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. 2008 Jun 24;105(25):8649-54.
doi: 10.1073/pnas.0802533105. Epub 2008 Jun 17.

Akt phosphorylation and nuclear phosphoinositide association mediate mRNA export and cell proliferation activities by ALY

Masashi Okada  1 , Sang-Wuk Jang, Keqiang Ye
Affiliations

Akt phosphorylation and nuclear phosphoinositide association mediate mRNA export and cell proliferation activities by ALY

Masashi Okada et al. Proc Natl Acad Sci U S A. .

Abstract

Nuclear PI3K and its downstream effectors play essential roles in a variety of cellular activities including cell proliferation, survival, differentiation, and pre-mRNA splicing. Aly is a nuclear speckle protein implicated in mRNA export. Here we show that Aly is a physiological target of nuclear PI3K signaling, which regulates its subnuclear residency, cell proliferation, and mRNA export activities through nuclear Akt phosphorylation and phosphoinositide association. Nuclear Akt phosphorylates Aly on threonine-219, which is required for its interaction with Akt. Aly binds phosphoinositides, and this action is regulated by Akt-mediated phosphorylation. Phosphoinositide binding but not Akt phosphorylation dictates Aly's nuclear speckle residency. Depletion of Aly results in cell growth suppression and mRNA export reduction. Inhibition of Aly phosphorylation substantially decreases cell proliferation and mRNA export. Furthermore, disruption of phosphoinositide association with Aly also significantly reduces these activities. Thus, nuclear PI3K signaling mediates both cell proliferation and mRNA export functions of Aly.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Aly is an Akt substrate in vitro and in vivo. (A) Schematic diagram of Aly. Aly is a 257-aa protein containing an RNA recognition motif (RRM). Aly has several Akt phosphorylation motifs. (B) Aly N terminus and C terminus are phosphorylated by Akt. Except GST-ALY 107–181 (RRM domain), both C and N termini of Aly were phosphorylated by Akt (Upper). Coomassie brilliant blue staining of used GST recombinant proteins is shown in Lower. (C) S34 and T219 residues in Aly are Akt phosphorylation sites. S34A and T219A mutation disrupted Aly phosphorylation by Akt in vitro. (D) ALY phosphorylation by EGF in vivo. The transfected HEK293 cells were serum-starved and fed with 32P-orthophosphate for 4 h. The cells were treated with 100 ng/ml EGF for 20 min after wortmannin (100 nM) or LY294002 (10 μM) treatment. GST-Aly was pulled down and separated on SDS/PAGE and analyzed by autoradiography or Western blotting. (E) T219 is the major phosphorylation site by Akt in vivo. GST-Aly wild type, S34A, and T219A construct transfected HEK293 cells were treated with NLS-Akt adenovirus. After 36 h of infection, cells were metabolically labeled. GST-Aly were pulled down and analyzed by autoradiography and immunoblotting. S34A mutation weakly decreased Aly phosphorylation, and T219A mutation substantially abolished Aly phosphorylation (first panel). The T219A mutant did not bind to Akt (second panel). (F) Ablation of Akt abolishes Aly phosphorylation. Knocking down of Akt eliminated Aly T219 phosphorylation, whereas overexpression of active myristoylated Akt enhanced Aly phosphorylation (top panel in Left). Aly T219 phosphorylation was selectively diminished in Akt1 but not Akt2 knockout MEF cells (top panel in Right).
Fig. 2.
Fig. 2.
Aly binds to phosphatidylinositol-(3,4,5)-Tris-phosphate. (A) Aly binds to phosphatidylinositol lipids. GFP-Aly transfected HEK293 cells were harvested, mixed with phosphatidylinositol lipid-conjugated beads, and incubated for 3 h at 4°C. Lipid-bound proteins were analyzed by immunoblotting. Exogenous and endogenous Aly associated with phospholipids, especially PI(4,5)P2 and PI(3,4,5)P3. (B) NGF mediates the interaction between PI(3,4,5)P3 and Aly. PC12 cells were treated with NGF for various time points. The cell lysates were incubated with PI(3,4,5)P3-conjugated beads. After extensive washing, the bead-bound proteins were analyzed by immunoblotting. (C) T219 phosphorylation is required for Aly/PI(3,4,5)P3 association. (D) The N terminus of Aly is the PI(3,4,5)P3 binding site. (E) Amino acid sequence of N-terminal Aly. Positive amino acid residues were indicated in gray. (F) R27/29/31 and R79/K81 are important for Aly's PI(3,4,5)P3 binding activity. R27/29/31A and R79A/K81A mutants lost its PI(3,4,5)P3 interaction activity.
Fig. 3.
Fig. 3.
Aly regulates cell cycle progression and cell proliferation. (A) Aly knockdown alters the expression of cell cycle regulators. HeLa cells were transfected with a control scrambled RNA or Aly siRNA and incubated for 48 h. The cell lysate was separated by SDS/PAGE and analyzed by immunoblotting. Cyclin A2 and cyclin B1 protein expression levels were selectively decreased. (B). siAly inhibits S phase progression. HeLa cells were transfected with control or siAly and incubated for 4 days. Cells were treated with BrdU (20 μM) for 1 h at 37°C and then fixed by 3% formaldehyde. The fixed cells were treated with 2 M HCl and neutralized by boric acid (pH 8.4); cells were analyzed by immunostaining analysis against BrdU antibody. Depletion of Aly decreases BrdU incorporation. Results are expressed as mean ± SEM from three independent experiments. (C) Aly knockdown prevents cell proliferation. Twenty-four hours after transfection, HeLa cells were split into six-well dishes (5 × 104) and incubated for 5 days. After cells were fixed by 3% formaldehyde, cells were stained with crystal violet solution. (D) Ablation of Aly decreases cell growth. (E) Knockdown of Aly decreases mRNA export. Results are expressed as mean ± SEM from three independent experiments (P < 0.005, Student's t test).
Fig. 4.
Fig. 4.
Akt phosphorylation mediates Aly's effect in cell proliferation. (A) Aly phosphorylation does not alter Aly nuclear speckle residency. (B and C) Akt phosphorylation on Aly T219 is required for mRNA export activity (B). Quantitative analysis of mRNA export is shown in C. (D) Akt phosphorylation regulates Aly's cell proliferation activity. T219 phosphorylation mimetic mutants enhanced BrdU incorporation, whereas unphosphorylated mutant T219A displayed decreased cell proliferation activity. Results are expressed as mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (Student's t test). (E) Cell proliferation assay. HeLa cells, transfected with various Aly phosphorylation mutants, grew in six-well plates. The cell numbers were counted at the indicated time points.
Fig. 5.
Fig. 5.
Phosphoinositide binding of Aly regulates its subnuclear residency, mRNA export, and cell proliferation. (A) PI(3,4,5)P3 binding mutants localize outside of nuclear speckles. Wild-type Aly distributed in the nuclear speckles, colocalizing with SC35. R27/29/31A and R79A/K81A partially localized in the nuclear speckles and aggregated in nucleoplasm. (B) BrdU incorporation assay. Aly mutants lacking PI(3,4,5)P3 binding affinity suppressed BrdU incorporation. Results are expressed as mean ± SEM from three independent experiments. *, P < 0.05 (Student's t test). (C) Aly/PI(3,4,5)P3 interaction is implicate in mediating cell proliferation. (D) Aly/PI(3,4,5)P3 interaction is essential for its mRNA export activity. GFP, GFP-Aly wild type, and mutants deficient of PI(3,4,5)P3 binding were transfected into HeLa cells, respectively, and then incubated for 48 h, followed by mRNA export assay. R79/K81A mutant displayed crippled mRNA export activity compared with wild type and R27/29/31A mutant. Results are expressed as mean ± SEM from three independent experiments. *, P < 0.001; **, P < 0.01 (Student's t test).
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