Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration
- Sungkyunkwan University School of Medicine, Republic of Korea
- Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Republic of Korea
- University of California, San Francisco Medical Center, United States
- University of Ulsan, College of Medicine, Republic of Korea
Figures
Figure 1 with 2 supplements
Akt binds to Id2 and phosphorylates Serine 14.
(A) Mouse brain lysates were subjected to immunoprecipitation (IP)/immunoblotting (IB) with the indicated antibodies. (B) GST pull-down assays with purified GST-Id2 protein and P1 mouse brain …
Figure 1—figure supplement 1
AKT interacts with Id2.
Lysate of PC12 cells stably transfected with myc-tagged a constitutively active (CA) form or a kinase-dead (KD) form of Akt were subject to immunoprecipitation with anti-myc antibody and silver …
Figure 1—figure supplement 2
Akt binds to Id2 and phosphorylates Serine 14 on Id2.
(A) Comparison of the mouse Id1, Id2, Id3, and Id4 proteins demonstrate significant homology. Id proteins contain a helix-loop-helix domain but lack the basic DNA-binding region. (B) Id 1–4 proteins …
Figure 2 with 1 supplement
Akt controls Id2 protein stability in the neuron.
(A–B) Lysates from mouse hippocampus of the indicated days were subjected to IB with the indicated antibodies. (C) PC12 cell were transfected with the indicated combination of HA-Akt or GFP-Cdh1 and …
Figure 2—figure supplement 1
Degradation of Id2 proteins by the ubiquitin-proteasome.
Id2 protein is degraded by the proteasome. PC12 cells were treated with the proteasome inhibitor MG132 for 12 hr. Id2 protein level was analyzed by immunoblotting. b-Actin was used as the internal …
Figure 3 with 1 supplement
Phosphorylation of Id2 by Akt is essential for augmentation of axon growth and branching.
(A) Representative merged image of localization of endogenous Akt and Id2 in the hippocampus neurons (DIV 4). The neurons stained for Id2 (red) and Akt (green). Right panel shows a higher …
Figure 3—figure supplement 1
Phosphorylation of Id2 by Akt is essential for augmentation of axon growth and branching.
Rat E18 hippocampal neurons were plated onto pre-coated coverslips. The neurons were fixed with 4% paraformaldehyde at the indicated time points and permeabilized and blocked. Neurons were incubated …
Figure 4 with 2 supplements
Akt regulates growth cone localization of Id2 in the developing neuron.
(A) Schematic diagram of growth cone, showing microtubule mostly in the central [C] region and F-actin based peripheral [P]region. (B) Representative image of Id2 or S14-phospho-Id2 (green) with …
Figure 4—figure supplement 1
Id2 and S14-phospho-Id2 localized in the axonal growth cone of developing neuron.
(A) Fluorescent image of beta-tubulin (Tuj1:green) with Id2 or S14-phospho-Id2 (red) in DIV1-3 neurons. Scale bar, 20 µm. Enlargement of boxed area is in the bottom of each DIV images. Scale bar, 5 …
Figure 4—figure supplement 2
Knockdown of Id2 or inhibition of Akt impairs Id2 phosphorylation and its roles in the growth cone.
(A) PC12 cells were transfected with several si-RNA for Id2. 24 hr after transfection, Id2 protein level was analyzed by immunoblotting. (B) Cells were infected with purified lenti-GFP or si-Id2-GFP …
Figure 5 with 1 supplement
Akt/Id2 signaling promotes axon growth by regulating growth cone development.
(A–B) E18 mouse brain lysates were subject to IP with anti-Id2 or anti-S14-phospho-antibody, followed by IB with anti-ERM antibody. (C–D) GST pull-down assay using cell lysates of PC12 cells …
Figure 5—figure supplement 1
Akt/Id2 signaling promotes axon growth by regulating growth cone development.
(A) Representative merged image of co-localization of Id2 and radixin in the growing hippocampal neuron. Neurons were fixed with 4% paraformaldehyde. Hippocampal neurons were stained with anti-Id2 …
Figure 6 with 3 supplements
Akt/Id2 signaling is critical for the proper function of radixin in the growth cone.
(A and B) Hippocampal neurons were treated with DMSO or AKT inhibitor for 4 hr. Enlargement of boxed area is in right. Scale bar, 50 µm or 5 µm. Arrows indicates radixin positive filopodia at the …
Figure 6—figure supplement 1
Selection of si-RNA for radixin.
PC12 cells were transfected with several si-RNA for radixin or negative control. 24 hr after transfection, radixin protein level was analyzed by immunoblotting.
Figure 6—figure supplement 2
Akt-mediated S14 phosphorylation is dispensable for the transcriptional repression activity of Id2.
(A) HA-E47 was co-transfected with GFP-Id2 WT or mutants(S14D or S14A) into PC12 cells and determined the interaction between E47 and Id2 constructs as indicated after immunoprecipitation with …
Figure 6—figure supplement 3
Akt-mediated S14 phosphorylation on Id2 is critical for its role in the growth cone.
(A) Alignment of HLH in the mouse Id1 and Id2 proteins. (B) The binding analysis with PC12 cells that transfected with HA-E47 and Id2 WT or HLH domain mutants (Id2-P51S and Id2-V68P) following by …
Figure 7 with 1 supplement
Akt/Id2 signaling contributes to axon regeneration in injured hippocampus slices.
(A) Diagram illustrating experimental time course. The hippocampal slices were axotomized at the perforant path (PP) after DIV 7, and a series of AAV2- Id2 (WT, S14A, or S14D) or control with or …
Figure 7—figure supplement 1
Akt/Id2 signaling contributes to axon regeneration through growth cone formation.
(A) 293T cells were infected with purified AAV2-GFP, Id2 WT, S14A or S14D viruses. Id2 protein level was analyzed by immunoblotting. (B) Hippocampal slice cultures were prepared from a postnatal …
Figure 8
Schematic diagram of Akt/Id2 signaling pathway in the neuron.
Akt-mediated S14 phosphorylation of Id2 augments its protein stability and growth cone localization, thereby promoting growth cone formation and axon growth in the developing neuron and contributing …
