Regulation Of Protein Synthesis Initiation Research Articles

Abstract 4390: Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation

Abstract We previously reported that dietary daidzein, a soy isoflavone, increased breast cancer progression in a nude mouse model of breast cancer metastasis. Further studies indicated that equol, a metabolite of the soy isoflavone daidzein is responsible for the cancer promoting effects of soy isoflavones in breast cancer cells in vitro. We reported that equol increases the expression of the eukaryotic protein synthesis initiation factor eIF4G1, and the transcription factor c-Myc. Increased eIF4G in response to equol resulted in the non-canonical protein synthesis of pro-cancer molecules such as Cyclin D, Bcl-XL, p120, matrix metalloproteinases, etc., as confirmed by ribosome profiling and eIF4G1 knockdown studies. This study tested the hypothesis that equol promoted breast cancer progression via eIF4G1 upregulation. Immunocompromised nude mice were used to establish mammary fatpad tumors from human metastatic breast cancer cells expressing eIF4G1 knockdowns via a Tetracycline-inducible promoter. Oral gavage of 50 mg/kg BW Doxycycline 3X a week for 4 weeks was enough to knockdown eIF4G1 expression by ~80%. Therefore, one week following tumor establishment, the mice were treated with vehicle (90% corn oil, 10% ethanol), equol, Doxycycline or, equol and Doxycycline. Data show that as expected, equol treatment increased tumor growth relative to vehicle treatment. Moreover, eIF4G1 knockdown abolished the effect of equol on tumor growth. Interestingly, the effect of eIF4G1 knockdown on mammary tumor growth was not evident until 55 days of doxycycline treatment. Therefore, we tested the cell and tumor lysates for potential expression of the eIF4G1 isoform Dap5 when eIF4G1 is knocked out. Dap5 protein was expressed in eIF4G1 knockdown cells and was increased by equol treatment, indicating that Dap5 may compensate for eIF4G1 knockdown by inducing alternative cap-mediated protein synthesis. In conclusion, the cancer promoting effect of equol is abolished in breast tumors where eIF4G1 expression was stably knocked down, thus, validating our hypothesis that equol increases breast cancer progression via upregulation of eIF4G1 dependent protein synthesis initiation. Citation Format: Ailed M. Cruz-Collazo, Columba De la Parra, Robert Schneider, Suranganie Dharmawardhane. Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4390.

Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.

PHAS-I phosphorylation in response to foetal bovine serum (FBS) is regulated by an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes

The phosphorylation state of PHAS-I is thought to be important in the regulation of protein synthesis initiation. PHAS-I phosphorylation significantly increases in response to growth factors and insulin. ERK1/ERK2 have previously been implicated as PHAS-I kinases. Present work utilised a specific phosphorothioate oligonucleotide antisense strategy against ERK1/ERK2 to determine whether ERK1/ERK2 mediate FBS-stimulated PHAS-I phosphorylation in vivo. Depleting >90% of cellular ERK1/ERK2 had no effect on FBS-stimulated PHAS-I phosphorylation. However, treatment of cells with a specific p70 S6k pathway inhibitor, rapamycin, markedly attenuated FBS-stimulated PHAS-I phosphorylation. These results indicate that PHAS-I phosphorylation in response to FBS occurs through an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes.

Translational initiation factor eIF-2 subcellular levels and phosphorylation status in the developing rat brain

We have quantified the levels of the α subunit of initiation factor 2 (eIF-2) in the postmicrosomal supernatant and the ribosomal salt wash fractions from suckling and adult rat brain. The levels of eIF-2 in the ribosomal salt wash decrease in adult with respect to that present in suckling rat brain, but the total amount remains fairly constant, and a very close parallelism exists between the eIF-2 associated with ribosomes and RNA levels in the microsomal fraction in the two age groups. The phosphorylation state of eIF-2α, as determined by isoelectric focusing followed by protein immunoblotting, in the same subcellular fractions, did not reveal the presence of the phosphorylated form in any of the fractions studied. These results suggest that phosphorylation of the α subunit is not implied in the regulation of protein synthesis initiation during brain development, and some other component regulates both the number of active ribosomes and eIF-2 levels in microsomes.

The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p67) plays a critical role in regulation of protein synthesis initiation in animal cells.

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) isolated from reticulocyte lysate protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p67 and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p67 and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p67 protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p67 is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 alpha-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p67 and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p67. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 alpha subunit because of the presence of p67. (ii) p67 is essential for protein synthesis as it protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p67 is both degradable and inducible. Only the p67 level correlates directly with the protein synthesis activity of the cell, indicating that p67 is a critical factor in protein synthesis regulation in animal cells.

Effect of vasopressin on the regulation of protein synthesis initiation in liver cells.

Vasopressin was found to be an effective inhibitor of protein labelling in isolated liver cells. Its effect shows the following distinct characteristics: (1) in contrast with alpha-adrenergic agonists, its effect is observable under a wide range of cellular Ca2+-loading conditions; (2) it is not influenced by the nutritional state of the animal. The lack of vasopressin effect on valine production, and its ability to decrease protein labelling from near-saturation concentrations of [3H]valine, indicate that the observed variations in protein labelling reflect actual changes in the rate of protein synthesis. The action of vasopressin is primarily exerted on the initiation step of protein synthesis and this effect is accompanied by a decreased activity of eukaryotic initiation factor 2. Activators of protein kinase C showed similar but not additive effects on protein synthesis, as did vasopressin. It seems plausible to conclude that protein kinase C activation may play an important regulatory role in hepatic protein synthesis as a transducer of hormonal and perhaps other type of signals.

The specific phosphorylation of a 40S ribosomal protein in growth-arrested tetrahymena is induced by sodium.

In the past few years, in vivo phosphorylation of ribosomal proteins has been the subject of extensive studies and the results have shown that reversible phosphorylation of small subunit ribosomal protein S6, ubiquitous in eukaryotic cells, is apparently related to regulation of protein synthesis initiation. Thus the level of protein synthesis under various conditions is correlated with the level of S6 phosphorylation. In exponentially growing Tetrahymena, however, such phosphorylation does not occur, but when these cells are transferred to starvation buffers, the rate of protein synthesis is drastically reduced and a 40S ribosomal protein analogous to S6 of higher eukaryotic cells is fully and rapidly phosphorylated in all the ribosomes. We have studied the conditions which lead to this phosphorylation in growth-arrested Tetrahymena, in order to understand the physiological significance of this process. Our results show that there is no obvious correlation between this phosphorylation and starvation. Moreover, it is not a developmentally regulated process related to the conjugation cycle, but a modification induced by the presence of sodium ions or high concentration of Tris in the starvation buffer. The physiological significance of this process is discussed in terms of accumulation of negative charge density probably required for initiation of protein synthesis in the growth-arrested cells starving in Na+-containing buffers.

Regulation of protein synthesis initiation in eucaryotes

Eukaryotic initiation factor (eIF)2B is a guanine nucleotide exchange factor (GEF) for a second initiation factor, eIF2. During the initiation of messenger RNA (mRNA) translation, eIF2 binds Guanosine-5'-triphosphate (GTP) and initiator methionyl- transfer RNA (tRNAi) (met-tRNAi) and the resulting ternary complex subsequently binds to the 40S ribosomal subunit. Alterations in ternary complex formation due to changes in eIF2B GEF activity not only produce alterations in global rates of protein synthesis but also cause specific changes in the translation of mRNAs encoding certain proteins, such as the transcription factors general control nonrepressed 2 (GCN2) and activating transcription factor 4 (ATF4). Studies have implicated changes in eIF2B GEF activity in alterations in protein synthesis in a number of pathophysiological and physiological conditions, such as diabetes, cancer, sepsis, vanishing white matter disease, and resistance exercise. The chapter discusses the state of knowledge concerning the function of eIF2B and its role in regulating mRNA translation.

Roles of Eukaryotic Initiation Factor 2 Ancillary Factors in the Regulation of Eukaryotic Protein Synthesis Initiation

This chapter discusses a possible mechanism of protein synthesis inhibition by eukaryotic initiation factor-2 (eIF-2) kinases, heme-regulated protein synthesis inhibitor, and double-stranded RNA-activated inhibitor; it discusses the roles of four eIF-2 ancillary protein factors in the complex regulation of protein synthesis initiation in animal cells. The loss of interaction of eIF-2α(P) with Co-eIF-2B and Co-eIF-2C has been suggested as the mechanism of protein synthesis inhibition by eIF-2 kinases. However, a direct evidence that the formation of eIF-2α(P) and consequent loss of interaction of eIF-2α(P) with Co-eIF-2B and Co-eIF-2C are the sole cause of protein synthesis inhibition in heme-deficient reticulocyte lysates is still lacking. The four eIF-2 ancillary factors, Co-eIF-2A, Co-eIF-2B, Co-eIF-2C, and reversal factor, have been characterized by their effects on eIF-2 activity in vitro . The requirement for Co-eIF-2A for protein synthesis in reticulocyte lysates is shown by using antibodies. It has not yet been possible to reconstitute from purified components an efficient protein-synthesizing system with which to examine the requirements for specific factors in overall peptide chain initiation.

Regulation of protein synthesis initiation in HeLa cells deprived of single essential amino acids.

In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.