Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species - PubMed (original) (raw)

Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species

Valentina Evdokimova et al. Mol Cell Biol. 2006 Jan.

Abstract

YB-1 is a broad-specificity RNA-binding protein that is involved in regulation of mRNA transcription, splicing, translation, and stability. In both germinal and somatic cells, YB-1 and related proteins are major components of translationally inactive messenger ribonucleoprotein particles (mRNPs) and are mainly responsible for storage of mRNAs in a silent state. However, mechanisms regulating the repressor activity of YB-1 are not well understood. Here we demonstrate that association of YB-1 with the capped 5' terminus of the mRNA is regulated via phosphorylation by the serine/threonine protein kinase Akt. In contrast to its nonphosphorylated form, phosphorylated YB-1 fails to inhibit cap-dependent but not internal ribosome entry site-dependent translation of a reporter mRNA in vitro. We also show that similar to YB-1, Akt is associated with inactive mRNPs and that activated Akt may relieve translational repression of the YB-1-bound mRNAs. Using Affymetrix microarrays, we found that many of the YB-1-associated messages encode stress- and growth-related proteins, raising the intriguing possibility that Akt-mediated YB-1 phosphorylation could, in part, increase production of proteins regulating cell proliferation, oncogenic transformation, and stress response.

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Figures

FIG. 1.

YB-1 phosphorylation is increased following activation of the PI3K-Akt pathway. (A and B) NIH 3T3 cells stably expressing HA-YB-1 were grown to ∼80% confluence, serum starved for 24 h, and then either not stimulated (−) or stimulated with 100 ng/ml EGF, 20% dialyzed fetal bovine serum (FBS), 50 ng/ml IGF-I, or 10 ng/ml TGF-β1 for 2 h (A) or treated with 20 ng/ml rapamycin, 0.1 μM wortmannin, 100 μM LY294002, 50 μM U0126, or 100 μM PD098059 for 1 h prior to stimulation with IGF-I (+) (B). The whole-cell extracts were analyzed by IB. The positions of phosphorylated Akt (pAkt), phosphorylated glycogen synthase kinase 3β (pGSK-3β), phosphorylated FKHR (pFKHR), phosphorylated mTOR (pmTOR), phosphorylated Erk1 (pErk1), pErk2, phosphorylated MEK1/2 (pMEK1/2), 4E-BP1, and YB-1 are shown to the right of the gels. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels. (C and D) Another set of plates was treated as described above for panel A in the presence of [32P]orthophosphate. YB-1 was immunoprecipitated from cytosolic cell extracts with anti-YB-1 antibodies and detected by autoradiography or IB using anti-HA antibodies. The bars in the graphs below the gels indicate the relative amounts of recovered 32P-labeled YB-1 (32P-YB-1) as measured from two independent experiments by phosphorimaging software and normalized to the total amounts of YB-1 in the immunoprecipitates.

FIG. 2.

YB-1 interacts with and is phosphorylated by Akt. (A) The domain organization of YB-1 with the indicated RNP1 and RNP2 consensus motifs and potential phosphorylation sites is shown at the top. The positions of the Ala-Pro-rich domain (AP), CSD, and C-terminal (C-TERM) portion are indicated. The electrical charges of portions of the C-terminal region are shown above the schematic. YB-1 and its derivatives (1 μg of each) used in the kinase assay are shown below. GST-AP/CSD, N-terminal fragment containing both the Ala-Pro-rich domain (AP) and CSD. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (B) In vitro kinase assay using YB-1 derivatives and activated or inactive Akt forms, as indicated (+, present; −, absent). (C) In vitro kinase assay using activated Akt or mTOR immunoprecipitate as a source of kinase activity to phosphorylate YB-1 or GST-tagged 4E-BP1 (right). Coomassie blue staining of recombinant YB-1 and GST-tagged 4E-BP1 (1 μg of each) utilized in the assay is shown to the left. (D) In vitro kinase assay using activated Akt and the recombinant wild-type (WT) or mutant YB-1 proteins. (E) NIH 3T3 cells were transiently transfected with constructs expressing the HA-tagged wild-type YB-1 (WT) or S102A mutant YB-1 protein. Following 24 h posttransfection, cells were treated with IGF-I (+) and simultaneously labeled with [32P]orthophosphate for 2 h. Cytosolic cell extracts were then subjected to IP using anti-HA antibodies (αHA). YB-1 proteins were detected by autoradiography (top) or IB (bottom). (F to H) YB-1 interacts with Akt in vivo and in vitro. MCF-7 (F) or K-Ras-NIH 3T3 (G) cytosolic cell extracts were utilized for IP using anti-HA (αHA) or anti-YB-1 (αYB-1) antibodies, as indicated. preim., preimmune rabbit antibodies; Wortm, wortmannin; pAkt, phosphorylated Akt; IgG, immunoglobulin G. (H) The GST-tagged YB-1 proteins were immobilized on glutathione-Sepharose and incubated with the activated or inactive Akt forms (+). Silver staining of proteins bound to the beads is shown. GST-Seph., GST-Sepharose.

FIG. 3.

YB-1 association with mRNAs but not its cellular localization or protein levels may be affected by activated Akt. (A) Whole-cell extracts from Rat1a cells expressing vector alone or MyrAkt (input) or cytosolic (C) and nuclear (N) fractions were analyzed by IB using anti-Akt or anti-YB-1 antibodies. The positions of YB-1 and Akt are shown to the right of the gel. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (B) Cytosolic and nuclear fractions from K-Ras-NIH 3T3 cells treated with wortmannin (Wortm) (0.1 μM) or IGF-I (50 ng/ml) for 2 h (+) were analyzed by IB. Histone H3 and BiP were chosen as nuclear and cytosolic markers, respectively. Nucleolin is an example of a nucleocytoplasmic shuttling RNA-binding protein with predominantly nuclear localization. pAkt, phosphorylated Akt. (C) Polysomal profiles derived from Rat1a cells expressing vector alone or MyrAkt. Cytosolic cell extracts were layered on 15 to 50% (wt/vol) sucrose gradients in detergent-free lysis buffer and centrifuged at 4°C in a Beckman SW41 rotor for 3 h at 50,000 rpm. Gradients were fractionated from the bottom of the gradient and scanned for absorbance at 254 nm (A254). (D) Following fractionation, 15 fractions were collected, concentrated by precipitation with ice-cold 5% trichloroacetic acid, and analyzed by IB. Note that in contrast to vector alone, in MyrAkt cells YB-1 is detected in the top fractions containing unbound protein (lanes 1 and 2). PABP, poly(A)-binding protein.

FIG. 4.

Akt is a component of postpolysomal mRNPs that may mediate YB-1 phosphorylation when it is activated. (A and B) Nontransformed (vector) and K-Ras-transformed NIH 3T3 cells were grown in monolayers or on soft agar plates and analyzed by light microscopy (A). Whole-cell extracts were also analyzed by IB using the corresponding antibodies (B). Wortm, wortmannin; pAkt, phosphorylated Akt. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (C) K-Ras-transformed NIH 3T3 cells were treated with 0.1 μM wortmannin (Wortm) or 50 ng/ml IGF-I for 2 h (+). Five percent of cytosolic cell extracts (input) or 20% of postpolysomal (post-Ps) or polysomal (Ps) mRNP preparations were used for IB. PABP, poly(A)-binding protein. (D) Coomassie blue staining of the mRNP preparations analyzed in panel C. (E) NIH 3T3 cells were transiently transfected with constructs expressing the HA-tagged wild-type (WT) or S102A mutant YB-1 protein. Following 24 h posttransfection, cells were treated with IGF-I (+) and simultaneously labeled with [32P]orthophosphate for 2 h. Postpolysomal or polysomal fractions were then utilized for IP with anti-HA antibodies (αHA). YB-1 proteins were detected by autoradiography (top) or IB using anti-YB-1 antibodies (bottom).

FIG. 5.

Phosphorylation by Akt reduces the abilities of YB-1 to bind to the capped 5′ end of mRNA and to inhibit cap-dependent translation. (A) Gel retardation assay using uncapped CAT mRNA (1 μg) and increasing amounts (in micrograms) of the wild-type (WT) or recombinant YB-1 proteins phosphorylated (phosph.) by the inactive or activated Akt forms in the presence of cold ATP. Ethidium bromide staining of 1% agarose gel is shown. mock, mock phosphorylated. (B) UV cross-linking of YB-1 proteins to the cap-labeled or 5′-labeled LUC mRNAs (top). Wild-type YB-1 (WT) or S102A mutant were phosphorylated (phosph.) by the activated or inactive Akt forms in the presence of cold ATP prior to incubation with the corresponding 32P-labeled LUC mRNAs. Coomassie blue staining of the same gel is shown below. mock, mock phosphorylated. (C) Translation of capped (cap+) CAT-PVI IRES-LUC mRNA (0.25 μg) in rabbit reticulocyte lysate in the presence of [35S]methionine and indicated amounts of YB-1 phosphorylated (phosph.) by the inactive or activated Akt forms as in panel A. After phosphorylation, YB-1 proteins were additionally purified using heparin-Sepharose to eliminate any traces of Akt. Following 60-min incubation of the translation reaction mixture, proteins were resolved by SDS-10% PAGE and visualized by autoradiography. The positions of LUC and CAT are shown to the right of the gel, and the positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (D) The results of panel C as measured from three independent experiments by phosphorimaging software with the means ± standard deviations from the means (error bars) shown. pYB1, phosphorylated YB-1. (E) YB-1 was phosphorylated by the inactive or activated forms of Akt in the presence of [γ-32P]ATP, purified using heparin-Sepharose, and incubated in rabbit reticulocyte cell-free translation system as described above for panel C. IB of the same membrane is shown below. (F) Northern blotting showing degradation kinetics of cap+ CAT-PVI-LUC in the presence of mock-phosphorylated or phosphorylated YB-1 proteins. This experiment was done as described for panel C except for [35S]methionine. Total RNAs were recovered from translation reactions at the times indicated and analyzed by Northern blot hybridization using the 32P-labeled CAT cDNA probe as described earlier (13).

FIG. 6.

Functional distribution of total and YB-1-bound transcripts derived from K-Ras-transformed NIH 3T3 cells. (A) Venn diagram showing overlap between total and YB-1-bound transcripts. (B) The major functional categories represented in total and YB-1-bound mRNA populations. Bind., binding.

FIG. 7.

Binding of different mRNAs to YB-1 is dynamic and depends on growth conditions. (A) Semiquantitative RT-PCR analysis of selected candidate mRNAs derived from postpolysomal RNA preparations, total or YB-1 bound RNAs. K-Ras-NIH 3T3 cells treated with 0.1 μM wortmannin (Wortm.) or 50 ng/ml IGF-I for 2 h were utilized in this assay. Similar results were produced in three independent experiments using independently isolated YB-1-bound and total postpolysomal RNAs; the representative data are shown. The positions of molecular size markers (in kilobases) are shown to the right of the gels. GADD45β, growth arrest- and DNA damage-induced 45β; Preim., preimmune rabbit antibodies. (B) Multiprobe RNase protection assay of mRNAs recovered from postpolysomal (Post-Ps.) complexes with YB-1 or from polysomal (Ps) fractions of K-Ras-transformed cells treated with wortmannin (Wortm) or IGF-I (+), as described above for panel A. A customized growth factor template set was used in this assay. Note that undigested probe derived from the plasmid template is always larger than the actual RNase-digested product. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Effect of YB-1 overexpression on translation efficiencies of certain candidate mRNAs. K-Ras-NIH 3T3 cells expressing vector alone or HA-YB-1 were treated with IGF-I or wortmannin as described above for panel A and metabolically labeled with [35S]methionine for the last 60 min. Cytosolic cell extracts were utilized for IPs using mixtures of cyclin D1 and cyclin E antibodies or c-jun and actin antibodies immobilized on protein A/G-Sepharose beads to ensure equal recovery of the proteins from the immunoprecipitates. Immunoprecipitated proteins were then detected by SDS-10% PAGE and autoradiography.

FIG. 8.

Model for translational regulation via Akt-mediated YB-1 phosphorylation. Under normal growth conditions, competition between mRNAs for available translational components excludes many of them from translation and causes their accumulation in the pool of inactive postpolysomal mRNPs. Growth-related mRNAs are primarily affected, owing to their highly structured 5′ untranslated regions and the small amount of active eIF4E in the cell. A great number of these messages are kept silent and stable in the complex with YB-1, which supposedly functions as a gatekeeper blocking access of eIF4E and degradation enzymes to the mRNA species (13). Upon activation, Akt phosphorylates YB-1 and reduces its affinity to the capped 5′ terminus of mRNA. Akt signaling also leads to phosphorylation of 4E-BP1-inhibitory protein, thereby increasing levels of active eIF4E. In turn, eIF4E is capable of displacing phosphorylated YB-1 and releasing the YB-1-inhibited mRNAs to polysomes. PABP, poly(A)-binding protein; PAIP2, PABP-interacting protein 2; P, phosphate group; Rs, ribosome.

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Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species - PubMed (original) (raw)