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. 2003 Jul;23(13):4417-27.
doi: 10.1128/MCB.23.13.4417-4427.2003.

Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation

Anne B Vojtek  1 , Jennifer Taylor, Stacy L DeRuiter, Jenn-Yah Yu, Claudia Figueroa, Roland P S Kwok, David L Turner
Affiliations

Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation

Anne B Vojtek et al. Mol Cell Biol. 2003 Jul.

Abstract

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

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Figures

FIG. 1.
FIG. 1.
Hairpin siRNA expression vectors against Akt1 and Akt2 reduce the endogenous levels of the Akt kinases and reduce GSK3α/β phosphorylation at Ser21/9. (A) Sequences and expected structures of hairpin siRNAs for Akt1 and Akt2 expressed from the mouse U6 promoter. Watson-Crick base pairs (|) and G:U base pairs (:) are shown. (B and C) P19 cells were transfected with expression vectors for biCS5puro/GFP and U6-driven hairpin siRNAs against Akt1 and/or Akt2, or XASH3 (a control hairpin siRNA). The caspase inhibitor z-VAD-FMK was added to cells at the time of transfection. Akt1 and Akt2 (B, upper panel) and GFP (transfection control) (B, lower panel) were detected by Western blot analysis of extracts prepared from transiently transfected puromycin-selected cells. The Akt antisera recognizes both Akt1 and Akt2, which migrate as a single band. Phospho-GSK3α/β is reduced when Akt1 and Akt2 expression is inhibited by RNAi (C, middle panel; upper band is GSK3α and lower band is GSK3β; other panels are as described for panel B).
FIG. 2.
FIG. 2.
Inhibition of endogenous Akt by hairpin siRNAs reduces neuron generation by ngn3. (A) P19 cells were transfected with expression vectors for ngn3, nlsGFP, and U6-driven hairpin siRNAs against Akt1 (Akt1 HP7), Akt2 (Akt2 HP5), both Akt1 and Akt2, or a control, XASH3 (XASH3 HP). (B) Cells were transfected as described for panel A, but the caspase inhibitor z-VAD-FMK was added to cells at the time of transfection. Cells were fixed 4 days after transfection, and a neuronal β-tubulin was detected by indirect immunofluorescence with the antibody TuJ1. GFP was detected by epifluorescence. (C and D) The average number of GFP-positive cells and TuJ1-positive cells per field of view in hairpin-transfected cells without (C) or with (D) caspase inhibitor was determined. Averages are derived from counting the number of cells from three fields of view per transfection, from three independent transfections (standard deviations are indicated).
FIG. 3.
FIG. 3.
Activated Akt cooperates with ngn3 to increase transcription. (A) P19 cells were transfected with expression vectors for ngn3, activated Akt1, and a multimerized E-box driving a luciferase reporter (E1X3-TATA). (B) Activated Akt does not alter the level of the ngn3 protein. P19 cells were transfected with expression vectors for Myc epitope-tagged (MT) ngn3 and increasing concentrations of activated Akt1. Extracts were prepared, and MT-Ngn3 levels were assessed by Western blot analysis with anti-Myc antibody. (C) The carboxyl-terminal transactivation domain of ngn3 mediates Akt-regulated transcriptional cooperativity. P19 cells were transfected with expression vectors for Gal4-DBD fused to the ngn3 carboxyl-terminal transactivation domain (G4D-ngn3-COOH), constitutively active Akt1, or vector control, and the pFR Gal4-responsive luciferase reporter. (D) ngn3-induced expression of NeuroD1 is reduced in the presence of the PI3K/Akt inhibitor LY294002. NeuroD1 and hypoxanthine phosphoribosyltransferase (control) mRNAs were quantitated by real-time RT-PCR from P19 cells transfected with an ngn3 vector, with or without LY294002, as indicated. The change in threshold-crossing cycle is shown for each mRNA relative to that for transfection of a control vector (a decrease in threshold crossing corresponds to an increase in the mRNA level). The reduction in NeuroD1 expression in the presence of LY294002 is significant (P < 0.02 by Student's t test). Standard deviations are indicated in panels A, C, and D.
FIG. 4.
FIG. 4.
Activated Akt1 enhances complex formation between p300 and bHLH proteins. P19 cells were transfected with expression vectors for Myc epitope-tagged ngn3 (MT-ngn3) (A) or MT-MASH1 (B) with and without constitutively active Akt1. Endogenous p300 was immunoprecipitated from extracts 24 h after transfection and subjected to SDS-PAGE. p300 and coassociated Myc epitope-tagged bHLH proteins were detected by Western blot analysis with antibodies directed against p300 or the Myc epitope tag.
FIG. 5.
FIG. 5.
Activated Akt1 enhances the transcriptional activity of ngn3 in complex with the transcriptional coactivators p300 and CBP. P19 cells were transfected with expression vectors for the Gal4-DBD p300 fusion protein (Gal-p300) or Gal4-DBP-CBP fusion protein (Gal-CBP), ngn3, the pFR Gal4-responsive luciferase reporter, and constitutively active Akt1 (C/A Akt) or constitutively active Sgk1 (C/A SGK). The PI3K/Akt inhibitor LY294002 (12.5 μM) was added at the time of transfection, as indicated. (A) ngn3 and C/A Akt1 cooperatively enhance the transcriptional activity of Gal-p300. (B) ngn3 and C/A Akt1 cooperatively enhance the transcriptional activity of Gal-CBP. (C) Inhibition of the endogenous PI3K/Akt signaling pathway with LY294002 decreases the transcriptional activity of Gal-p300 in combination with ngn3, but not in the presence of C/A Akt1. (D) C/A Akt1, but not C/A SGK, enhances the transcriptional activity of ngn3 and Gal-p300.
FIG. 6.
FIG. 6.
Akt and CBP coassociate in vivo. Flag-CBP or Flag (expressed from Flag vector [control]) was immunoprecipitated from lysates prepared from HEK293 cells transfected with the indicated constructs. The immunoprecipitates were subjected to SDS-PAGE followed by Western blotting with antibodies directed against CBP or the epitope tag on Akt (GST).
FIG. 7.
FIG. 7.
Mutation of the Akt consensus phosphorylation site in p300 does not alter the cooperativity between ngn3, p300, and Akt. P19 cells were transfected with expression vectors for Gal4-p300 or Gal4-p300 S1834A, ngn3, constitutively active (C/A) Akt1 or vector control, and the pFR Gal4-responsive luciferase reporter.
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