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Results: 7

1.
FIG. 7.

FIG. 7. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

A model for Etk activation by TNF/TNFR2 in EC angiogenesis. Etk forms a preexisting complex with TNFR2, and Etk remains in a closed inactive form. TNF induces trimerization or conformational change of TNFR2, leading to an open conformation of Etk followed by Etk activation (tyrosine phosphorylation) and EC angiogenesis.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
2.
FIG. 5.

FIG. 5. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

TRAF2 is not involved in TNF-induced Etk activation in EC. BAEC were transfected with WT-TRAF2 or DN-TRAF2. Expression of TRAF2 was determined by Western blot with anti-Flag (a). Twenty-four hours posttransfection, cells were treated with human TNF (1 ng/ml for 15 min) or were left untreated. Etk (b) and JNK (c) activation were determined as described in the legend to Fig. 1. IP, immunoprecipitation; IB, immunoblot.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
3.
FIG. 2.

FIG. 2. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

Association of TNFR2 with Etk in EC is ligand independent. BAEC were either treated with TNF (1 ng/ml for 15 min) or were left untreated. An antibody against either Etk, TNFR1, or TNFR2 was used to examine its ability to precipitate a complex of Etk and TNFRs. A total of 40 μg of cell lysate and 6 μg of antibody was used for each immunoprecipitation (IP). (a) Etk associates with TNFR2 but not with TNFR1 in EC. Anti-Etk was used as a positive control for immunoprecipitation. (b) Association of Etk with TNFR2 is TNF independent in EC. Normal goat sera (NS) were used as controls. IgG, immunoglobulin G; IB, immunoblot; R1, TNFR1; R2, TNFR2.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
4.
FIG. 1.

FIG. 1. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

TNF activates Etk specifically through TNFR2. (a) TNFR2 (R2) agonist, but not TNFR1 (R1) agonist, activates Etk in EC. BAEC were treated with human TNF (1 ng/ml), anti-TNFR1 (αR1; 5 μg/ml), anti-TNFR2 TNFR1 (αR2; 5 μg/ml), or a normal goat serum (5 μg/ml; NS; TNFR1) for 15 min or were left untreated. Etk activation was detected by immunoprecipitation with anti-Etk followed by Western blot with antiphosphotyrosine (pY). Total Etk was detected by Western blot with anti-Etk. IgG, immunoglobulin G; IB, immunoblot.(b) JNK activation was determined by an in vitro kinase assay with GST-c-Jun as a substrate. JNK1 protein in the immunoprecipitates was determined by Western blot with anti-JNK1. (c and d) TNF fails to activate Etk in TNFR2-deficient cells. Lung EC from wild type (WT), TNFR1−/−, or TNFR1−/−TNFR2−/− mice were treated with murine TNF at 1 ng/ml for 15 min, and Etk (c) and JNK (d) activation was determined.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
5.
FIG. 3.

FIG. 3. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

Multiple domains (the PH, TH, and SH2 domains) of Etk are responsible for association with TNFR2 in EC. (a) Schematic diagram of Etk domains and expression constructs. The kinase-dead Etk (Etk-KD) has a mutation at the kinase domain (K444Q). The dominant-negative Etk (Etk-DN) has mutations at the kinase domain (K444Q) and the phospholipid-binding PH domain (E42K). Etk-ΔSH3 has a deletion of the SH3 domain. Etk-SK contains the SH2 and the kinase domains. Etk-K contains the kinase domain only. Etk-PTH contains the PH and TH domains. Etk-DK has a deletion of the kinase domain. Etk-N contains the PTH and SH3 domains. Etk-SH3/2 contains the SH3 and SH2 domains. A summary of interaction with TNFR2 is shown on the right. Etk-WT, wild-type Etk; aa, amino acid. (b) Mapping the Etk domain interacting with TNFR2. Flag-tagged Etk-SK, Etk-K, Etk-PTH, and Etk-DK were transfected in EC, and expression was determined by anti-Flag. Interaction of Etk domains with TNFR2 was examined by immunoprecipitation with TNFR2 or normal serum (NS) followed by Western blot with anti-Flag as indicated. TNFR2 protein in the immunoprecipitate was detected by Western blot with anti-TNFR2. (c) Mapping the TNFR2-interacting domain in the N-terminal portion of Etk. Flag-tagged TNFR2 was cotransfected in EC with Etk-DK, Etk-N, or Etk-SH3/2. Expression was determined by anti-Flag (lanes 1 to 3). Interaction of Etk domains with TNFR2 was determined by immunoprecipitation with TNFR2 followed by Western blot with anti-Flag (lanes 4 to 6). (d) Determining the role of the SH3 domain in Etk for TNFR2 association. T7-tagged Etk-WT, DN, and Etk-ΔSH3 were cotransfected with Flag-TNFR2 in EC, and interaction of Etk with TNFR2 was examined by immunoprecipitation with TNFR2 followed by Western blot with anti-T7. TNFR2 was detected by Western blot with anti-Flag. IP, immunoprecipitation; IB, immunoblot; R2, TNFR2.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
6.

FIG. 4. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

The C-terminal 16-amino-acid residues in TNFR2 are critical for association and activation of Etk. (a) Schematic diagram of TNFR2 domains and the deletion mutants at the C terminus. TNFR2(−16) and TNFR2(−37) are mutants lacking 16- and 37-amino-acid residues at the C terminus, respectively. The black box indicates the TRAF2-binding domain. TNFR2(mTR2) is a mutant containing mutations at the TRAF2-binding motif. All TNFR2 expression constructs are Flag-tagged at the N termini. (b) Flag-tagged Etk-DK or the VC was cotransfected in EC with TNFR2 expression constructs, and expression was determined by anti-Flag. (c) Interaction of Etk-DK with full-length TNFR2 and TNFR2(mTR2) but not with TNFR2(−16) or TNFR2(−37). Interaction was determined by immunoprecipitation with anti-TNFR2 (recognizes the extracellular domain of TNFR2) followed by Western blot with anti-Flag. (d) Interaction of TRAF2 with TNFR2-WT but not with TNFR2(mTR2) or TNFR2(−37). Flag-tag TNFR2-WT, TNFR2(mTR2), or TNFR2(−37) was cotransfected in EC with a TRAF2 expression construct. Association of TRAF2 with TNFR2 or TNFR2(−37) was determined by immunoprecipitation with anti-TNFR2 followed by Western blot with anti-TRAF2. (e) Association of Etk with TNFR2 mutants. BAEC were transfected with T7-tag Etk-WT and Flag-TNFR2 constructs (WT, −16, or mTR2) as indicated. Association of Etk with TNFR2 proteins was determined by immunoprecipitation with anti-TNFR2 followed by Western blot with anti-T7. TNFR2 protein was determined by Western blot with anti-Flag. (f) Activation of Etk by TNFR2 mutants. Etk activation was determined by immunoprecipitation with anti-T7 followed by Western blot with anti-pY. Total Etk was determined by Western blot with anit-T7. (g) Schematic diagram of Etk-binding/activating motif and TRAF2-binding motif in the C-terminal intracellular domain of human and murine TNFR2. The Etk-binding/activating motif is highly conserved between the human and murine TNFR2 and differs in only one residue (in bold). IP, immunoprecipitation; IB, immunoblot; R2, TNFR2; aa, amino acid.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.
7.

FIG. 6. From: Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis.

A critical role of Etk in TNF-induced angiogenesis. BAEC in a 6-well plate were transfected with VC, Etk-SK, or Etk-DK, which coexpresses an enhanced green fluorescent protein. Twenty-four hours postinfection, transfection efficiency and transgene expression were determined. Cells were split into a 24-well plate to reach 100% confluency for migration and tube formation assays (see Materials and Methods). (a) Expression of Etk-SK and Etk-DK was determined by Western blot with anti-Flag. (b) Etk-SK functions as a constitutive active form, whereas Etk-DK functions as a dominant-negative form of Etk. VC, Etk-SK, and Etk-DK-expressing cells were treated with TNF (1 ng/ml for 15 min), and activation of endogenous Etk was determined by immunoprecipitation with anti-Etk followed by Western blot with anti-phosphotyrosine. (c) Etk-SK promotes, while Etk-DK inhibits, TNF-induced EC migration. Transfected BAEC were subjected to injury, and wound healing was measured in the absence or presence of TNF (1 ng/ml) at 0, 12, and 24 h postinjury. Zero and 24 h in the presence of TNF are shown. Triplets for each sample were performed. Experiments were repeated two times. (d) Quantitation of EC migration. Image capture and analyses were performed as described in Materials and Methods. Data presented are means ± standard errors of the means of the triplicate in one experiment. Similar results from two other experiments were obtained. (e) Etk-SK promotes, while Etk-DK inhibits, TNF-induced EC tube formation. Transfected cells were seeded in collagen gels as described in Materials and Methods in the presence or absence of TNF (1 ng/ml), and tubes were visualized on day 5. Image capture and analyses were performed as described in Materials and Methods. Triplets for each sample were performed. Experiments were repeated one time. (f) Quantitation of EC tube formation. Three images from each well were captured, and total tube areas were measured in each field (30 mm2). Data presented are means ± standard errors of the means of the two triplicates from two independent experiments. IP, immunoprecipitation; IB, immunoblot.

Shi Pan, et al. Mol Cell Biol. 2002 November;22(21):7512-7523.

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