylated high-molecular-weight forms of 4E-BP1, an important question is why very little of the phosphorylated protein occurs in these forms under normal circumstances. One possibility is that hyperphosphorylation of 4E-BP1


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Figure 5 Effects of calyculin A and MG132 on the stability of 4E-BP1 and conversion to high-molecular-weight forms. (a) Jurkat cells were incubated with or without calyculin A for 1 h and cycloheximide was then added for the times indicated. Upper panel: cytoplasmic extracts were analysed by gel electrophoresis and immunoblotting using antibody against total 4E-BP1. The sizes of molecular weight markers (in kDa) are indicated. The regions of the blots corresponding to the a, b and g forms of 4E-BP1 are shown. (Note that the band below the a form of 4E-BP1 is a smaller fragment that is often observed in Jurkat cell extracts.) Bottom panel: the combined relative intensities of the a, b and g forms of 4E-BP1 (determined by quantitative densitometry) are plotted on a log scale as a function of time with cycloheximide. The intensity corresponding to 50% of the initial value is indicated by the horizontal line. (b) The extracts analysed in (a) above were re-probed using antibody against phospho-Thr37/46. (c) Jurkat cells were pre-incubated with zVAD-FMK for 1 h to prevent subsequent apoptosis and then treated with or without calyculin A and/or MG132 in the presence of cycloheximide for 16 h. Cytoplasmic extracts were analysed by gel electrophoresis and immunoblotting for total 4E-BP1 and a-tubulin. The a, b and g forms of normal-sized 4E-BP1 and higher-molecular-weight forms of the protein are indicated. 4E-BP1, 4E-binding protein 1.

induced by calyculin A enhances the accumulation of polyubiquitinated forms to an extent that exceeds the capacity of the protein degradation machinery to rapidly degrade the protein. This would be consistent with the calyculin-induced decrease in the level of normal sized 4E-BP1 (Figures 2 and 4) and the limited ability of MG132 to increase further the levels of highmolecular-weight phosphorylated 4E-BP1 (Figure 2). Alternatively, an additional site on 4E-BP1, which is not normally phosphorylated (or is phosphorylated in only a small fraction of the protein), may become more extensively phosphorylated in the presence of calyculin A and act as a signal for polyubiquitination or other modifications. The ability of rapamycin partially to impair the effect of calyculin A in inducing the loss of

normal-sized 4E-BP1 (Figure 6) argues against less direct effects of the protein phosphatase inhibitor, for example on the enzymes involved in the ubiquitination or degradation of 4E-BP1. However, the presence of a substantial amount of 4E-BP1 that is phosphorylated on Thr37/46, Ser65 and Thr70 but which remains of normal size in the presence of calyculin A (Figures 2, 4–6) suggests that, although phosphorylation may be necessary, it is not sufficient for polyubiquitination. It remains to be determined whether the effects of calyculin A are due to inhibition of the dephosphorylation of 4E-BP1 itself or to an ability to enhance the activity of mTOR (Carroll et al., 2006). Our data suggest that, under some circumstances, stimuli that promote the phosphorylation of 4E-BP1 Oncogene


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Figure 6 Effects of rapamycin on calyculin A-induced changes in the level and phosphorylation of 4E-BP1. Jurkat cells were preincubated for 1 h in the presence or absence of rapamycin and/or calyculin A. Cycloheximide was then added to the cells and the incubations were continued for a further 16 h. Cell extracts were prepared and immunoblotted for total 4E-BP1, phosphorylated 4E-BP1 (Ser65, Thr70 and Thr37/46) and a-tubulin as indicated. The a, b and g forms of normal-sized 4E-BP1 and higher-molecularweight forms of the protein are indicated.

(for example, growth factors or other mitogenic agents) may increase the ubiquitination and turnover of this factor. Consistent with this, phosphorylation correlates with a dramatic loss of the protein following the fertilization of sea urchin eggs (Cormier et al., 2001; Salau¨n et al., 2003), and rapamycin has an inhibitory effect on the loss of 4E-BP in this system. In contrast, in adipocytes insulin (which stimulates the phosphorylation of 4E-BP1) has been reported to increase the halflife of the protein (Lin et al., 1995). However, this conclusion was based on pulse–chase studies of [35S]methionine-labelled cells over a prolonged time period and it is possible that insulin may have directly inhibited protein degradation in these cells (Li et al., 2000; Fawcett et al., 2001a, b). Conversely, physiological stresses that cause dephosphorylation of 4E-BP1, such as amino-acid starvation, DNA damage or the activation of p53 might be expected to stabilize the protein. Recent data do indeed suggest that induction of p53 leads to the accumulation of Oncogene

hypophosphorylated 4E-BP1 (Tilleray et al., 2006) and a similar outcome has been observed in cells treated with the proapoptotic cytokine TRAIL (IW Jeffrey and MJ Clemens, unpublished data). The effects of genotoxic stress on 4E-BP1 are further discussed below. There are many precedents in the literature for the stimulation of protein ubiquitination and degradation by phosphorylation (reviewed in Fuchs et al., 1998). However, there are also cases where phosphorylation and ubiquitination of a protein appear to be mutually exclusive. Of interest in the present context is the finding that a ubiquitinated form of initiation factor eIF4E is never phosphorylated (Murata and Shimotohno, 2006). This suggests that the ubiquitin-dependent turnover of eIF4E could be blocked by phosphorylation. Thus the phosphorylation of both eIF4E and 4E-BP1 that occurs in response to mitogenic and growth-promoting stimuli (Proud, 2002; Mamane et al., 2006) may have opposite consequences for the stability and levels of these two proteins. This would allow increased rates of protein synthesis under growth-stimulatory conditions as a result of both enhanced stability of eIF4E and inactivation or loss of its inhibitor. Conversely, physiological stresses that cause dephosphorylation of both eIF4E and 4E-BP1 may result in a decreased level of eIF4E and an increased level of 4E-BP1 due to opposite effects on the turnover of these proteins. Consistent with this, exposure of cells to ionizing radiation or DNAdamaging agents, which causes dephosphorylation of 4E-BP1 (Kumar et al., 2000; Tee and Proud, 2000), increases the stability of the protein (Schneider et al., 2005; Le Bouffant et al., 2006). On the other hand, physiological stresses such as heat shock or cadmium chloride increase the ubiquitination and turnover of eIF4E (Othumpangat et al., 2005a; Murata and Shimotohno, 2006). However, DNA damage has no apparent effect on eIF4E turnover (Paglin et al., 2005). Elevated levels of eIF4E result in inhibition of apoptosis (Clemens, 2004; Mamane et al., 2004), whereas overexpression of 4E-BP1 is proapoptotic (Li et al., 2002; Proud, 2005). It is therefore of interest that calyculin A is also antiapoptotic (Song and Lavin, 1993). Clearly, this protein phosphatase inhibitor is likely to affect the phosphorylation of a wide range of additional target proteins that may have regulatory effects on apoptosis (for example, IkB; Harhaj and Sun, 1997). Nevertheless, the regulation of 4E-BP1 may contribute to the inhibition of cell death by calyculin A. There are certain similarities between the regulation of eIF4E function by 4E-BP1 and the control of nuclear factor-kB (NF-kB) activity by IkB (Chen, 2005; Scheidereit, 2006). Both IkB and 4E-BP1 are phosphorylated in response to extracellular signals, favouring the ubiquitination and inactivation/degradation of these proteins. The loss of IkB allows NF-kB to enter the nucleus and stimulate the transcription of genes with antiapoptotic activity (Graham and Gibson, 2005). Likewise, the inactivation or loss of 4E-BP1 releases active eIF4E, which stimulates post-transcriptionally the expression of antiapoptotic or oncogenic proteins (Rosenwald et al., 1995; Rousseau et al., 1996; Hoover et al., 1997; Carter


819 4E

4E

(3)

4E

4E

4E-BP1

rapamycin

(2)

eIF4G eIF4G eIF (4)4G

mTOR/ other kinases

(1)

(5)

4E-BP1

Translation

S P

PT

4E-BP1

protein phosphatase(s)

++ MG132

4A

(6)

ubiquitination

calyculin A P T

(High levels of eIF4E favour the translation of mRNAs encoding growth-promoting and anti-apoptotic proteins)

S P

4E-BP1

(7) proteasome

(8) S P

P T

DEGRADATION OF 4E4E-BP1

4E-BP1

(9)

OTHER FUNCTIONS OF 4E-BP1?

Figure 7 Model for the regulation of 4E-BP1 by phosphorylation and polyubiquitination. In its hypophosphorylated state, 4E-BP1 binds to eIF4E (1) and sequesters the latter in an inactive complex. Phosphorylation of 4E-BP1 on multiple sites, by mTOR and other protein kinases (2), releases the eIF4E which is then available to interact with eIF4G (3) and initiate the translation of capped mRNAs (4). Phosphorylation is inhibited by rapamycin. High levels of active eIF4E preferentially favour the translation of mRNAs with extensive secondary structure, which encode growth-promoting and antiapoptotic proteins. Phosphorylated 4E-BP1 is dephosphorylated by protein phosphatase PP2A (5), thus allowing 4E-BP1 to become inhibitory for translation again. Dephosphorylation is inhibited by calyculin A. Our data suggest that phosphorylated 4E-BP1 can also be (poly)ubiquitinated (6). This may target some forms of the protein for degradation by the proteasome (7). Proteasome-mediated degradation is inhibited by MG132; this agent additionally causes dephosphorylation of normal-sized 4E-BP1 (presumably by an indirect mechanism) and this also stabilizes the protein. Some highermolecular-weight forms of 4E-BP1 may subsequently undergo further modifications (8) to generate species that could have other functions in the cell (9). 4E-BP1, 4E-binding protein 1; mTOR, mammalian target of rapamycin.

et al., 1999; Nimmanapalli et al., 2003; Othumpangat et al., 2005b). Since the overactivity or overexpression of either NF-kB or eIF4E inhibits apoptosis and promotes malignant transformation (Topisirovic et al., 2003; Clemens, 2004; Ren et al., 2006), the eIF4E/4E-BP1 system and its regulatory mechanisms can be viewed as a translational parallel to the NF-kB/IkB system.

Antibodies Antibodies against total 4E-BP1 and a-tubulin were from Santa Cruz Technology (Santa Cruz, CA, USA) and Sigma, respectively. Antibodies specific for phosphorylation sites in 4E-BP1 (Ser64, Thr36/45 and Thr69 in mouse; Ser65, Thr37/46 and Thr70 in human) were from Cell Signalling Technology. Soluble anti-ubiquitin, anti-ubiquitin-agarose beads and unmodified agarose beads were from Calbiochem.

Materials and methods

Preparation of cell extracts Cytoplasmic extracts were prepared by washing cells in phosphate-buffered saline and re-suspending the pellets in cell lysis buffer (50 mM 4-morpholinepropanesulphonic acid (MOPS), 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM ethylene glycol bis(b-aminoethylether)-N,N,N0 ,N0 ,tetraacetic acid (EGTA), 7 mM 2-mercaptoethanol, 40 mM b-glycerophosphate, 0.5% NP-40) containing the following phosphatase and proteinase inhibitors: Complete Mini, EDTAfree protease inhibitor cocktail (Roche, Lewes, Sussex, UK), 2 mM sodium vanadate, 1 mM microcystin, 1 mM phenylmethylsulphonylfluoride and 2 mM benzamidine. After 10 min at 41C the cell lysates were centrifuged for 15 min at 12 000 g to pellet the nuclei.

Cell culture and treatments Murine embryonic fibroblasts, MEL cells expressing a temperature-sensitive form of p53 (Johnson et al., 1993) and Jurkat human T lymphoma cells were grown as described (Juo et al., 1998; Jeffrey et al., 2002; Constantinou and Clemens, 2005). Where specified, cells were treated with calyculin A (Cell Signaling Technology, Hitchin, Herts, UK) (10 nM), MG132 (Cell Signaling Technology) (30 mM), zVAD-FMK (Calbiochem, Nottingham, UK) (10 mM), rapamycin (Calbiochem) (30 nM) or cycloheximide (Sigma, Poole, Dorset, UK) (20 mg/ml) for the times shown. Control cells received an equal volume of dimethylsulphoxide or water.

Oncogene


820 Immunoprecipitation and eIF4E affinity purification Immunoprecipitation was carried out in the presence of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% (v/v) Triton X-100, 2.5 mM Na pyrophosphate, 1 mM b-glycerophosphate, 1 mM sodium vanadate and 1 mg/ml leupeptin, using either monoclonal anti-4E-BP1 and protein A-agarose beads or anti-ubiquitin-agarose beads. Unmodified agarose beads were used as a control for non-specific immunoprecipitation. After five washes in the above buffer bound proteins were eluted with 2 sodium dodecyl sulphate (SDS) sample buffer. The proteins were then analysed by gel electrophoresis and immunoblotting as described below. Purification of eIF4E and associated proteins was performed by affinity chromatography on m7GTP-Sepharose beads (GE Healthcare, Amersham, Bucks, UK) as described previously (Constantinou and Clemens, 2007).

difluoride membranes. After fixation for 10 min with 0.05% glutaraldehyde in phosphate-buffered saline the blots were blocked with skimmed milk powder (5%, w/v) and incubated with the appropriate primary antibodies. All blots were developed with horseradish peroxidase-linked secondary antibodies using enhanced chemiluminescence (Cell Signaling Technology). Levels of a-tubulin were determined as a loading control. Molecular weight markers were a biotinylated protein ladder (10–200 kDa, from Cell Signaling Technology) and were detected with horseradish peroxidaselinked anti-biotin. Quantitative densitometry was performed using Scion Image software (Scion Corporation, Frederick, Ma, USA).

Immunoblotting Cytoplasmic extracts were analysed by SDS gel electrophoresis, loading equal amounts of protein (10–20 mg per sample). The proteins were transferred to poly(vinylidene)

We thank Dr Simon Morley (University of Sussex) for providing Jurkat cells and antibody to eIF4E. This work was supported by the Association for International Cancer Research and the Cancer Prevention Research Trust.

Acknowledgements

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