The C. elegans Discoidin Domain Receptor DDR-2 Modulates the Met-like RTK-JNK Signaling Pathway in Axon Regeneration

Abstract

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2-JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.

Fig 1. DDR-2 is required for efficient axon regeneration in C. elegans.

(A) SVH-2–JNK MAPK pathway required for axon regeneration in C. elegans. The growth factor SVH-1 and its receptor tyrosine kinase SVH-2 promote axon regeneration through tyrosine phosphorylation of MLK-1 in the KGB-1 JNK pathway. (B) Structures of DDR-1 and DDR-2. Schematic diagrams of DDR-1, DDR-2 and their mammalian counterpart DDR2 are shown. Domains are shown as follows: a signal sequence (SS), a discoidin domain (DS), a DS-like domain, a transmembrane domain (TM), and a tyrosine kinase domain (Kinase). The bold lines underneath indicate the extent of the deleted region in each deletion mutant. The ddr-2(tm797) mutation causes a premature translation stop (indicated by asterisk) in the extracellular domain. (C) Representative D-type motor neurons in wild-type and ddr-2 mutant animals 24 hr after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In ddr-2 mutants, proximal ends of axons failed to regenerate (arrowheads). Scale bar = 10 μm. (D,F) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% CI. *P<0.05, ***P<0.001. NS, not significant. (E) Percentages of axons that initiated regeneration 24 or 72 hr after laser surgery. Error bars indicate 95% CI. **P<0.01, ***P<0.001. NS, not significant.

Fig 2. Tyrosine kinase activity of DDR-2.

(A) Schematic diagrams of DDR-2, DDR-2C and Tpr-DDR-2C. LZ, leucine zipper. (B,C) Auto-tyrosine-phosphorylation of DDR-2. COS-7 cells were transfected with control vector or the plasmids encoding FLAG-DDR-2C, FLAG-Tpr-DDR-2C (WT) and FLAG-Tpr-DDR-2C (K554E) (KN), as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted with anti-phospho-tyrosine (pY) and anti-FLAG antibodies.

Fig 3. Localization of DDR-2.

(A,B) Localization of DDR-2::GFP in D-type motor neurons after axon injury. Fluorescent images of severed axons in wild-type animals carrying Punc-25::ddr-2::gfp and Punc-47::mcherry are shown. Each image was taken at the indicated times after laser surgery. Arrowheads indicate the ends of proximal axons (A). The image was taken at 4 hr after axon injury. Arrows indicate the tip of the proximal injured axon (B). Scale bars = 2 μm. (C) Quantification of the relative fluorescence levels of DDR-2::GFP on the tips of the severed axons. The relative fluorescent intensities of severed axons in animals carrying Punc-25::ddr-2::gfp and Punc-47::mcherry::caax were compared. “N” = number of axons. Error bars indicate 95% CI. *P<0.05, **P<0.01.

Fig 4. The relationship between DDR-2 and SVH-2 in axon regeneration.

(A,C) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% CI. *P<0.05, **P<0.01, ***P<0.001. NS, not significant. (B) Tyrosine phosphorylation of MLK-1. COS-7 cells were transfected with plasmids encoding FLAG-Tpr-SVH-2C, FLAG-Tpr-DDR-2C and HA-MLK-1, as indicated. Complexes immunoprecipitated with anti-HA or anti-FLAG antibody were analyzed by immunoblotting with anti-phospho-tyrosine (pY), anti-HA and anti-FLAG antibodies.

Fig 5. Interactions of SHC-1 with SVH-2 and DDR-2.

(A) The relationship among EMB-9–DDR-2, SHC-1 and SVH-1–SVH-2 in the JNK signaling pathway. (B) Structure of SHC-1. Dark and hatched boxes represent the PTB and SH2 domains, respectively. Essential Arg residues required for binding to phospho-tyrosine in PTB and SH2 domains are indicated by asterisks. (C,D) Interactions of SHC-1 with SVH-2 and DDR-2. COS-7 cells were transfected with plasmids encoding FLAG-Tpr-SVH-2C (WT), FLAG-Tpr-SVH-2C(K767R) (KN), FLAG-Tpr-DDR-2C (WT), FLAG-Tpr-DDR-2C (K554E) (KN), T7-SHC-1 (WT), T7-SHC-1 (R136K), T7-SHC-1 (R234K) and T7-SHC-1 (R136K; R234K), as indicated. Whole-cell extracts and immunoprecipitated complexes obtained with anti-FLAG antibody (IP: FLAG) were analyzed by immunoblotting. (E) Percentages of axons that initiated regeneration 24 hr after laser surgery. Error bars indicate 95% CI. **P<0.01, ***P<0.001. NS, not significant.

Fig 6. DDR-2 modulates SVH-1–SVH-2 signaling to regulate axon regeneration after neuron injury.

HGF/plasminogen-like protein SVH-1 is constitutively expressed in, and secreted from ADL sensory neurons in the head. Expression of SVH-2 is induced by axon injury. Following axon injury, DDR-2 accumulates at the severed end and is activated by EMB-9. DDR-2 facilitates the efficient activation of SVH-2 by SVH-1. Activated SVH-2 phosphorylates MLK-1 MAPKKK at a tyrosine residue, creating a docking site for SHC-1. SHC-1 constitutively forms a complex with MEK-1 MAPKK, and thereby functions to connect MLK-1 and MEK-1. SHC-1 participates in restricting the SVH-1–SVH-2 signal to the MLK-1–MEK-1 pathway.