![Figure 3. Mutation of Y1311 and Y1320 impairs DEP-1–mediated activation of Src in HEK 293T cells, without affecting PTP activity. (A) HEK 293T cells were transfected with increasing amounts of WT DEP-1 cDNA vector. The phosphorylation level of Src on Y529 and Y418 was determined by immunoblotting total cell lysates (TCL) with the corresponding phospho-specific antibodies. (Bottom panel) Src was immunoprecipitated from the corresponding cell lysates shown in panel A with the Src GD11 antibody and its kinase activity determined in vitro using a Src peptide substrate with [γ-32P]ATP as previously done. Based on these results, all other transfection experiments were performed with 3-4 μg of DEP-1 plasmids to achieve optimal Src activation in these cells. (B) HEK 293T cells were transfected with empty vector (pmT2), WT DEP-1 and the indicated mutants, and the phosphorylation level of Src on Y529 and Y418 analyzed as described in panel A. (C) DEP-1 is constitutively tyrosine phosphorylated in HEK 293T cells. Total cell lysates of HEK 293T cells transfected with equal levels of WT DEP-1 and the various mutants were immunoblotted with a phospho-specific antibody detecting pY1320 DEP-1. (D) The catalytic activity of DEP-1 is not affected by the various mutations reducing its tyrosine phosphorylation. WT DEP-1 and mutants were coexpressed with activated Src Y527F in HEK 293T cells to enhance DEP-1 phosphorylation. DEP-1 was immunoprecipitated from 200 μg of HEK 293T cell lysates (not containing phosphatase inhibitors) and submitted to a pNPP hydrolysis assay. Empty vector (pmT2) and DEP-1 catalytically inactive C/S mutant represent negative controls. pNPP hydrolysis after 5 minutes of reaction is reported in this graph. (Bottom panel) Western blot analysis reveals similar levels of expressed WT DEP-1, mutants, and constitutively active Src in the various conditions tested; *P < .05. (E) Mutant DEP-1 is as effective as WT DEP-1 at dephosphorylating VEGFR2. HEK 293T cells cotransfected with VEGFR2 and either empty vector (pmT2), WT DEP-1, DEP-1 Y1311F/Y1320F (YY/FF), or the C/S mutant were stimulated with VEGF (50 ng/mL) for 5 minutes. Phosphorylation of immunoprecipitated VEGFR2 was detected using the general PY99 phosphotyrosine antibody (PY). Similar DEP-1 expression levels are shown. This result is representative of 2 independent experiments. All other results are representative of at least 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/13/10.1182_blood-2011-12-398040/4/zh89991297130003.jpeg?Expires=1735821599&Signature=uHRFmf1GNV9LnZHEMMZO5k4F9KTv0kXUJCwOYXYaCk46ZoA0xm-S7g2UZtkkrM-sx9bwCrfo4mhhud0jW9ExBti85FBhn-XMb-mSE-0b7uZ1eF8uU6f1WIcxlOOUJ3sgtB~IEmhl4rTcnoOzp7CnBsEl2X4fjVjytgb~BsXmDzjyxu4p0W5eE5GGYieqKfjLRdOeTdasVCJwA~NgjaUztS0TG4v3uXNPzJaPkSaPQrPEUsA0fE~aOYP3h0Lsvu032c-t2bFuiE~1R7bJ0fUjtwkuZ5woLDUOtrRNAHvLr7AncSHPoHWCTzUZPG~mhdQxNv77ssOTL7XX-PnkaBwZBw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mutation of Y1311 and Y1320 impairs DEP-1–mediated activation of Src in HEK 293T cells, without affecting PTP activity. (A) HEK 293T cells were transfected with increasing amounts of WT DEP-1 cDNA vector. The phosphorylation level of Src on Y529 and Y418 was determined by immunoblotting total cell lysates (TCL) with the corresponding phospho-specific antibodies. (Bottom panel) Src was immunoprecipitated from the corresponding cell lysates shown in panel A with the Src GD11 antibody and its kinase activity determined in vitro using a Src peptide substrate with [γ-32P]ATP as previously done. Based on these results, all other transfection experiments were performed with 3-4 μg of DEP-1 plasmids to achieve optimal Src activation in these cells. (B) HEK 293T cells were transfected with empty vector (pmT2), WT DEP-1 and the indicated mutants, and the phosphorylation level of Src on Y529 and Y418 analyzed as described in panel A. (C) DEP-1 is constitutively tyrosine phosphorylated in HEK 293T cells. Total cell lysates of HEK 293T cells transfected with equal levels of WT DEP-1 and the various mutants were immunoblotted with a phospho-specific antibody detecting pY1320 DEP-1. (D) The catalytic activity of DEP-1 is not affected by the various mutations reducing its tyrosine phosphorylation. WT DEP-1 and mutants were coexpressed with activated Src Y527F in HEK 293T cells to enhance DEP-1 phosphorylation. DEP-1 was immunoprecipitated from 200 μg of HEK 293T cell lysates (not containing phosphatase inhibitors) and submitted to a pNPP hydrolysis assay. Empty vector (pmT2) and DEP-1 catalytically inactive C/S mutant represent negative controls. pNPP hydrolysis after 5 minutes of reaction is reported in this graph. (Bottom panel) Western blot analysis reveals similar levels of expressed WT DEP-1, mutants, and constitutively active Src in the various conditions tested; *P < .05. (E) Mutant DEP-1 is as effective as WT DEP-1 at dephosphorylating VEGFR2. HEK 293T cells cotransfected with VEGFR2 and either empty vector (pmT2), WT DEP-1, DEP-1 Y1311F/Y1320F (YY/FF), or the C/S mutant were stimulated with VEGF (50 ng/mL) for 5 minutes. Phosphorylation of immunoprecipitated VEGFR2 was detected using the general PY99 phosphotyrosine antibody (PY). Similar DEP-1 expression levels are shown. This result is representative of 2 independent experiments. All other results are representative of at least 3 independent experiments.
