TBC1D3 reduces S6K activation, but does not affect mTOR-C1 pathway.

<p>(A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of S6K:T389 phosphorylation normalized to S6K protein levels (** <i>p</i><0.01). (B) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, pre-treated with Rapamycin (50 nM) for 2 h, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of 4EBP1 and S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of 4EBP1 phosphorylation normalized to GAPDH protein levels. The data are presented as means ± SD of three independent experiments.</p

Model for the regulation of IRS-1 degradation by TBC1D3 expression.

<p>We propose that TBC1D3 suppresses the degradation of IRS-1 by regulating the phosphorylation of S6K at T389. In this model, mTOR phosphorylates S6K in response to insulin signaling. TBC1D3 interacts, directly or indirecly, with PP2A B56γ to enhance the dephosphorylation of S6K:T389 thereby reducing the S6K-dependent phosphorylation of IRS-1 at key sites which are required for IRS-1 ubiquitination and degradation.</p

PP2A B56γ mediates S6K phosphorylation in response to TBC1D3 expression.

<p>(A) DU145 cells with GFP (control) or PP2A B56γ knockdown were transfected with myc-TBC1D3 or empty vector, serum-starved, and stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of S6K were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of S6K:T389 phosphorylation normalized to S6K protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). (B) Reduction of IRS-1:S636/639 phosphorylation induced by TBC1D3 expression is rescued by a rapamycin-resistant mutant of S6K. pCIS2-IRS-1, myc-TBC1D3 or empty vector were co-transfected with HA-S6K-ED₃E or vector in DU145 cells. Cells were stimulated with insulin (10 nM) for 0, 5 or 30 min after serum-starvation. Phosphorylation and protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1:S636/639 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). The data are presented as means ± SD of three independent experiments. (C) TBC1D3 co-immunoprecipitates with PP2A B56γ. Hek293 cells were transfected with myc-TBC1D3 or empty vector. Cell lysates were immunoprecipitated with anti-myc or anti-B56γ antibodies, respectively. Co-IP samples were separated by SDS-PAGE and analyzed by Western blotting with specific antibodies.</p

TBC1D3 expression blocks IRS-1 degradation.

<p>IRS-1 degradation is delayed in cells expressing TBC1D3. DU145 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 normalized to GAPDH protein levels. The value of IRS-1 at time 0 was set at 1.0. The data are presented as means ± SD of three independent experiments.</p

TBC1D3 selectively decreases IRS-1 Serine phosphorylation.

<p>(A) Empty vector or myc-TBC1D3 was co-transfected with pCIS2-IRS-1 in Hek293 cells. Cells were stimulated with insulin (10 nM) for 30 min. Phosphorylation and protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). (B) Hek293 cells were transfected with myc-TBC1D3 or empty vector, serum-starved and stimulated with insulin (10 nM) for 5 or 30 min. Phosphorylation of endogenous IRS-1:S636/639 was analyzed by Western blotting. (<i>Right panel</i>) Quantification data of endogenous IRS-1 phosphorylation normalized to IRS-1 protein levels (* <i>p</i><0.05). The data are presented as means ± SD of three independent experiments.</p

TBC1D3 increases signaling through the activation of insulin pathway.

<p>(A) HepG2 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Phosphorylation and protein levels of Akt were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of Akt:S473 phosphorylation normalized to Akt protein levels (* <i>p</i><0.05, ** <i>p</i><0.01). (B) HepG2 cells were transfected with two different TBC1D3 siRNA oligos (50 nM) (si#1 and si#2), scramble siRNA (scr) or untransfected (ctr). After 36 h, cells were serum-starved and stimulated with 10 nM insulin for the indicated times and analyzed by Western blotting with the listed antibodies. (<i>Right panel</i>) Quantification data of Akt:S473 phosphorylation normalized to Akt protein levels (* <i>p</i><0.05). The data are presented as means ± SD of three independent experiments.</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-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

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