Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2005 Aug;25(16):6889–6898. doi: 10.1128/MCB.25.16.6889-6898.2005

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

PMC Copyright notice

FIG. 4. — Semaphorin 4D induces the tyrosine phosphorylation of PYK2 and Src in endothelial cells. (A) Treatment of endothelial cells with Semaphorin 4D and cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs with NGF induces changes in tyrosine phosphorylation. Endothelial cells and cells stably expressing Trk-A/Plexin-B1 chimeric receptors were treated with 200 ng/ml Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF), respectively, for the indicated periods of time, lysed, and analyzed for the presence of tyrosine phosphoproteins (WB: P-Y). Changes in tyrosine phosphorylation of proteins of approximately 110 and 60 kDa are detected. (B) PYK2 is activated in endothelial cells in response to S4D. Endothelial cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoblotted with antibodies specific for different sites of tyrosine phosphorylation on PYK2. The levels of phosphorylation on Y579 and 580, residues that reflect kinase activity, and Y881, corresponding to the proposed Grb-2 binding site in the C-terminal domain, are observed starting at 1 min and 3 min after treatment with Semaphorin 4D, respectively. (C) Src is found in tyrosine-phosphorylated molecular complexes in endothelial cells in response to S4D. Cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoprecipitated for tyrosine-phosphorylated proteins. The detection of Src in the tyrosine immunoprecipitated fraction dramatically increased at 1 min after treatment (IP: P-Y and WB: Src; upper panel). Total Src was used as a loading control (WB: Src; lower panel). (D) Blockade of Src has no effect on Semaphorin 4D-induced PYK2 activation. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for PYK2 (IP: PYK2), and immunoblotted for the presence of the phosphorylated tyrosine residues Y579 and Y580, located in the activation loop (WB: P-PYK2 Y579/580). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in both control and PP2 treated populations. Total PYK2 was used as a loading control (WB: PYK2). (E) Blockade of Src prevents Semaphorin 4D-induced phosphorylation of p85 PI3K subunit. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for p85, and immunoblotted for the presence of the phosphorylated tyrosine residues (IP: p85 and WB: P-Y). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in the DMSO populations but not in cells incubated with PP2 (upper panel). Total p85 was used as a loading control (WB: p85; lower panel). MW, molecular mass.

FIG. 4. — Semaphorin 4D induces the tyrosine phosphorylation of PYK2 and Src in endothelial cells. (A) Treatment of endothelial cells with Semaphorin 4D and cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs with NGF induces changes in tyrosine phosphorylation. Endothelial cells and cells stably expressing Trk-A/Plexin-B1 chimeric receptors were treated with 200 ng/ml Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF), respectively, for the indicated periods of time, lysed, and analyzed for the presence of tyrosine phosphoproteins (WB: P-Y). Changes in tyrosine phosphorylation of proteins of approximately 110 and 60 kDa are detected. (B) PYK2 is activated in endothelial cells in response to S4D. Endothelial cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoblotted with antibodies specific for different sites of tyrosine phosphorylation on PYK2. The levels of phosphorylation on Y579 and 580, residues that reflect kinase activity, and Y881, corresponding to the proposed Grb-2 binding site in the C-terminal domain, are observed starting at 1 min and 3 min after treatment with Semaphorin 4D, respectively. (C) Src is found in tyrosine-phosphorylated molecular complexes in endothelial cells in response to S4D. Cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoprecipitated for tyrosine-phosphorylated proteins. The detection of Src in the tyrosine immunoprecipitated fraction dramatically increased at 1 min after treatment (IP: P-Y and WB: Src; upper panel). Total Src was used as a loading control (WB: Src; lower panel). (D) Blockade of Src has no effect on Semaphorin 4D-induced PYK2 activation. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for PYK2 (IP: PYK2), and immunoblotted for the presence of the phosphorylated tyrosine residues Y579 and Y580, located in the activation loop (WB: P-PYK2 Y579/580). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in both control and PP2 treated populations. Total PYK2 was used as a loading control (WB: PYK2). (E) Blockade of Src prevents Semaphorin 4D-induced phosphorylation of p85 PI3K subunit. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for p85, and immunoblotted for the presence of the phosphorylated tyrosine residues (IP: p85 and WB: P-Y). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in the DMSO populations but not in cells incubated with PP2 (upper panel). Total p85 was used as a loading control (WB: p85; lower panel). MW, molecular mass.

HHS Vulnerability Disclosure