An evolutionarily conserved mechanism for cAMP elicited axonal regeneration involves direct activation of the dual leucine zipper kinase DLK
- University of Michigan, United States
- Washington University School of Medicine, United States
- University of Pennsylvania, United States
- University of Michigan School of Medicine, United States
Figures
Figure 1
PKA stimulates and is required for axonal regeneration in Drosophila motoneurons.
Single motoneuron axons were labeled by expression of UAS-mCD8-GFP using the m12-Gal4 driver and imaged either 0 hr or 15 hr after nerve crush injury. Representative images are shown in (A), while (B…
Figure 2
PKA modulates the levels of Wnd protein and downstream signaling in Drosophila neurons.
(A) The puc-lacZ transcriptional reporter for Wnd/JNK signaling indicates that activated PKA stimulates Wnd signaling. A pan-neuronal driver (BG380-Gal4) is used to express UAS-dnc-RNAi, UAS-PKACA …
Figure 3 with 2 supplements
PKA activates DLK via phosphorylation of its activation loop.
(A-B) Changes in endogenous DLK abundance in response to treatment with forskolin (30 µM) (A) or the PKA inhibitor H-89 (5 µM) (B) for 6 hr in cultured rat embryonic cortical neurons. Quantification …
Figure 3—figure supplement 1
PKA can directly phosphorylate DLK at S302.
(A) PKA can induce DLK S302 phosphorylation in vitro. Flag-DLK was purified from HEK293 cells by anti-Flag immunoprecipitation and used for an in vitro kinase activity with purified PKA catalytic …
Figure 3—figure supplement 2
DLK activation by PKA does not require TORC1.
(A-B) The phosphorylation level of DLK S302 is not sensitive to treatments of TORC1 inhibitors. HEK293 cells were either untransfected or transfected with Flag-DLKWT + empty plasmid or Flag-DLKWT + …
Figure 4 with 1 supplement
PKA promotes the stability of DLK independently of DLK downstream signaling.
(A-B) Activation of PKA promotes DLK stability independently of JNK. HEK293 cells were transiently transfected with Flag-DLKWT, and (A) treated with forskolin (6 hr, 30 µM) or (B) co-transfected …
Figure 4—figure supplement 1
Summary of predicted PKA phosphorylation sites on DLK/Wnd in different species.
The sequence of DLK homologues in different species (human, mouse, Drosophila and C. elegans) were analyzed by Group-based Prediction System (GPS) to computationally predict PKA phosphorylation …
Figure 5
PKA stimulates axonal regeneration via DLK in adult DRG neurons.
(A-D) Induction of regeneration by forskolin requires PKA. Experimental design (A and also see Materials and Methods). To demonstrate that forskolin-induced neurite outgrowth is mediated by PKA, we …
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Figure 5—source data 1
Measurements of the longest neurite length for 100 neurons after replating in each condition.
The longest neurite length for 100 WT neurons after replating in four different conditions (DMSO+Vehicle, DMSO+forskolin, PKA inhibitor+Vehicle, PKA inhibitor+forskolin) are shown in Table ‘PKAi’. This table contains data from 4 independent experiments. The longest neurite length for 100 DLK KO neurons after replating in two different conditions (DMSO and forskolin) are shown in Table ‘DLK KO’. The table contains data from 3 independent experiments.
- https://doi.org/10.7554/eLife.14048.011
