Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak

Abstract

Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Ang1) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Ang1 stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Pak1) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Ang1-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak.

Fig. 1. Ang1-MH stimulation induces EC migration and tyrosine phosphorylation and association of a Tek/Dok-R–Nck complex. (A) DMEM alone (–) or Ang1-MH (+)-conditioned medium was used to stimulate SVR ECs for 10 min in the absence of phosphatase inhibitors. Proteins from Tek immunoprecipitates were separated by SDS–PAGE and immunoblotted with antibodies recognizing phosphotyrosine (pY), Tek and Dok-R, which revealed that Ang1-MH can stimulate the tyrosine phosphorylation of Tek and the co-immunoprecipitation of tyrosine phosphorylated Dok-R. Both lysates contained equal concentrations of Tek receptor. (B) Ang1-MH (+) stimulation of SVR ECs results in the tyrosine phosphorylation of Dok-R and co-immunoprecipitation of tyrosine phosphorylated Tek and Nck when compared with DMEM-stimulated lysates (–). Immunoprecipitations of Dok-R (first two lanes, anti-Dok-R blot) and Nck (last two lanes, anti-Nck blot) with their respective antibodies revealed that equal amounts of protein were immunoprecipitated from both stimulated (+) and unstimulated (–) lysates. Immunoprecipitation of tyrosine phosphorylated Nck from Ang1-MH-stimulated lysates could co-immunoprecipitate more tyrosine phosphorylated Dok-R than unstimulated (–) lysates (last two lanes). (C) SVR ECs were seeded in the top chamber of a modified Boyden chamber while chemoattractants (Mock, Ang1-MH or 50 ng/ml VEGF) were placed in the bottom well. Stimulation with Ang1-MH and VEGF resulted in a 5-fold increase in migration over Mock-stimulated cells. Data points are represented as the number of migrated cells per field. All experiments were performed in triplicate and differences between Mock-stimulated and ligand-stimulated migration values were statistically significant (P <0.05).

Fig. 2. Dok-R recruits Nck through tyrosine phosphorylation of Y351. (A) The positions of three tyrosine residues (276, 304 and 351) in Dok-R found within the context YxxP are indicated. Double mutations in Y276 and Y304 to phenylalanine (F) abolish Dok-R binding to RasGAP (Dok-R^DM) while mutation of Y351 to F abrogates binding to Nck (Dok-R^Y351F). (B) Overexpression of Tek with Dok-R, or its mutants, in HEK 293T cells resulted in the tyrosine phosphorylation of Dok-R, Dok-R^DM and Dok-R^Y351F and their co-immunoprecipitation with tyrosine phosphorylated Tek. Nck could co-immunoprecipitate with Dok-R or Dok-R^DM, but this interaction was abrogated in Dok-R^Y351F-transfected cells. Equal amounts of Tek and Dok-R/mutant were observed in transfected cell lysates (CL). (C) Lysates prepared from Tek-Dok-R and Tek-Dok-R^Y351F cell lines stimulated with Mock or Ang1-MH for 1 h in the absence of phosphatase inhibitors were immunoprecipitated with anti-Tek antibodies. Ang1-MH can stimulate the tyrosine phosphorylation of Tek in stable cell lines and the co-immunoprecipitation of tyrosine phosphorylated Dok-R or its mutants. Mock stimulation of these cells resulted in basal levels of Tek tyrosine phosphorylation. Immunoprecipitation of Tek also resulted in the co-immunoprecipitation of Nck in Ang1-MH-stimulated lysates of Tek-Dok-R cells, but not Tek-Dok-R^Y351F mutant cells. Immunoblotting with anti-Tek antibodies revealed equal amounts of Tek protein expression in all lysates.

Fig. 3. Nck recruitment to Dok-R is required for Ang1-mediated migration. (A) Lysates prepared from Tek-Dok-R or mutant cell lines were subjected to immunoblot analysis using anti-HA antibodies that recognized transfected Dok-R/mutant (inset). Equal Dok-R-expressing clones of Tek-Vec, Tek-Dok-R, Tek-Dok-R^DM and Tek-Dok-R^Y351F stable cell lines (inset) were placed in a migration assay as described previously using Mock, Ang1-MH or 30 ng/ml bFGF in Mock media as chemoattractants. Ang1-MH stimulation of Tek-Vec cells resulted in a 3-fold increase in migration over Mock, and expression of Dok-R or Dok-R^DM potentiated this Ang1-dependent motility. However, Tek-Dok-R^Y351F cells displayed a dramatic reduction in migration. Stimulation of cells with bFGF resulted in relatively equal migration levels. All experiments were performed in triplicate using three different clones (data not shown). Migration differences between Mock and Ang1-MH stimulations of all cell lines, and of Ang1-MH stimulations of Tek-Dok-R^Y351F compared with Tek-Vec/Tek-Dok-R/Tek-Dok-R^DM were all found to be statistically significant (P <0.05). (B) Tek-Dok-R and Tek-Dok-R^Y351F stable cell lines seeded on coverslips were stimulated with Mock- or Ang1-MH-conditioned media for 4 h and then immunostained using antibodies against actin. Mock-stimulated cells displayed a diffused dispersion of actin at the cell membrane with no membrane protrusions. However, cells stimulated with Ang1-MH displayed localized regions of actin and Tek-Dok-R cells displayed a greater frequency and extent of membrane projections (arrowheads) than did Tek-Dok-R^Y351F cells.

Fig. 4. Pak colocalizes with Tek through a Dok-R–Nck interaction. Antibodies recognizing Tek, Dok-R and Pak were used to detect these proteins in HEK 293T cells overexpressing Tek with either Dok-R or Dok-R^Y351F by confocal microscopy. Cellular distributions of Tek (red) were found at the cell membrane and were colocalized with Dok-R/Dok-R^Y351F (green) as seen by the merged image (yellow). The colocalization seen between Tek and Dok-R, or its mutants, specifically required an active Tek kinase, since cells co-transfected with Tek^K853A and Dok-R display diffuse staining throughout the cytoplasm rather than at the membrane (inset). Similarly, in cells overexpressing activated Tek and Dok-R, Pak (green) was enriched at the cell membrane (white arrows), which colocalized with Tek, as indicated by the merged image. However, cells expressing Tek and Dok-R^Y351F did not have this enriched localization of Pak at the membrane and no colocalization with Tek can be seen (merge).

Fig. 5. Activated Tek promotes the formation of a Pak, Nck and Dok-R ternary complex in vivo and results in Pak activation. (A) SVRs were stimulated with Ang1-MH or DMEM in the presence of sodium orthovanadate. Immunoprecipitation of Dok-R resulted in co-immunoprecipitation of Pak in lysates from Ang1-MH-stimulated cells. Reprobing the membrane with anti-Dok-R antibodies revealed equal amounts of immunoprecipitated Dok-R from both stimulated and unstimulated lysates. (B) Lysates from HEK 293T cells overexpressing Pak, Tek/Tek^K853A and Dok-R/Dok-R^Y351F were immunoprecipitated with antibodies that specifically recognize Dok-R, Pak or Tek. In cells overexpressing Tek + Dok-R, both Dok-R and Pak could co-immunoprecipitate with one another. Interaction of Dok-R with Pak was not observed in cells expressing either a kinase inactive Tek (Tek^K853A) or Dok-R^Y351F. Immunoblotting of cell lysates (CL) revealed equal expression of Pak, Dok-R and Tek. (C) Pak immunoprecipitates from the above transfectants were subjected to an in vitro kinase assay using MBP as substrate. Tek-mediated MBP phosphorylation by Pak was 40% greater from cells that expressed Dok-R than from cells expressing Dok-R^Y351F or Tek^K853A with either Dok-R or Dok-R^Y351F. The graph represents MBP phosphorylation values after normalization for Pak immunoprecipitation. Immunoprecipitation of Tek and Tek^K853A with anti-Tek antibodies revealed that Tek, but not Tek^K853A, was tyrosine phosphorylated. Data shown are average of two independent experiments ± SD.

Fig. 5. Activated Tek promotes the formation of a Pak, Nck and Dok-R ternary complex in vivo and results in Pak activation. (A) SVRs were stimulated with Ang1-MH or DMEM in the presence of sodium orthovanadate. Immunoprecipitation of Dok-R resulted in co-immunoprecipitation of Pak in lysates from Ang1-MH-stimulated cells. Reprobing the membrane with anti-Dok-R antibodies revealed equal amounts of immunoprecipitated Dok-R from both stimulated and unstimulated lysates. (B) Lysates from HEK 293T cells overexpressing Pak, Tek/Tek^K853A and Dok-R/Dok-R^Y351F were immunoprecipitated with antibodies that specifically recognize Dok-R, Pak or Tek. In cells overexpressing Tek + Dok-R, both Dok-R and Pak could co-immunoprecipitate with one another. Interaction of Dok-R with Pak was not observed in cells expressing either a kinase inactive Tek (Tek^K853A) or Dok-R^Y351F. Immunoblotting of cell lysates (CL) revealed equal expression of Pak, Dok-R and Tek. (C) Pak immunoprecipitates from the above transfectants were subjected to an in vitro kinase assay using MBP as substrate. Tek-mediated MBP phosphorylation by Pak was 40% greater from cells that expressed Dok-R than from cells expressing Dok-R^Y351F or Tek^K853A with either Dok-R or Dok-R^Y351F. The graph represents MBP phosphorylation values after normalization for Pak immunoprecipitation. Immunoprecipitation of Tek and Tek^K853A with anti-Tek antibodies revealed that Tek, but not Tek^K853A, was tyrosine phosphorylated. Data shown are average of two independent experiments ± SD.

Fig. 6. Ang1 stimulates the activation of Pak kinase in ECs. SVRs were stimulated with Mock, Ang1-MH or 30 ng/ml EGF in Mock medium for 0, 5 or 10 min. Lysates were immunoprecipitated with anti-Pak antibodies and then subjected to an in vitro kinase assay using MBP. Pak immunoblots revealed relatively equal Pak immunoprecipitations. The graph represents MBP phosphorylation values after normalization for Pak immunoprecipitation. Data shown are average of two independent experiments ± SD.

Fig. 7. Ang1-mediated cell migration is potentiated by overexpression of Pak. Tek-Dok-R and Tek-Dok-R^Y351F alone or transfected with Pak, were subjected to a migration assay using Mock or Ang1-MH as chemoattractant. Cells stimulated with Ang1-MH resulted in migration similar to that detected previously with Tek-Dok-R and Tek-Dok-R^Y351F cells. However, overexpression of Pak in Tek-Dok-R cells resulted in a potentiation of Ang1-mediated cell migration, which was not seen in Tek-Dok-R^Y351F cells. Cell lysates prepared from these cells revealed equal expression of Dok-R or Dok-R^Y351F and a 2-fold increase in Pak when overexpressed (inset).

Fig. 8. Ang1-mediated migration. A schematic representation of the signaling pathways mediated through Ang1/Tek signaling that promote cell migration. Ang1 regulates two pathways that mediated cell motility, the first being through activation of the PI3-kinase pathway and the second involving the phosphotyrosine-dependent recruitment of Dok-R, Nck and activation of Pak at the cell membrane. PI3-kinase comprises a p85 regulatory subunit, which binds the p110 catalytic subunit. Wort (wortmannin)/LY (LY294002) are pharmacological inhibitors of PI3-kinase.