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The 60S subunits isolated fromArtemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by "diagonal" (two-dimensional nonreducing-reducing) NaDodSO4/polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO4 gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, P0. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDod-S04/polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)2 and (P2)2 dimers, each of which is anchored to P0. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.The large ribosomal subunits of Escherichia coli contain multiple copies of a protein, L7/L12, that is present as two dimers, each of which is anchored to the ribosome through interaction with a common protein, L10 (1-4). The four copies of L7/L12 are easily and selectively removed from the ribosome and can be reconstituted (5). The proteins are involved in the GTP-dependent reactions of protein synthesis-in particular, translocation (6-8). A model has been proposed recently, showing different orientation for the two L7/L12 dimers on the ribosome surface (9).The large subunits of eukaryotic ribosomes also contain multicopy acidic proteins (10, 11) that are structurally and functionally related to L7/L12 (12-15). These proteins exist in phosphorylated states and have been designated P proteins (16-18). Two different P proteins having slightly different amino acid compositions, electrophoretic mobilities, and molecular weights have been designated P1 and P2 (16). Anti-ribosome antibodies from certain patients with systemic lupus erythematosus react exclusively with P1 and P2 and, in addition, with a third acidic protein designated PO (19,20). These three proteins also react with a mouse monoclonal antibody raised against chicken ribosomes (17).The spatial arrangement of the P proteins in the eukaryotic ribosome is not yet well established. We have investigated the topography of P1 and P2 [designated eL12' and eL12 in Artemia ribosomes (10) Elution of the proteins from CM-cellulose at pH 5.0 (6 M urea/10 mM sodium acetate/0.1% methylamine/3 mM iodoacetamide, adjusted to pH 5.0 with acetic acid) instead of pH 7.2 was also used for some experiments.Abbreviations: P proteins, eukaryotic ribosomal proteins PO, P1, and P2; NEPHGE, nonequilibrium pH gradient electrophoresis.tPresent address:

We have demonstrated that the purified guanine nucleotide exchange factor (GEF) may be isolated as a complex with NADPH. Complete inhibition of the GEFcatalyzed exchange of eukaryotic initiation factor 2-bound GDP for GTP was observed in the presence of either 0.5-0.75 mM NAD+ or NADP+. Incubation of GEF with ATP results in the phosphorylation of its Mr 82,000 polypeptide. This phosphorylation is strongly inhibited by heparin but is not affected by heme or H8 {N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride}, an inhibitor of cAMP-and cGMPdependent protein kinases and protein kinase C. The purification of GEF was modified to eliminate any contaminating kinase activity and the isolated protein appears to be homogeneous as judged by NaDodSO4/polyacrylamide gel electrophoresis and silver staining. The Mr 82,000 subunit of GEF is phosphorylated only upon addition of ATP and casein kinase II. The extent of phosphorylation is =0.55 mol of phosphate per mol of GEF, and this results in a 2.3-fold increase in the guanine nucleotide exchange activity. Following treatment of the phosphorylated GEF with alkaline phosphatase, the activity of the protein is reduced by a factor of 5. Rephosphorylation of GEF increases its specific activity to that of the phosphorylated protein. The results of this study suggest that phosphorylation/dephosphorylation of GEF plays a role in regulating polypeptide chain initiation.During the initiation of protein synthesis in eukaryotic cells, the formation of a ternary complex [eIF-2-GTP-Met-tRNAf; where eIF-2 is eukaryotic initiation factor (eIF) 2] is followed by the transfer of this complex to a 40S ribosomal subunit (1, 2). Upon joining the 60S subunit to give the complete 80S initiation complex, GTP is hydrolyzed, and eIF-2 is released as the eIF-2-GDP binary complex (3, 4). In mammalian systems, this binary complex is stable in the presence of Mg2+ and is functionally inactive (5-7). Regeneration of the eIF-2-GTP Met-tRNAf species requires the guanine nucleotide exchange factor (GEF), which facilitates nucleotide exchange and recycling of eIF-2 (7-10). Phosphorylation of the a subunit of eIF-2 [eIF-2(a)] by either the hemecontrolled repressor (4, 11) or the double-stranded RNA induced kinase (12,13) is associated with the cessation of protein synthesis and is due to the inability of GEF to catalyze the GDP/GTP exchange from eIF-2(a-P)-GDP (8,10,14). Studies from this laboratory have demonstrated that the redox state of the cell may also influence GEF activity (15).Phosphorylation plays a major role in the regulation of eukaryotic protein synthesis. Initiation factors, ribosomal proteins, messenger ribonucleoprotein particles, and aminoacyl-tRNA synthases have been shown to be modified by phosphorylation (16-22). In addition to eIF-2(a), the phosphorylation of other initiation factors [eIF-2(p8), eIF-4B, and eIF-4F] has been reported in vivo under conditions of heat shock (19) and of nutrient deprivation (20) and in vitro by cAMP-independent kinase (21). In contras...

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. Significance: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.

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Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma (GBM) to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Whereas cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for GBM radiosensitization.

Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization.