Figure 1.
TTRAP interacts with TGF-β receptors.
A) TTRAP associates with endogenous TβRI. NMuMG cells stably expressing FLAG-TTRAP were treated with 4 ng/ml of TGF-β for one hour or left untreated. Cellular lysates were prepared and TTRAP was precipitated with FLAG affinity beads. The precipitated proteins and 1/30th of the input lysates were analyzed by western blotting. B) Co-IP analysis of the TTRAP-TGF-β receptor interaction. The indicated proteins were co-expressed in HEK293T cells. Total cellular lysates were prepared and the TGF-β receptors were precipitated with a FLAG antibody. The precipitated proteins and 1/20th of the input lysates were analyzed by western blotting. C) Analysis of the binding of TTRAP to membrane associated TGF-β receptors. To label surface receptors cells were incubate with [125I]-TGF-β, cross-linked with DSS and TTRAP was pulled down. The precipitated receptors were detected by autoradiography. D) EGFP-TTRAP and FLAG-TβRI-KR were co-expressed in AML12 cells and their localizations were monitored by fluorescence microscopy. A juxta-membrane region of the cell was zoomed out at the bottom. Co-localized foci are indicated by arrowheads. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). E) Mapping of the TGF-β receptor binding domain of TTRAP by co-IP. The precipitated proteins and 1/20th of the input lysates were analyzed by western blotting using HA and FLAG antibodies.
Figure 2.
TTRAP associates with the TAK1 complex.
A) TTRAP associates with TRAF6. The indicated proteins were expressed in HEK293T cells. Total cellular lysates were prepared and TRAFs were pulled down. The precipitated complexes were analyzed by western blotting. B) TTRAP binds to TAK1. The indicated proteins were co-expressed in HEK293T cells. TTRAP was precipitated from the cellular lysates and the co-precipitation of TAK1 was analyzed by western blotting. C) TAK1 kinase activity is not required for TTRAP binding. Transfected HEK293T cells were treated with 0.5 µM (5Z)-7-oxozeaenol. TTRAP was precipitated from the lysates and the co-precipitating TAK1 molecules were detected. D) TTRAP associates with endogenous TAK1. An NMuMG cell population was established stably expressing FLAG-TTRAP. FLAG-TTRAP was precipitated from the TGF-β treated cells and the co-purifying endogenous TAK1 was detected by western blotting. E) TTRAP interacts with TAB2. TAB2 was precipitated from transfected HEK293T cells and the protein complexes were analyzed by western blotting. F) Ternary complex formation of TTRAP, TAK1 and TRAF6. TAK1 or TTRAP was precipitated from transfected HEK293T cells with a FLAG antibody and the co-precipitation of the other two molecules were analyzed by an HA antibody. In the co-IP - indicated by a dashed box - the TTRAP complexes were eluted from the agarose beads by a large excess of FLAG peptide and subjected to a second round of IP with a TAK1 antibody. Co-precipitation of TAK1 and TRAF6 was monitored by western blotting. G) TRAF2 can not substitute for TRAF6 in the TAK1-TTRAP-TRAF6 complex. FLAG-TAK1 containing complexes were pulled down from transfected HEK293T cells. The precipitated proteins were analyzed by western blotting.
Figure 3.
TTRAP is ubiquitylated by TRAF6 and promotes TRAF6 dependent ubiquitylation of TAK1.
A) FLAG tagged proteins were pulled down from transfected HEK293T cells and the precipitating TTRAP protein was detected by western blotting using an HA antibody. B) Tranfected HEK293T cells were lysed in 0.5% hot SDS. The lysates were diluted with IP buffer and TAK1 was pulled down. Ubiquitylated TAK1 was detected by western blotting using a His-tag antibody. The input lysates were also analyzed by western blotting using the indicated antibodies.
Figure 4.
The TAK1-TTRAP-TRAF6 complex is stabilized by ubiquitylation and recruited to TβRI.
A) FLAG-TRAF6 was precipitated from transfected HEK293T cells and the co-precipitation of TAK1 and TTRAP was examined by western blotting. B, C) The indicated epitope tagged proteins were co-expressed in HEK293T cells. TβRI was pulled down from the lysates and the co-precipitating TTRAP, TRAF6 and TAK1 were analyzed by western blotting.
Figure 5.
TTRAP is involved in non-canonical TGF-β signaling.
A) Stable NMuMG cell populations expressing GFP, GFP-tagged full-length TTRAP (TTRAP) and GFP-tagged 1–123 aa TTRAP (N-TTRAP) proteins were generated by retroviral transduction. Transduced, GFP positive cells were sorted by FACS. Expressions of the introduced genes were verified by western blotting (top left). The above NMuMG cell lines were transfected with Smad (3TP-lux) and NF-κB reporters (top right). The cells were treated with 4 ng/ml of TGF-β for 16 hours and firefly luciferase activites were measured. To take into account the different transfection efficiencies, a Renilla luciferase expressing plasmid was co-transfected with the reporters. Subsequently, the firefly luciferase activities were normalized for Renilla luciferase activities. The error bars represent standard deviations. Smad2 phosphorylation was monitored in the parental and TTRAP expressing NMuMG cells by western blotting (bottom). A non-specific band is shown as a loading control. B) Endogenous TTRAP level was reduced in AML12 cells by transduction of lentiviruses expressing shRNAs specific for the murine TTRAP mRNA. Transduced, GFP positive cells were enriched by FACS. Lentiviruses expressing three different TTRAP shRNAs were used to rule out off-target effects. A virus vector expressing a non-target shRNA was also employed as a control. In the transduced cell populations TTRAP level was monitored by western blotting (left). Smad and NF-κB transcriptional activities were measured as above in parental and shRNA expressing AML12 cells (right). C) Transfected HEK293T cells were treated with 4 ng/ml of TGF-β for 30 minutes before cell lysis. TAK1 and p38 were precipitated from the lysates and their phosphorylation status was monitored by western blotting. D) Smad2 and p38 phosphorylation were examined in non-target and TTRAP shRNA expressing AML12 cells. Comparable sample loading was also monitored using p38 and Smad2/3 antibodies.
Figure 6.
TTRAP is involved in TGF-β induced apoptosis.
The stable NMuMG cell populations described in Figure 5 were treated in 0.2% (A) or 10% (B) FBS containing medium as indicated and cell viability was assessed using two different methods. PI uptake of cells, as a measure of membrane integrity, was monitored by FACS (left panels). The experiments were repeated at least twice with similar outcome. On the right side MTT assays were used to measure cell viability. The chemicals used at the following concentrations: TGF-β 4 ng/ml; SB431542, SB203580 and SP600125 were all used at 10 µM. The error bars represent standard deviations. C) NMuMG cells stably expressing TTRAP or EGFP were treated as indicated and integrity of their membranes was monitored by PI uptake. The chemicals used at the following concentrations: TGF-β 4 ng/ml, staurisporine (STS) 1 µM, MG132 2.5 µM. Experiments were repeated several times and a representative result is shown. D) Non-target and TTRAP shRNA lentivirus transduced AML12 cells were treated as indicated in 10% FBS medium. After 24 hours, cells were stained with annexin V and analyzed by FACS.