Regulation of translation factors eIF4GI and 4E-BP1 during recovery of protein synthesis from inhibition by p53

Abstract

Activation of the tumour suppressor protein p53 rapidly inhibits protein synthesis. This is associated with dephosphorylation and cleavage of initiation factor eIF4GI and the eIF4E-binding protein 4E-BP1. When the activation of p53 is reversed within 16 h 4E-BP1 becomes rephosphorylated, the level of intact eIF4GI slowly increases and protein synthesis gradually recovers. The recovery of protein synthesis is partially blocked by rapamycin and wortmannin but not by the protein kinase inhibitors PD98059 and CGP74514A. Both rapamycin and wortmannin, but not PD98059 or CGP74514A, delay the reappearance of eIF4GI. In contrast, full-length 4E-BP1 rapidly becomes rephosphorylated and this process is partially inhibited by rapamycin, PD98059 and CGP74514A. Thus, activation of p53 results in the inhibition of distinct rapamycin-and wortmannin-sensitive pathways that target eIF4GI, and rapamycin-sensitive and -insensitive pathways that target 4E-BP1. Following inactivation of p53 the gradual recovery is determined largely by the kinetics of restoration of eIF4GI rather than by the rephosphorylation of full-length 4E-BP1. These findings suggest that the ability of cells to rephosphorylate 4E-BP1, resynthesise eIF4GI and restore the rate of protein synthesis after inactivation of p53 is an important aspect of recovery following the relief of physiological stress. Activation of the tumour suppressor protein p53 causes a rapid inhibition of protein synthesis which precedes the induction of a cell cycle block and subsequent apoptosis. 1-3The inhibition of translation is accompanied by early dephosphorylation and cleavage of the eukaryotic initiation factor (eIF)4E-binding protein 4E-BP1 as well as cleavage of polypeptide chain initiation factor eIF4GI and dephosphorylation of this protein at Ser 1108 . 3 The cleaved products of 4E-BP1 and eIF4GI, which are generated by a caspaseindependent mechanism, associate with eIF4E and there is a correspondingly decreased extent of interaction of eIF4E with full-length eIF4GI. These changes are likely to play an important role in impairing the initiation of protein synthesis.In this study, we have examined whether the inhibition of protein synthesis can be reversed when a temperaturesensitive form of p53 is inactivated, prior to the irreversible commitment to cell death, 2 by return of the cells to the non-permissive temperature (381C). We have also investigated the requirement for various signalling pathways that are known to regulate protein synthesis and have analysed the integrity and state of phosphorylation of eIF4GI and 4E-BP1 during the recovery phase. The results indicate a multifactorial mode of regulation of translation following the inactivation of p53 that is partially dependent on the activities of the protein kinase mammalian target of rapamycin (mTOR) and phosphatidylinositol-3-kinase (PI3k) but is independent of the protein kinases Mek1/2 and cdc2. Furthermore, our findings suggest that the rate of recovery of protein synthesis is limited by the kinetics of reappearance ...

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

“…To determine whether this pathway is also able to regulate the expression of TCTP, we made use of a pair of mouse erythroleukaemia cells expressing either a temperature-sensitive mutant form of p53 or a non-activatable mutant p53 (Constantinou and Clemens, 2007). Our results (Figure 2a) clearly show that activation of p53 results in downregulation of TCTP levels, similar to the effects of cellular Ca þ þ -stress conditions (Figure 1).…”

“…MEFs from PKR-knockout mice (PKR À/À cells) and from the corresponding wild-type mice (PKR þ / þ cells), provided by the laboratory of Dr C Weissmann, were cultivated with the addition of 0.1 mM b-mercaptoethanol. Cultivation of mouse erythroleukaemia cells was performed as described earlier (Constantinou and Clemens, 2007). MEFs from mutant eIF2a (S51A)-knock-in mice and from wild-type animals were kindly provided by Drs D Scheuner and R Kaufman.…”

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

“…Purification of eIF4E and associated proteins was performed by affinity chromatography on m 7 GTP-Sepharose beads (GE Healthcare, Amersham, Bucks, UK) as described previously (Constantinou and Clemens, 2007).…”

Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas

“…The importance of p53 in this process is demonstrated by the fact that inactivation of p53 occurs in >50% of human cancers, and loss of p53 function is known to be essential for carcinogenesis (Vogelstein et al 2000;Vousden and Prives 2009). Upon activation, p53 functions as a transcription factor to induce a number of genes, such as p21 (el-Deiry et al 1993), MDM2 (Barak et al 1993;Wu et al 1993), and TIGAR (Bensaad et al 2006). These p53 target genes mediate diverse biological functions of p53, including cell cycle arrest and apoptosis (Harms et al 2004;Riley et al 2008).…”