Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser 235/236 in vitro and in vivo using an mTORindependent mechanism. Mutation of rpS6 at Ser 235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTORindependent pathway linking the Ras/ERK signaling cascade to the translational machinery.In eukaryotic cells, the main rate-limiting step of translation is initiation, which is controlled by an array of proteins that respond to signaling cascades activated by extracellular signals (reviewed in Refs. 1-3). The mammalian target of rapamycin, mTOR, 4 is a conserved serine/threonine kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate mammalian cell growth (reviewed in Refs. 4, 5-8).Under conditions of nutrient and energy sufficiency and insulin or mitogen stimulation, mTOR stimulates two important translational regulators, the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E). eIF4E is crucial for ribosome recruitment as it binds to the 7-methylguanosine cap structure (m7GpppN, where N is any nucleotide) at the 5Ј-end of nearly all transcribed mRNAs to initiate cap-dependent translation (reviewed in Ref. 7). When mTOR is active, eIF4E nucleates the assembly of the translation preinitiation complex through recruitment of numerous initiation factors, resulting in association of the ribosomal subunits to the mRNA. S6K1 and S6K2 are serine/threonine kinases directly stimulated by mTOR which in turn, phosphorylate substrates involved in cell and body size (5, 6). S6K1 phosphorylates several substrates located in the cytoplasm and the nucleus, including the ribosomal protein (rp) S6 (reviewed in Ref. 9).Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and represents the most extensively studied substrate of S6K1 (10). Because the initial discovery that liver-derived rpS6 was phosphoryla...

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner.Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5 0 untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum-responsive eIF4B phosphorylation site, Ser422, located in an RNA-binding region required for eIF4A helicase-promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide-3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin-resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.

The translation initiation step in eukaryotes is highly regulated and rate-limiting. During this process, the 40S ribosomal subunit is usually recruited to the 5' terminus of the mRNA. It then migrates towards the initiation codon, where it is joined by the 60S ribosomal subunit to form the 80S initiation complex. Secondary structures in the 5' untranslated region (UTR) can impede binding and movement of the 40S ribosome. The canonical eukaryotic translation initiation factor eIF4A (also known as DDX2), together with its accessory proteins eIF4B and eIF4H, is thought to act as a helicase that unwinds secondary structures in the mRNA 5' UTR. Growing evidence suggests that other helicases are also important for translation initiation and may promote the scanning processivity of the 40S subunit, synergize with eIF4A to 'melt' secondary structures or facilitate translation of a subset of mRNAs.

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of inflammation and profound immune suppression. Here we identify Yes-associated protein (Yap) as a critical regulator of the immunosuppressive microenvironment in both mouse and human PDAC. Within Kras:p53 mutant pancreatic ductal cells, Yap drives the expression and secretion of multiple cytokines/chemokines, which in turn promote the differentiation and accumulation of Myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Pancreas-specific knockout of Yap or antibody-mediated depletion of MDSCs promoted macrophage reprogramming, reactivation of T cells, apoptosis of Kras mutant neoplastic ductal cells, and pancreatic regeneration after acute pancreatitis. In primary human PDAC, YAP expression levels strongly correlate with a MDSC gene signature, and high expression of YAP or MDSC-related genes predicts decreased survival in PDAC patients. These results reveal multifaceted roles YAP in PDAC pathogenesis and underscore its promise as a therapeutic target for this deadly disease.

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5 untranslated region (5UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecininduced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.Targeting the translation initiation pathway is emerging as a potential therapy for inhibiting cancer cell growth (35,38). Ribosome recruitment to the 5Ј ends of eukaryotic mRNAs proceeds via translation initiation mechanisms that are dependent either on the 5Ј cap structure (m 7 GpppN, where N is any nucleotide) or an internal ribosome entry site (IRES). The majority of translation initiation events in eukaryotes are mediated through cap-dependent translation whereby the 40S ribosomal subunit is recruited to the vicinity of the mRNA 5Ј cap structure by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of eIF4E (the cap-binding subunit), eIF4A (an RNA helicase), and eIF4G (a large scaffolding protein for eIF4E, eIF4A, and other initiation factors). Once assembled at the 5Ј cap, the 40S ribosomal subunit in association with several initiation factors scans the 5Ј untranslated region (5ЈUTR) of the mRNA until it encounters a start codon in a favorable context, followed by polypeptide synthesis (37).Early in vitro studies have shown that the initiation factor eIF4B acts to potentiate ribosome recruitment to the mRNA (3, 45). eIF4B stimulates translation of both capped and uncapped mRNAs in vitro (1,36). This function is exerted through stimulation of the helicase activity of eIF4A (43), possibly through direct interactions with eIF4A (44) or with mRNA, the ribosome-associated eIF3, and 18S rRNA (28,29,44). Thus, eIF4B is thought to form auxiliary bridges between the mRNA and the 40S ribosomal subunit. Toeprinting studies using mammalian eIF4B underscored its importance in the assembly of the 48S initiation complex, especially on mRNAs harboring secondary structures in the 5ЈUTRs (11).In vivo studies of eIF4B are limited. Ectopic expression of eIF4B in cultured Drosophila melanogaster cells and in developing eye imaginal discs stimulated cell proliferation (16). Enhanced cell proli...

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5 UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.I nitiation is a tightly regulated rate-limiting step in the translation of eukaryotic mRNAs. Ribosome recruitment to the mRNA commences with binding of translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap structure, which is present at the 5Ј end of all nuclear-encoded eukaryotic mRNAs (1). eIF4F (comprising the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A and eIF4G, a scaffold for binding eIF4E and eIF4A) binds to the cap, unwinds (with the aid of eIF4A) the cap-proximal region of the mRNA, and, through interaction with the ribosome-bound eIF3, recruits the 40S ribosomal subunit to the mRNA (2-4). The 40S subunit then scans the 5Ј UTR in a 5Ј to 3Ј direction until it encounters an initiation codon. A subsequent joining of the 60S ribosomal subunit and release of eIFs result in formation of an elongationcompetent 80S ribosome.Secondary structures in 5ЈUTRs of mRNAs are thought to become unwound to allow ribosomal complexes to move along the mRNA in search of the initiation codon. Thus, in addition to its role in the initial attachment of ribosomal complexes to mRNA, eIF4A is believed to assist ribosomal complexes during scanning (5). Recent observations suggest that the process of eukaryotic initiation requires additional members of the DEAD/DExH-box protein family; for instance, a DEAD-box protein, yeast Ded1, and its mammalian homologue, DDX3, are biochemically and genetically implicated in translation initiation on long structured 5ЈUTRs (6), and another DExH-box protein, DHX29, strongly stimulates cap-dependent initiation on mRNAs with structured 5ЈUTRs in vitro (7). Here we studied the importance of DHX29 for translation in vivo and characterized it as a novel factor required for cell proliferation. Results DHX29 Is a Ubiquitously Expressed Cytoplasmic Protein That Associ-ates with the 40S Ribosomal Subunit. DHX29 has been found to interact with the 40S ribosomal subunit in vitro and to associate with 40S ribosomal complexes in a rabbit reticulocyte lysate (7). To determine how general these findings are, we examined the distribution of DHX29 in polysome preparations from HeLa cells using 2 commercial DHX29 antibodies (Fig. 1A). The specificity of these antibodies to human DHX29 was confirmed by immunoblotting against the purified native protein [supporting information (SI) Fig. S1] and recombinant DHX29, as well as by the loss of immunoreactivity following DHX29 RNAi ...