Dissociation of double stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay

Tg, Lawson; Ka, Lee; Mm, Maimone; Rd, Abramson; Dever, Thomas E.; Merrick, WC; Re, Thach

doi:10.1021/bi00437a033

Cited by 92 publications

(64 citation statements)

References 39 publications

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“…Previous studies have suggested that RSKs directly phosphorylate rpS6 at Ser 235/236 and eIF4B at Ser 422 . The former promotes binding of rpS6 to the 7-methylguanosine cap complex and enables cap-dependent translation to proceed, while the latter is critical for eIF4B binding to the cap complex and enhanced helicase activity of eIF4A and increased cellular translation (64)(65)(66). In agreement with these results, we observed that RSK4-overexpressing cells exhibited elevated levels of overall translation, which are maintained in the presence of PI3K inhibitors ( Figure 7F).…”

Section: Rsk Expression Promotes Resistance To Pi3k Inhibitors In Vivosupporting

confidence: 83%

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

Serra¹,

Eichhorn²,

García-García³

et al. 2013

J. Clin. Invest.

113

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The PI3K signaling pathway regulates diverse cellular processes, including proliferation, survival, and metabolism, and is aberrantly activated in human cancer. As such, numerous compounds targeting the PI3K pathway are currently being clinically evaluated for the treatment of cancer, and several have shown some early indications of efficacy in breast cancer. However, resistance against these agents, both de novo and acquired, may ultimately limit the efficacy of these compounds. Here, we have taken a systematic functional approach to uncovering potential mechanisms of resistance to PI3K inhibitors and have identified several genes whose expression promotes survival under conditions of PI3K/mammalian target of rapamycin (PI3K/mTOR) blockade, including the ribosomal S6 kinases RPS6KA2 (RSK3) and RPS6KA6 (RSK4). We demonstrate that overexpression of RSK3 or RSK4 supports proliferation upon PI3K inhibition both in vitro and in vivo, in part through the attenuation of the apoptotic response and upregulation of protein translation. Notably, the addition of MEK-or RSK-specific inhibitors can overcome these resistance phenotypes, both in breast cancer cell lines and patient-derived xenograft models with elevated levels of RSK activity. These observations provide a strong rationale for the combined use of RSK and PI3K pathway inhibitors to elicit favorable responses in breast cancer patients with activated RSK.

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Section: Rsk Expression Promotes Resistance To Pi3k Inhibitors In Vivosupporting

confidence: 83%

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

Serra¹,

Eichhorn²,

García-García³

et al. 2013

J. Clin. Invest.

113

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“…Because some DEAD-box proteins have the ability to unwind a short RNA duplex, DEAD-box proteins are often referred to as RNA helicases. Nevertheless, only a third of yeast DEAD-box proteins have been tested for unwinding activity (26,29,31,(48)(49)(50)(51)(52)(53)(54), and several are not efficient unwinding enzymes (21,55). In contrast, some have the ability to promote duplex formation and to dissociate RNA-protein complexes or function as ATP-dependent RNA-binding proteins (56)(57)(58)(59).…”

Section: Resultsmentioning

confidence: 99%

Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

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“…However, the role of Ser-235/Ser-236 compared with Ser-240/Ser-244 in the regulation of terminal oligopyrimidine mRNA translation is unknown, and this idea will need to be tested experimentally. Similarly, although it is known that eIF4B stimulates the RNA helicase activity of eIF4A (51)(52)(53) and may thereby modulate the mRNA binding step in mRNA translation, the effect of phosphorylation of eIF4B in altering its function is relatively unexplored.…”

Section: Discussionmentioning

confidence: 99%

Glucagon Represses Signaling through the Mammalian Target of Rapamycin in Rat Liver by Activating AMP-activated Protein Kinase

Kimball

Siegfried

Jefferson

2004

Journal of Biological Chemistry

120

104

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The opposing actions of glucagon and insulin on glucose metabolism within the liver are essential mechanisms for maintaining plasma glucose concentrations within narrow limits. Less well studied are the counterregulatory actions of glucagon on protein metabolism. In the present study, the effect of glucagon on amino acid-induced signaling through the mammalian target of rapamycin (mTOR), an important controller of the mRNA binding step in translation initiation, was examined using the perfused rat liver as an experimental model. The results show that amino acids enhance signaling through mTOR resulting in phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP)1, the 70-kDa ribosomal protein (rp)S6 kinase, S6K1, and rpS6. In contrast, glucagon repressed both basal and amino acid-induced signaling through mTOR, as assessed by changes in the phosphorylation of 4E-BP1 and S6K1. The repression was associated with the activation of protein kinase A and enhanced phosphorylation of LKB1 and the AMP-activated protein kinase (AMPK). Surprisingly, the phosphorylation of two S6K1 substrates, rpS6 and eukaryotic initiation factor 4B, was not repressed but instead was increased by glucagon treatment, regardless of the amino acid concentration. The latter finding could be explained by the glucagoninduced phosphorylation of the ERK1 and the 90-kDa rpS6 kinase p90 rsk . Thus, glucagon represses phosphorylation of 4E-BP1 and S6K1 through the activation of a protein kinase A-LKB-AMPK-mTOR signaling pathway, while simultaneously enhancing phosphorylation of other downstream effectors of mTOR through the activation of the extracellular signal-regulated protein kinase 1-p90 rsk signaling pathway. Amino acids also enhance AMPK phosphorylation, although to a lesser extent than glucagon and amino acids combined.The counter-regulatory action of glucagon on insulin-stimulated glucose metabolism is well documented in the literature (1). In contrast, few studies have examined the effects of the hormone on protein metabolism. Reports suggest that in vivo glucagon has an overall catabolic effect on protein metabolism, although the mechanisms through which it acts are unclear (2-5). Studies using a perfused rat liver preparation show that glucagon stimulates protein degradation, inhibits protein synthesis, and prevents completely the stimulation of protein synthesis induced by elevated levels of perfusate amino acid concentration (6). Studies using rat hepatocytes show that glucagon attenuates the stimulatory effect of epidermal growth factor on phosphorylation of the ribosomal protein (rp) 1 S6 kinase S6K1 (7).In rat liver, the stimulation of S6K1 phosphorylation by hormones and nutrients is regulated through a signal transduction pathway involving a protein kinase referred to as the mammalian target of rapamycin (mTOR). Phosphorylation activates S6K1, which then phosphorylates rpS6 (8) and eukaryotic initiation factor (eIF)4B (9), resulting in an enhanced translation of subsets of mRNA such as those containing an oligopyri...

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Dissociation of double stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay

Cited by 92 publications

References 39 publications

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer

Cofactor-dependent specificity of a DEAD-box protein

Glucagon Represses Signaling through the Mammalian Target of Rapamycin in Rat Liver by Activating AMP-activated Protein Kinase

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