Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

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.

FIGURE 1.

Conditionally active AKT regulates rapamycin hypersensitivity and cyclin D1 and c-MYC IRES activities. A, immunoblot analysis of LN229 and MEF cells expressing the myr-AKT-MER fusion protein or EV control transfectants. The indicated cells were treated with the ligand 4OHT (1 μm, 24 h), and the extracts were prepared and separated by SDS-PAGE. The blots were incubated with anti-AKT, anti-Ser(P)⁴⁷³-AKT, and anti-β-actin antibodies. The black and gray arrowheads represent the myr-AKT-MER and endogenous AKT, respectively. B, LN229_AKT-MER and LN229_EV (left panel) and MEF_AKT-MER and MEF_EV (right panel) were treated in the presence or absence of 4OHT and rapamycin as shown and subjected to propidium iodide staining followed by flow cytometry. The means ± S.D. are shown for three independent experiments. C, AKT-dependent differential cyclin D1 and c-MYC IRES activities in myr-AKT-MER-expressing cell lines following 4OHT and rapamycin treatment as shown (LN229_AKT-MER and LN229_EV, left bottom panel; MEF_AKT-MER and MEF_EV, right bottom panel). The top panel depicts schematic diagrams of the dicistronic vectors. The relative fold change in firefly luciferase activity is shown as compared with activities obtained in the absence of rapamycin and normalized to values obtained for pRF in each cell line. The means ± S.D. are shown for three independent experiments.

FIGURE 2.

Recombinant hnRNP A1 S199E does not support IRES activity in vitro. Translation-competent extracts were prepared from LN229 cells in which hnRNP A1 expression had been knocked down via siRNA exposure, and the indicated proteins were added to the extracts prior to programming the extracts with either in vitro transcribed dicistronic cyclin D1 (left panel) or dicistronic c-MYC (right panel) reporter mRNAs. Translations were performed at 30 °C for 40 min. An irrelevant ITAF, GAPDH was added as a negative control. Renilla (black bars) and firefly (open bars) luciferase activities were determined and normalized to values obtained for extracts alone. The means ± S.D. are shown for three independent experiments.

FIGURE 3.

Phosphomimetic hnRNP A1 S199E mutant in vitro IRES RNA binding characteristics and ability to compete with native hnRNP A1 for IRES binding. A and B, hnRNP A1 (closed circles) and S199E mutant (open circles) binding curves for the cyclin D1 (165 nucleotides) (A) and c-MYC (233 nucleotides) (B) IRES RNAs. C and D, mutant hnRNP A1 (open circles) competes with native protein for binding to cyclin D1 (C) or c-MYC (D) IRES RNAs. Native hnRNP A1 was adsorbed to the ELISA well and the binding reaction initiated by the addition of biotinylated IRES RNA and mutant hnRNP A1. IRES RNA binding was determined via colorimetry. The irrelevant ITAF, GAPDH (closed circles in C and D, was included as a negative control and did not bind either the cyclin D1 or c-MYC IRES RNAs. The results are expressed as percentages of the value obtained in the absence of competitor. The means ± S.D. are shown (n = 3).

FIGURE 4.

hnRNP S199E mutant displays reduced annealing activity. A, recombinant native or S199E hnRNP A1 was purified and analyzed for reannealing activity. The amount of protein used in the annealing reactions is shown above the panel, and the migration positions of the indicated species are also displayed. B and C, subcellular localization of GFP-tagged native (B) and S199E hnRNP A1 (C) in LN229 cells following the indicated treatments. The cells were transfected with GFP-hnRNP A1 or GFP-hnRNP A1 S199E plasmids and treated with 10 nm rapamycin (rap) for 24 h and GFP visualized using fluorescence microscopy.

FIGURE 5.

The hnRNP A1 S199E mutant inhibits IRES activity in cells. Rapamycin stimulated IRES activity is inhibited in LN229_AKT-MER, LN229_EV, MEF_AKT-MER, and MEF_EV cells expressing the hnRNP A1 S199E mutant. The indicated cell lines expressing empty vector, GST-hnRNP A1 (GST-A1), or GST-hnRNP A1 S199E were transfected with the dicistronic reporter plasmids shown in the absence or presence of rapamycin. A, without 4OHT; B, with 4OHT. C and D are identical to A and B, respectively, except using the MEF_AKT-MER and MEF_EV lines as indicated. Relative fold change in firefly activity is shown as compared with activities obtained in the absence of rapamycin and normalized to values obtained for pRF in each cell lines. The means ± S.D. are shown (n = 3).

FIGURE 6.

Overexpressing the hnRNP A1 S199E mutant confers sensitivity to TORC1 inhibition. The indicated lines were stably transfected with vector alone, GST-hnRNP A1, and GST-hnRNP A1 S199E and treated without or with rapamycin (rapa) as shown. The percentage of cells in the S phase of the cell cycle was subsequently determined on propidium iodide-stained cells by flow cytometry. A, without 4OHT; B, with 4OHT in LN229_AKT-MER and LN229_EV cells. C and D are identical to A and B, respectively, except using the MEF_AKT-MER and MEF_EV lines. The means ± S.D. are shown (n = 3).

FIGURE 7.

The hnRNP A1 S199E mutant confers TORC1 inhibitor sensitivity in GBM xenografts. Inhibition of tumor growth by CCI-779 in xenografts of LN229_AKT-MER and LN229_EV cells overexpressing the hnRNP A1 mutant in SCID mice. The mice were treated with CCI-779 for 5 days with the indicated doses of drug and tumor growth assessed at day 8 (D8) or day 12 (D12) after initial treatment. The mice were treated with 4OHT as indicated via daily maintenance injections of 4OHT. The horizontal bars indicate 50% reduction in control tumor growth (vehicle-treated). The data represent tumor volumes (means ± S.D. of three experiments).

FIGURE 8.

GBM displaying elevated levels of AKT activity harbor high levels of phosphorylated Ser¹⁹⁹ hnRNP A1. A, representative immunohistochemical analysis of Ser(P)¹⁹⁹-hnRNP A1, activated AKT, and activated ERK in a panel of 22 human glioblastoma samples. The sections were prepared and stained with antibodies to the indicated proteins followed by an appropriate secondary antibody. The sections were subsequently incubated with streptavidin-horseradish peroxidase and developed with 3,3′-diaminobenzidine reagent. Magnified images of the indicated regions are shown to the right. NB, normal brain. The arrows indicate regions of significant staining. Bar, 10 μm. B, glioblastoma samples stained with the indicated antibodies following preabsorption with the relevant reacting phosphorylated peptide (left panels) or corresponding nonphosphorylated peptide (right panels). The arrows indicate areas of significant staining in samples stained with antibodies preabsorbed with the corresponding nonphosphorylated peptides. C, representative immunoblot analysis of glioblastoma samples displaying elevated Ser(P)⁴⁷³-AKT levels. Normal brain and tumor lysates were analyzed using antibodies against the indicated proteins.