Oncogene (2008) 27, 811–822 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc 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 structure by polypeptide chain initiation factor eIF4E initiation factor 4E (eIF4E) for protein synthesis is (Mamane et al., 2004; von der Haar et al., 2004). The regulated by the 4E-binding proteins (4E-BPs), which availability of the latter for formation of the active act as inhibitors of cap-dependent mRNA translation. The eIF4F complex is regulated by the 4E-binding proteins ability of the 4E-BPs to sequester eIF4E is regulated by (4E-BP1 and 4E-BP2) (Clemens, 2001; Mamane et al., reversible phosphorylation at multiple sites. We show here 2006). The most thoroughly studied of these is 4E-BP1, that, in addition, 4E-BP1 is a substrate for polyubiquiti- which in its hypophosphorylated state, binds and nation and that some forms of 4E-BP1 are simultaneously sequesters eIF4E. Phosphorylation of 4E-BP1, at multi- polyubiquitinated and phosphorylated. In Jurkat cells ple sites, decreases the affinity of this protein for eIF4E inhibition of proteasomal activity by MG132 enhances the and thus releases the cap-binding protein for eIF4F level of hypophosphorylated, unmodified 4E-BP1 but only complex formation. The phosphorylation of 4E-BP1 is modestly increases the accumulation of high-molecular- influenced by a wide range of extracellular stimuli. In weight, phosphorylated forms of 4E-BP1. In contrast, general, conditions that promote cell growth enhance inhibition of protein phosphatase activity with calyculin A the phosphorylation of 4E-BP1 and are associated with reduces the level of unmodified 4E-BP1 but strongly increased rates of overall protein synthesis; conversely, enhances the amount of phosphorylated, high-molecular- growth inhibitory conditions and physiological stresses weight 4E-BP1. Turnover measurements in the presence result in dephosphorylation of the protein and cause of cycloheximide show that, whereas 4E-BP1 is normally downregulation of protein synthesis (Mamane et al., a very stable protein, calyculin A decreases the apparent 2006). The protein kinase mammalian target of rapa- half-life of the normal-sized protein. Affinity chromato- mycin (mTOR) plays a crucial role in the phosphoryla- graphy on m7GTP-Sepharose indicates that the larger tion of 4E-BP1, although other protein kinases have also forms of 4E-BP1 bind very poorly to eIF4E. We suggest been implicated (Herbert et al., 2002; Gingras et al., that the phosphorylation of 4E-BP1 may play a dual role 2004; Tee and Blenis, 2005). There is evidence that in the regulation of protein synthesis, both reducing the 4E-BP1 associates with protein phosphatase 2A (Peter- affinity of 4E-BP1 for eIF4E and promoting the conver- son et al., 1999), and it is likely that the depho- sion of 4E-BP1 to alternative, polyubiquitinated forms. sphorylation of 4E-BP1 is catalysed by this enzyme. Oncogene (2008) 27, 811–822; doi:10.1038/sj.onc.1210678; The availability of eIF4E has differential effects on published online 23 July 2007 the translation of different mRNAs, with those contain- ing complex, structured 50-untranslated regions being Keywords: calyculin A; eIF4E; protein degradation; protein particularly sensitive to changes in the level of the factor phosphatases; protein synthesis (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 Introduction with malignant transformation (De Benedetti and Graff, 2004), probably because the factor promotes the The translation of most mRNAs in eukaryotic cells synthesis of antiapoptotic proteins (Clemens, 2004; requires recognition of the 50 m7GTP-containing cap Mamane et al., 2004), and many naturally occurring tumours contain elevated levels of eIF4E. Conversely, enhanced expression of 4E-BP1 can reverse the trans- Correspondence: Professor MJ Clemens, Translational Control Group, Division of Basic Medical Sciences, Centre for Molecular formed phenotype and increase the susceptibility of cells and Metabolic Signalling, St George’s, University of London, to apoptosis (Li et al., 2002; Proud, 2005). In naturally Cranmer Terrace, London SW17 0RE, UK. occurring tumours, the level of phosphorylation of E-mail: [email protected] 4E-BP1 correlates with tumour stage and prognosis 1Current address: Yasoo Health Ltd, 1 Poseidon Street, PO Box 25193, 1307 Nicosia, Cyprus. (Castellvi et al., 2006). Received 22 February 2007; revised 23 May 2007; accepted 13 June 2007; Whereas a large body of work has been devoted to the published online 23 July 2007 phosphorylation of 4E-BP1, very few studies have Phosphorylation, ubiquitination and stability of 4E-BP1 A Elia et al 812 examined regulation of the stability of the protein. 1995; Gingras et al., 1996; Mendez et al., 1996), mouse Following fertilization of sea urchin eggs, there is a rapid and human 4E-BP1 were detected as multiple bands on loss of 4E-BP which occurs in parallel with dramati- gels in the vicinity of 20 kDa. These correspond to cally enhanced rates of protein synthesis (Cormier differentially phosphorylated forms, with hyperpho- et al., 2001; Salau¨ n et al., 2003). This loss is inhibited sphorylated (g) 4E-BP1 migrating more slowly than by rapamycin, suggesting a role for protein phosphory- the less phosphorylated (b and a) forms. In addition, lation in the degradation of 4E-BP. In a mammalian analysis of extracts from mouse embryonic fibroblasts cell system dephosphorylation of 4E-BP1 induced by showed that regions of the blots corresponding to the tumour suppressor protein p53 is accompanied higher-molecular-weight proteins contain several other by an increase in the level of the 4E-BP1, in parallel with bands that are recognized by antibodies against total inhibition of protein synthesis (Tilleray et al., 2006). 4E-BP1 (Figure 1a). At least some of these forms of the However, the mechanisms regulating 4E-BP1 levels have protein are ubiquitinated, as revealed by immunopreci- not so far been extensively investigated. pitation with anti-4E-BP1 followed by immunoblotting Many proteins of regulatory significance are targeted with anti-ubiquitin. As shown in Figure 1b (left panel), a for degradation by the 26S proteasome as a result of band that migrates at around 50 kDa was identifiable their polyubiquitination (Fang and Weissman, 2004). by this approach. When the converse experiment was Moreover, the susceptibility of such proteins to be performed, immunoprecipitating a cell extract with anti- processed by this pathway is often controlled by protein ubiquitin-coated agarose beads and blotting with anti- phosphorylation. In addition to the now classical cases 4E-BP1, a similar sized band was seen (Figure 1b, right of the inhibitor of nuclear factor kB(IkB) family (Karin panel). The mobility of this protein suggests that it contains and Ben-Neriah, 2000; Chen, 2005; Krappmann and three or four ubiquitin moieties. The co-precipitation Scheidereit, 2005) and cyclins (Lin et al., 2006a), other reaction was specific since no anti-4E-BP1 reactive material examples of proteins whose (poly)ubiquitination is was observed when unmodified agarose beads were used stimulated by their phosphorylation include the type I (Figure 1b, right panel, lane 2) and no unmodified interferon receptor IFNAR1 (Kumar et al., 2004), the (20 kDa) 4E-BP1 was detected after anti-ubiquitin transcription factor STAT1 (Kim and Maniatis, 1996), precipitation. the antiapoptotic protein Bcl-2 (Lin et al., 2006b) and b- catenin (Aberle et al., 1997; Orford et al., 1997). On the other hand, phosphorylation can inhibit the ubiquitina- kDa M tion of other proteins such as the proto-oncogenes c-jun 100 80 (Musti et al., 1997; Fuchs et al., 1998), c-fos (Okazaki 60 Higher molecular and Sagata, 1995) and c-mos (Nishizawa et al., 1993). 50 40 Recent studies have shown that initiation factor forms 30 eIF4E can undergo ubiquitination and proteasome- γ β mediated turnover (Othumpangat et al., 2005a; Murata 20 α and Shimotohno, 2006). In contrast, although there have been indications that 4E-BP1 is also degraded by 10 the proteasome (Walsh and Mohr, 2004; Walsh et al., 1 MkDa M 2 3 2005; Wan et al., 2005), no information has been 60 50 polyUb-4E-BP1 published on the possible ubiquitination of this protein. polyUb-4E-BP1 40 In this paper, we present evidence from a variety of cell 30 lines indicating that human and mouse 4E-BP1 can be 20 polyubiquitinated, giving rise to high-molecular-weight 10 Anti-4E-BP1 Anti-UbIP forms. Our data suggest that this process is controlled IPAnti-Ubblot Anti-4E-BP1 blot by phosphorylation of the protein. Moreover, we show Figure 1 Evidence for polyubiquitination of 4E-BP1. (a)A that enhancing the phosphorylation of 4E-BP1 both cytoplasmic extract from murine embryonic fibroblasts was decreases the level of the normal-sized protein and subjected to gel electrophoresis followed by immunoblotting for converts the latter into high-molecular-weight forms total 4E-BP1. Molecular weight markers were separated in a that are unable to associate with eIF4E. The implica- 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 tions for the regulation of protein synthesis and cell higher-molecular-weight cross-reacting bands are shown. The transformation by eIF4E and 4E-BP1 are discussed. 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.