In this report, we have demonstrated that netrin stimulation results in the activation of FAK and Src tyrosine kinases, as well as tyrosine phosphorylation of DCC, in dorsal spinal neurons, cortical neurons and transfected HEK293 cells. Experiments with an axon outgrowth assay using rat spinal neurons and in vitro
axon turning of X. laevis
spinal neurons suggest that SFK is centrally involved in DCC-mediated netrin signaling. SFK activity is required for both axon growth and attraction in response to netrin stimulation. We propose that DCC functions as a tyrosine kinase–coupled receptor by directly interacting and activating FAK and Src. This conclusion is supported by independent and complementary studies29,30
. Interestingly, another recent report shows that clr-1
, a protein tyrosine phosphatase, is involved in UNC-6/netrin–mediated axon attraction in C. elegans31
. Similarly to high eukaryotes, the UNC-40/DCC receptor is required for axon attraction induced by UNC-6/netrin in C. elegans
. This study suggests that clr-1
probably dephosphorylates a key protein in the netrin signaling pathway, possibly UNC-40/DCC. These findings are consistent with our conclusions that DCC functions as a tyrosine kinase–coupled receptor and that tyrosine phosphorylation of DCC is critical for netrin signaling.
Signal transduction via tyrosine kinase–coupled receptors has been widely used by many transmembrane receptors, such as cytokines and T-cell receptors. Our study supports a model by which DCC may also use a similar mechanism (Supplementary Fig. 2). Three motifs in the intracellular domain, known as P1, P2 and P3, are conserved among DCC family members from different species7,12
. The P1 domain has been implicated in the interaction with UNC-5 and is important for the functional complex of DCC with UNC-5 (ref. 12
). The DCC/UNC-5 receptor complex is important for netrin to induce growth cone repulsion. The P2 domain, which is rich in prolines, may play a role in protein-protein interactions. The P3 domain has been shown to interact with the Slit receptor, Robo32
. The association between Robo and DCC may be important for Slit to silence the netrin-induced growth cone attraction. We demonstrated that FAK and Src interact with DCC under physiological conditions, via different mechanisms and through different regions of DCC. FAK interacts via its C-terminal domain, with the C-terminal half of the P3 domain of DCC (Supplementary Fig. 2). The SH3 domain of Src interacts with a single PXXP motif, beginning at residue Pro1400, of DCC. Furthermore, the interaction between Src and DCC is stimulated by netrin and partially inhibited by PP2, indicating a role of tyrosine phosphorylation in the interaction between Src and DCC. In contrast, the interaction between FAK and DCC is not stimulated by netrin nor inhibited by PP2.
Our results suggest a model in which different regions of DCC are responsible for interacting with FAK and Src (Supplementary Fig. 2). We propose that netrin binding to DCC activates the associated FAK. FAK autophosphorylates on Tyr397 and recruits Src (or enhances the interaction between Src and DCC). Thus, FAK also shows a scaffolding function, likely by recruiting Src to the receptor complex. FAK and Src in the receptor complex positively stimulate each other. The active FAK and Src then phosphorylate target molecules, including the DCC receptor, which in turn induce downstream signaling events.
The importance of SFK in netrin signaling is demonstrated by our axon turning experiments using the Src inhibitor PP2 and dominant-negative Src mutants. Furthermore, the importance of DCC tyrosine phosphorylation is supported by our observation that the DCC-Y1420F mutant interferes with netrin-induced axon attraction. Therefore, in addition to functioning as an upstream receptor, DCC also serves as a critical downstream substrate of FAK and Src in netrin signaling. This mechanism of action is similar to that used by cytokine receptors, where phosphorylation of the receptor by Jak family kinases is important for cytokine signaling. However, DCC tyrosine phosphorylation is not required for the activation of Src and FAK. Tyrosine-phosphorylated DCC may create binding sites for SH2-containing downstream signaling molecules. Future studies will be needed to identify substrates phosphorylated by Src and FAK, as well as proteins that interact with phospho-DCC—these studies will provide further insights into the mechanism of DCC signaling in response to netrin stimulation.
Several proteins have been reported to participate in netrin signaling, including PI3K33
, Rho family GTPases34–36
and ERK MAP kinase37,38
. How DCC receptor activation leads to modulation of these downstream molecules is currently unknown. Both FAK and Src are known to regulate PI3K, Rho family small GTPases and ERK16,25
. Therefore, our study provides a possible mechanism by which DCC could modulate these downstream signaling molecules.
Netrin can stimulate both axon growth and turning initiated by the same netrin receptor, DCC. However, the intracellular signaling pathways for axon growth and turning may diverge after the receptor-proximal events. For example, the nuclear factor of activated T cells (NFAT) is involved in netrin-induced axon growth but not the turning response39
. In contrast, the Slit receptor Robo silences netrin-induced turning but not the outgrowth response32
. We observed that expression of the kinase-inactive Src mutant, Src-KM, blocked both axon outgrowth and turning in response to netrin (). These data suggest that Src activation is a receptor-proximal event and important for both axon outgrowth and the turning responses. Notably, mutations in the SH2 and SH3 domains preferentially inhibited netrin-induced axon outgrowth and turning, respectively. These observations suggest that Src-SH2M and Src-SH3M may selectively block downstream signaling pathways leading to axon outgrowth or turning. Future experiments are required to test whether Src-SH2M blocks the NFAT pathway and whether Src-SH3M mimics the effect of Robo silencing.