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Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance.

Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N - J. Cell Biol. (2004)

Bottom Line: Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function.Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants.We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada.

ABSTRACT
Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

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DCC phosphorylation on tyrosine 1418 is critical for neurite outgrowth in N1E-115 cells. (A) Immunocytochemistry of N1E-115 cells expressing DCC, Y1261F-, Y1272F-, Y1361F-, or Y1418F-DCC mutant proteins. The cells were costained with anti-DCC antibodies and with rhodamine-conjugated phalloidin to stain F-actin. Cells expressing DCC were treated with either PP2 or SU6656. Bar, 20 μm. (B) Quantitative analysis of the percent transfected N1E-115 cells exhibiting neurites shown in A (n = 4). The percent transfected N1E-115 cells exhibiting neurites was determined by counting >100 expressing cells exhibiting at least one neurite per cell. A neurite was defined as a process that measured at least the length of one cell body. Error bars represent SD.
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fig7: DCC phosphorylation on tyrosine 1418 is critical for neurite outgrowth in N1E-115 cells. (A) Immunocytochemistry of N1E-115 cells expressing DCC, Y1261F-, Y1272F-, Y1361F-, or Y1418F-DCC mutant proteins. The cells were costained with anti-DCC antibodies and with rhodamine-conjugated phalloidin to stain F-actin. Cells expressing DCC were treated with either PP2 or SU6656. Bar, 20 μm. (B) Quantitative analysis of the percent transfected N1E-115 cells exhibiting neurites shown in A (n = 4). The percent transfected N1E-115 cells exhibiting neurites was determined by counting >100 expressing cells exhibiting at least one neurite per cell. A neurite was defined as a process that measured at least the length of one cell body. Error bars represent SD.

Mentions: To further demonstrate the importance of the tyrosine kinase activity of Fyn on the neurite outgrowth function of DCC, N1E-115 cells were cotransfected with DCC and either Fyn-CA or Fyn-DN. We show that the expression of Fyn-DN completely inhibits neurite extensions induced by DCC, whereas the expression of Fyn-CA leads to a small increase in cells exhibiting neurites compared with cells expressing DCC alone (Fig. 5, E and F). Likewise, treatment of N1E-115 cells expressing DCC with PP2 or SU6656 abolishes the ability of DCC to induce neurite extensions, demonstrating the essential role of Src family kinases in the neurite outgrowth function of DCC (Fig. 7, A and B). The expression of Fyn-CA by itself induces the formation of neurites in N1E-115 cells, as demonstrated previously (Suetsugu et al., 2002), but to a lesser extent than that induced by DCC expression (Fig. 5, E and F). In comparison, the expression of Src-CA or Src-DN in N1E-115 cells did not affect the neurite outgrowth function of DCC (Fig. 5, E and F). These results show that the kinase activity of Fyn, but not of Src, is required for the neurite outgrowth function of DCC.


Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance.

Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N - J. Cell Biol. (2004)

DCC phosphorylation on tyrosine 1418 is critical for neurite outgrowth in N1E-115 cells. (A) Immunocytochemistry of N1E-115 cells expressing DCC, Y1261F-, Y1272F-, Y1361F-, or Y1418F-DCC mutant proteins. The cells were costained with anti-DCC antibodies and with rhodamine-conjugated phalloidin to stain F-actin. Cells expressing DCC were treated with either PP2 or SU6656. Bar, 20 μm. (B) Quantitative analysis of the percent transfected N1E-115 cells exhibiting neurites shown in A (n = 4). The percent transfected N1E-115 cells exhibiting neurites was determined by counting >100 expressing cells exhibiting at least one neurite per cell. A neurite was defined as a process that measured at least the length of one cell body. Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172574&req=5

fig7: DCC phosphorylation on tyrosine 1418 is critical for neurite outgrowth in N1E-115 cells. (A) Immunocytochemistry of N1E-115 cells expressing DCC, Y1261F-, Y1272F-, Y1361F-, or Y1418F-DCC mutant proteins. The cells were costained with anti-DCC antibodies and with rhodamine-conjugated phalloidin to stain F-actin. Cells expressing DCC were treated with either PP2 or SU6656. Bar, 20 μm. (B) Quantitative analysis of the percent transfected N1E-115 cells exhibiting neurites shown in A (n = 4). The percent transfected N1E-115 cells exhibiting neurites was determined by counting >100 expressing cells exhibiting at least one neurite per cell. A neurite was defined as a process that measured at least the length of one cell body. Error bars represent SD.
Mentions: To further demonstrate the importance of the tyrosine kinase activity of Fyn on the neurite outgrowth function of DCC, N1E-115 cells were cotransfected with DCC and either Fyn-CA or Fyn-DN. We show that the expression of Fyn-DN completely inhibits neurite extensions induced by DCC, whereas the expression of Fyn-CA leads to a small increase in cells exhibiting neurites compared with cells expressing DCC alone (Fig. 5, E and F). Likewise, treatment of N1E-115 cells expressing DCC with PP2 or SU6656 abolishes the ability of DCC to induce neurite extensions, demonstrating the essential role of Src family kinases in the neurite outgrowth function of DCC (Fig. 7, A and B). The expression of Fyn-CA by itself induces the formation of neurites in N1E-115 cells, as demonstrated previously (Suetsugu et al., 2002), but to a lesser extent than that induced by DCC expression (Fig. 5, E and F). In comparison, the expression of Src-CA or Src-DN in N1E-115 cells did not affect the neurite outgrowth function of DCC (Fig. 5, E and F). These results show that the kinase activity of Fyn, but not of Src, is required for the neurite outgrowth function of DCC.

Bottom Line: Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function.Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants.We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada.

ABSTRACT
Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

Show MeSH
Related in: MedlinePlus