Abstract
The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.
Highlights
The ability of internal ribosome entry site (IRES)-mediated protein synthesis to contribute to aberrant gene expression in cancer and during integrated cell stress responses is well documented (6 – 8); the processes regulating IRES function are poorly defined
We describe a phosphomimetic mutant of the ITAF heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (S199E), which is able to bind to the cyclin D1 and c-MYC IRESs normally but is deficient in nucleic acid annealing activity
The relative amount of firefly luciferase activity is indicative of IRES-mediated protein synthesis directed by either the cyclin D1 or c-MYC IRESs, whereas Renilla luciferase activity is a readout of cap-dependent initiation [4]
Summary
Cell Lines, Constructs, and Transfections—The glioblastoma line LN229 was obtained from ATCC (Manassas, VA), and mouse embryonic fibroblasts (MEFs) were generously provided by Dr Hong Wu (Department of Molecular and Medical Pharmacology, UCLA) These lines were transfected with a myristoylated AKT-estrogen receptor ligand-binding domain fusion (myr-AKT-MER) cloned into pTracer-SV40 and stably expressing clones isolated [2]. Filter Binding Assays and ELISA—The indicated amounts of GST-hnRNP A1 and GST-S199E-hnRNP A1 were added to in vitro transcribed 32P-labeled RNAs corresponding to either the cyclin D1 or c-MYC IRESs in separate reactions in a volume of 10 ml in buffer containing 5 mM HEPES (pH 7.6), 30 mM KCl, 2 mM MgCl2, 200 mM DTT, 4% glycerol, and 10 ng of yeast tRNA for 10 min at room temperature [5]. Statistical analysis was done with Student’s t test and analysis of variance models using Systat 13 (Systat Software, Chicago, IL)
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