The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis
2020; Elsevier BV; Volume: 295; Issue: 29 Linguagem: Inglês
10.1074/jbc.ra120.012979
ISSN1083-351X
AutoresXin Jin, Jianling Xie, Michael Zabolocki, Xuemin Wang, Tao Jiang, Dong Wang, Laurent Désaubry, Cédric Bardy, Christopher G. Proud,
Tópico(s)RNA and protein synthesis mechanisms
ResumoFluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival. Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival. Dysregulation of protein synthesis (mRNA translation) plays an important roles in many diseases, in particular, cancers (1Robichaud N. Sonenberg N. Ruggero D. Schneider R.J. Translational control in cancer.Cold Spring Harb. Perspect. Biol. 2019; 11 (29959193): a03289610.1101/cshperspect.a032896Crossref PubMed Scopus (63) Google Scholar). The rate of protein synthesis can be modulated by changes in the phosphorylation of proteins involved in the initiation and elongation stages of the process of mRNA translation; these are termed eukaryotic initiation or elongation factors (eIFs, eEFs), respectively. For example, phosphorylation of the heterotrimer eIF2 on its α-subunit (eIF2α) leads to inhibition of translation initiation and thus general protein synthesis (2Wek R.C. Role of eIF2α kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10 (29440070): a03287010.1101/cshperspect.a032870Crossref PubMed Scopus (119) Google Scholar). Several kinases can perform this modification and are generally activated by stress conditions such as the accumulation of unfolded proteins in the endoplasmic reticulum or absence of a given amino acid. eEF2 also undergoes phosphorylation, which inhibits its activity and thus slows down translation elongation (3Kenney J.W. Moore C.E. Wang X. 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Chem. 2018; 293 (29523683): 8285-829410.1074/jbc.RA118.002316Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). PHBs may be useful targets for cancer therapy (33Ortiz M.V. Ahmed S. Burns M. Henssen A.G. Hollmann T.J. MacArthur I. Gunasekera S. Gaewsky L. Bradwin G. Ryan J. Letai A. He Y. Naranjo A. Chi Y.Y. LaQuaglia M. et al.Prohibitin is a prognostic marker and therapeutic target to block chemotherapy resistance in Wilms' tumor.JCI Insight. 2019; 4 (31391345): e12709810.1172/jci.insight.127098Crossref PubMed Scopus (9) Google Scholar); however, there remains a paucity of information about the effects of FLZ and of its targets, the PHBs. Here we built upon earlier data indicating that PHBs (in particular PHB2) interact with certain components of the translational (protein synthesis) machinery, including ribosomal proteins, or kinases that act upon them (RRID:SCR_018711), and with some signaling proteins involved in the regulation of translation such as ERK (MAP kinase), the AMP-activated protein kinase (AMPK) or mTORC1. We now show that FLZ induces the phosphorylation of two key components of the protein synthetic machinery, initiation factor eIF2 and elongation factor eEF2. The phosphorylation of each of these general translation factors inhibits its function. Consistent with this, FLZ induces the inhibition of overall protein synthesis. FLZ also elicits a marked increase in intracellular calcium ion levels, which activate eEF2 kinase and evoke endoplasmic reticulum (ER) stress, which causes enhanced phosphorylation of eIF2. FLZ also inhibits of mTORC1 signaling and thus affects S6K and eIF4E-BP1, all of which effects likely contribute to the inhibition of overall protein synthesis caused by FLZ. FLZ also induces cell death, and this appears to involve the phosphorylation of both eEF2 and eIF2. Our data substantially extend understanding of the physiological roles of PHBs. We began by testing a range of PHB-binding compounds for their effects, in A549 lung carcinoma cells, on translation factors or signaling pathways linked to them. As shown in Fig. 1A, FLZ caused a very marked increase in the phosphorylation of eEF2 rapidly by 15 min, whereas the other compounds had little or no effect, even after 60-min exposure. By 15 min, FLZ also elicited a rise in the phosphorylation of the AMPK, a metabolic sensor which can activate eEF2K (34Horman S. Browne G.J. Krause U. Patel J.V. Vertommen D. Bertrand L. Lavoinne A. Hue L. Proud C.G. Rider M.H. Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis.Curr. Biol. 2002; 12 (12194824): 1419-142310.1016/S0960-9822(02)01077-1Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 35Browne G.J. Finn S.G. Proud C.G. Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398.J. Biol. Chem. 2004; 279 (14709557): 12220-1223110.1074/jbc.M309773200Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The effect on phosphorylated (P)-eEF2 occurred at concentrations lower than 20 μM, but the increased phosphorylation of AMPK did not (Fig. S1A), suggesting the change in eEF2 phosphorylation was not because of activation of AMPK. Further experiments to test the role of eEF2K are described below. At 60 min, FLZ did not exert a noticeable effect on other proteins tested, apart from causing a small shift in the electrophoretic behavior of 4E-BP1 toward faster-migrating species, which correspond to less phosphorylated variants of this protein (cf. decrease in signal for P–Ser-65 in 4E-BP1 (Fig. 1A). This suggests that FLZ causes mild impairment of mTORC1 signaling, which was confirmed by a large decrease in the phosphorylation of p70 S6 kinase, a direct substrate for mTORC1 (Fig. 1A and Fig. S1B). However, little if any change was seen for S6, a substrate for p70 S6K (Fig. 1A and Fig. S1B). A slight increase in the phosphorylation of ERK was consistently observed (Fig. 1A), which likely accounts for the small rise in the phosphorylation of the translation initiation factor eIF4E, which is linked to ERK signaling via the eIF4E kinases, the MNKs (9Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. Mitogen-activated kinases activate the serine/threonine kinases Mnk1 and Mnk2.EMBO J. 1997; 16 (9155017): 1909-192010.1093/emboj/16.8.1909Crossref PubMed Scopus (755) Google Scholar, 36Wang X. Flynn A. Waskiewicz A.J. Webb B.L.J. Vries R.G. Baines I.A. Cooper J. Proud C.G. The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses and cytokines is mediated by distinct MAP kinase pathways.J. Biol. Chem. 1998; 273 (9545260): 9373-937710.1074/jbc.273.16.9373Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). When treatment with FLZ was extended to 120 min, it was clear that the increased phosphorylation of eEF2 was sustained (Fig. 1B and Fig. S2, A–C) and that FLZ also induced, albeit more slowly, the phosphorylation of eIF2α (Fig. 1B). Activation of AMPK (as judged from P-ACC (acetyl-CoA carboxylase) levels) declined by the final time point (Fig. 1B). The FLZ-induced rise in the phosphorylation of eEF2 was very fast, being evident as early as 1 min (Fig. S2A). Given these striking effects of FLZ on phosphorylation of regulators of mRNA translation in A549 cells, we considered it important to assess its effects in other cancer cell lines. In MDA-MB-231 breast cancer cells FLZ also increased the phosphorylation of eEF2 and eIF2 (Fig. 1C and Fig. S2, D and E), but had little or no effect on P-ERK or P-eIF4E. Other compounds tested had little, if any, effect on the proteins studied in MDA-MB-231 cells, apart from some activation of ERK by FL3 (and FLZ) (Fig. S3A). The FLZ-induced phosphorylation of eEF2 was transient, being maximum at 30-60 min (Fig. 1C and Fig. S2, D and E); eIF2α phosphorylation again rose markedly but more slowly; it was still elevated at 120 min (Fig. 1, B–D). FLZ had little effect on the AKT/mTORC1 pathway in MDA-MB-231 cells, apart from a minor inhibitory effect on P-AKT (Ser-473), a substrate for mTORC2 (Fig. S3B). FL3 but not the other PHB ligands inhibited the phosphorylation of S6K1 (Fig. S3, A and B), a readout of mTORC1 activity in MDA-MB-231 cells, concomitant with a reduction in the phosphorylated levels of eEF2 (Fig. S3, A and B). A broadly similar pattern of changes in the phosphorylation of eEF2 and eIF2 was also observed in a third cell line, cervical cancer HeLa cells (Fig. 1D and Fig. S2, F and G). These data show that FLZ rapidly enhances the phosphorylation of eIF2 and eEF2 in all three cell lines, with the effect on P-eEF2 being considerably faster (already higher at 5 min in all three lines) than the rise in P-eIF2 (evident by 30 min). Because FLZ increases the phosphorylation of eIF2α and eEF2 and these modifications impair the functions in the elongation and initiation phases of mRNA translation (2Wek R.C. Role of eIF2α kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10 (29440070): a03287010.1101/cshperspect.a032870Crossref PubMed Scopus (119) Google Scholar, 37Proud C.G. Control of the elongation phase of protein synthesis.in: Sonenberg N. Hershey J.W.B. Mathews M.B. Translational control of gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 719-739Google Scholar), we asked whether FLZ affected the rate of protein synthesis. To do this, we employed the widely used SunSET assay (38Schmidt E.K. Clavarino G. Ceppi M. Pierre P. SUnSET, a nonradioactive method to monitor protein synthesis.Nat. Methods. 2009; 6 (19305406): 275-27710.1038/nmeth.1314Crossref PubMed Scopus (737) Google Scholar), whereby cells are incubated with puromycin, which becomes incorporated into newly made proteins. Proteins are then resolved by SDS-PAGE and analyzed by immunoblotting using an anti-puromycin antibody. The signal observed reports newly made proteins. The assay was configured such that puromycin was added after a 15- or 45-min pretreatment of the cells with FLZ to allow us to assess effects of protein synthesis over two different time windows, 15–30 or 45–60 min. FLZ modestly reduced puromycin incorporation over the 15- to 30-min interval in A549, MDA-MB-231 or HeLa cells (Fig. 2, A–C) and more markedly decreased it over the 45- to 60-min time period. Indeed, when corrected for nonspecific labeling by comparing with the signal seen in the presence of an inhibitor of translation, cycloheximide (CHX), net puromycin incorporation was very low over the 45- to 60-min time period in FLZ-treated A549 or HeLa cells (Fig. 2, A–C). Thus, FLZ inhibits protein synthesis and does so more strongly at time points later than the initial increase in eEF2 phosphorylation. The timing of this inhibition correlates with the increased phosphorylation of eIF2, which is well-known to cause a general inhibition of translation (2Wek R.C. Role of eIF2α kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10 (29440070): a03287010.1101/cshperspect.a032870Crossref PubMed Scopus (119) Google Scholar). This suggested that the inhibition of protein synthesis caused by FLZ was not only, or not primarily, because of the rise in P-eEF2. To assess whether the inhibition of protein synthesis caused by FLZ did indeed involve the phosphorylation of eEF2, we made use of MDA-MB-231 cells in which the gene for eEF2K had been knocked out (39Xie J. Shen K. Lenchine R.V. Gethings L.A. Trim P.J. Snel M.F. Zhou Y. Kenney J.W. Kamei M. Kochetkova M. Wang X. Proud C.G. Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis.Int. J. Cancer. 2018; 142 (29235102): 1865-187710.1002/ijc.31210Crossref PubMed Scopus (17) Google Scholar); in such cells, as expected, no phosphorylation of eEF2 was observed (Fig. 2D). When compared with WT MDA-MB-231 cells, it was evident that the rapid phase of FLZ-induced inhibition was essentially lost, whereas the inhibition seen at later times was hardly affected (Fig. 2, E and F; compare quantification with that in Fig. 2B). Taken together, these data are consistent with the conclusion that the rapid phase of the inhibition of protein synthesis by FLZ involves the phosphorylation of eEF2, and the longer-term inhibition (at 45-60 min) involves additional effects, which likely include the increased phosphorylation of eIF2α and/or accumulation of 4E-BPs (see below). Lastly, we noted that the early phase of the inhibition of protein synthesis caused by FLZ was stronger in HeLa cells than in A549 cells (Fig. 2, A and C). Interestingly, direct comparison on the same immunoblots showed that FLZ induced higher levels of P-eEF2 in HeLa than in A549 cells, which is consistent with eEF2 phosphorylation being largely responsible for the rapid inhibition of translation by FLZ in HeLa cells but playing a lesser role in A549 cells (Fig. S4). FLZ can also inhibit 4E-BP1 phosphorylation over longer (i.e. 60 min) periods of time (Fig. 1 and Fig. S1), dephosphorylated 4E-BP1 associates with eIF4E and hence interferes with cap-dependent translation. It was therefore important to determine whether FLZ affected 4E-BP1–eIF4E binding. There was an increase in the signal for total 4E-BP1 in response to FLZ in A549 cells, which probably reflects an accumulation of dephosphorylated 4E-BP1 (cf. decreased phosphorylation of 4E-BP1 at Thr-37/46) (Fig. 3A). Dephosphorylated 4E-BP1 is stabilized by its association with eIF4E (40Yanagiya A. Suyama E. Adachi H. Svitkin Y.V. Aza-Blanc P. Imataka H. Mikami S. Martineau Y. Ronai Z.A. Sonenberg N. Translational homeostasis via the mRNA cap-binding protein, eIF4E.Mol. Cell. 2012; 46 (22578813): 847-85810.1016/j.molcel.2012.04.004Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and the collapse of multiple differentially phosphorylated bands into one major hypophosphorylated species. As shown by m7GTP affinity chromatography, although FLZ did not alter the association of 4E-BP1 with eIF4E at 15 min, it did increase 4E-BP1-eIF4E binding after 60 min (Fig. 3, B–D). However, FLZ did not alter levels of 4E-BP1 phosphorylation or total 4E-BP1 in MDA-MB-231 cells (Fig. S3, C and D). These data imply that inhibition of 4E-BP1 phosphorylation, and increased binding of this protein to eIF4E are likely to contribute to the inhibitory effect of FLZ, at least in A549 cells, on the late (60 min) but not the rapid (15 min
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