CUL7-mediated TBC1D3 degradation requires a CUL7-associated Fbw8.

<p>HeLa cells were transiently transfected with Myc-TBC1D3 along with mock, full-length CUL7 (1–1698) or truncated CUL7-mutant (268–1698) that is unable to recruit Fbw8. The cells were starved and stimulated with 10% FCS for different times. Lysates were resolved by SDS-PAGE at the indicated times. Immunoblotting was carried out with anti-Myc, -tubulin and -HA antibodies (left panel). Right panel shows mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

Interactions between expressed Fbw8, CUL7 and TBC1D3.

<p>HeLa cells were transfected with HA-CUL7, Flag-TBC1D3 and Myc-Fbw8 constructs. At 18 h after transfection, cell lysates (200 µg) were immunoprecipitated with anti-TBC1D3 (2C7) (left panel) or anti-Myc (right panel) antibodies, separated by SDS-PAGE and immunoblotted as indicated.</p

CUL7 mediates the degradation and ubiquitination of TBC1D3.

<p>(A) CUL7 increased TBC1D3 ubiquitination. HeLa cells were transfected with Myc-TBC1D3 and with or without HA-CUL7. TBC1D3 was immunoprecipitated with anti-Myc antibody. Polyubiquitination of TBC1D3 was analyzed by immunoblotting, using anti-ubiquitin antibody. (B) CUL7 expression enhances TBC1D3 degradation. HeLa cells were transfected with Myc-TBC1D3 and with or without HA-CUL7. Cells were starved for 3 h and stimulated with 10% FCS. Lysates were separated by SDS-PAGE and immunoblotted with anti- TBC1D3, CUL7 and tubulin antibodies. (C) Summary of results. Mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

CUL7 siRNA silencing delays TBC1D3 degradation.

<p>(A) HeLa cells were transfected with scramble siRNA or CUL7 siRNA. At 24 h post-transfection, Myc-TBC1D3 was expressed in the cells for another 18 h. The cells were then starved for 3 h and stimulated with 10% FCS for different time periods. Cell lysates were prepared, separated by SDS-PAGE and immunoblotted with anti- TBC1D3, CUL7 and tubulin antibodies. (B) Summary of results. Mean ± SE of 3 independent experiments. TBC1D3 expression is normalized by tubulin. The initial level of TBC1D3 expression in each group is set to 100%.</p

TBC1D3 interacts with Fbw8 in a phosphorylation-dependent manner.

<p>(A) <i>In vitro</i> interaction between TBC1D3 and Fbw8. Separate sets of HeLa cells were transfected with GFP-TBC1D3 or Myc-Fbw8. At 18 h after transfection, the GFP-TBC1D3-expressing cells were starved and stimulated with 10% FCS. To prepare TBC1D3-beads, GFP-TBC1D3 was immunoprecipitated with mouse anti-GFP antibody on Protein G beads. The TBC1D3-beads were treated with or without alkaline phosphatase (AP) for 1 h at 37°C. TBC1D3-beads were incubated with Fbw8-cytosol prepared from HeLa cells expressing Myc-Fbw8. Following incubation at 4°C, the beads were washed and the bound proteins were separated and analyzed by immunoblot using anti-Myc antibody. TBC1D3-beads pulled down Fbw8 and alkaline phosphatase treatment blocked TBC1D3-Fbw8 interaction. (B) <i>In vivo</i> interactions between TBC1D3 and Fbw8. HeLa cells were co-transfected with GFP-TBC1D3 and Myc-Fbw8. After 18 h, the cells were starved and stimulated with 10% FCS. Cell lysates were treated with and without AP and immunoprecipitated with anti-GFP antibody. The precipitates were separated by SDS-PAGE followed by immunoblot analysis. (C) GST-Fbw8-Skp1 pull-down of TBC1D3 is phosphorylation dependent. Glutathione beads with bound GST-Fbw8-Skp1 complex or GST alone were incubated with GFP-TBC1D3 expressing lysates, treated with or without AP for 1 hour at 37°C. The beads were washed and eluted proteins were separated by SDS-PAGE. Immunoblotting with monoclonal anti-TBC1D3 antibody (2C7) showed that GST-Fbw8-Skp1 pull-down of TBC1D3 was nearly abolished by prior alkaline phosphatase treatment (right panel). The right panel shows the Ponceau S staining of the transferred membrane. The experiments were repeated three times.</p

The proposed model for CUL7-mediated TBC1D3 degradation.

<p>TBC1D3 facilitates the propagation of GF-receptor signaling. In turn, GF stimulation induces TBC1D3 phosphorylation and phosphorylated TBC1D3 is recruited to Fbw8. CUL7 provides the structural backbone for the assembly of the CUL7-E3 ligase. The N-terminal portion of CUL7 binds to the adapter protein Skp1 that in turn binds the substrate recognition component, Fbw8. The RING E3 ligase associated with the C-terminus of CUL7 is called RBX1 or ROC1. It recruits an E2 ligase that provides the charged ubiquitin for transfer to the Fbw8-bound TBC1D3. TBC1D3 binds to CUL7 directly through the CPH domain as well as to Fbw8. TBC1D3 is degraded by CUL7-E3 ligase. This creates an inactivation loop that suppresses the effect of TBC1D3 on GF-receptor signaling. TBC1D3 binding to the CPH domain may also allow TBC1D3 to recruit CUL7 to unknown regulatory complexes where additional CUL7 substrates could be more efficiently ubiquitinated.</p

TBC1D3 interacts with CUL7.

<p>(A) <i>In vitro</i> transcription/translation assay. Plasmid inserts encoding putative TBC1D3 binding partners were transcribed and translated <i>in vitro</i> using a coupled, T7 polymerase-driven reaction with ³⁵S-methionine. The SARA and Rab5a are the full-length human proteins while the CUL7 is an amino-terminal portion of the human CUL7. The proteins were resolved using SDS-PAGE, and TBC1D3 binding was visualized by autoradiography. The migration of molecular weight standards is indicated. (B) TBC1D3 co-immunoprecipitates with CUL7. HeLa cells were transfected with plasmids encoding HA-CUL7 and Myc-TBC1D3. Cells were lysed and immunoprecipitated with anti-Myc antibody. The immunoprecipitates were resolved by SDS-PAGE and blotted with anti-HA and anti-Myc antibodies.</p