A regulatory mechanism involving TBP-1/Tat-Binding Protein 1 and Akt/PKB in the control of cell proliferation

Abstract

TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells.

Figure 1. Characterization of TBP-1 silenced clones.

A, B: Cells stably transfected with TBP1 sh-RNA plasmid or control cells (wt T11hT, Human Primary Fibroblasts Immortalized by hTERT) were cultured in DMEM+10%FBS for 24 hrs. Levels of TBP-1 expression was evaluated by Western Blot with anti-TBP-1 on whole protein lysates. B: As control, protein levels of other proteasome components (two 19S-ATPases, Rpt-1 and Rpt-6, and a 20S component, C8) was evaluated in the clones T1, T10C and T10E. Bands intensity was evaluated by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point (i.e. TBP-1 levels in T11hT cells), arbitrarily set to 1. Representative of at least four independent experiments.

Figure 2. TBP-1 knockdown determines an increase in the growth properties.

A, B: Cells from the T1, T10C and control cells (wt T11hT) were cultured in DMEM either in the presence (A) or in absence (B) of 10% FBS. Cells were collected at the time points indicated and counted in a Burker chamber. The values are the mean ± SE of three experiments performed in triplicate. C, D: wt T11hT cells, cells from control cell pool or from the sh-TBP-1 cell pool were cultured in DMEM either in the presence (C) or in absence (D) of 10% FBS. Cells were collected at the time points indicated and counted in a Burker chamber. The values are the mean ± SE of three experiments performed in triplicate. E, F: Cells from the T1 clone were transfected by electroporation with empty vector (indicated just as T1) or TBP-1 expression plasmid (indicated as T1+TBP-1); cells were then cultured either in the presence (E) or in absence (F) of 10%FBS and collected at the time points indicated (being T₀ the time at 24 hours after transfection). Cells from each time point have been counted in a Burker chamber. Values are mean ± SE of two experiments performed in triplicate and are indicated as values relative to the reference point (T₀). E, F lower panels: TBP-1 expression and Akt activation have been evaluated by Western Blot with anti-TBP-1, anti-Phospho-Akt Ser473, anti-Akt and anti-actin, as loading control, on whole protein lysates of cells collected at each time point, as indicated.

Figure 3. TBP-1 knockdown reduces sensitivity to serum starvation.

A, B: Cells from the T1 clone and control cells (wt T11hT) were plated at different cell densities as indicated, either in the presence or absence of 10% FBS. After six hrs from plating, cell viability was measured by MTS assay. In the histograms, cell viability is expressed as relative to controls, arbitrarily set to 100 (%). The values are the mean ± SE of three experiments performed in quintuplicate. C, D: 1.8×10⁵ cells/35 mm plates from the T1 clone and control cells (wt T11hT) were grown for 24 hrs, in the presence or absence of 10% FBS. Apoptosis was checked by detection of Caspase-3 (C) and PARP-1 (D) cleavage in Western Blot. Detection with anti-actin was included for control of equal loading. Bands Intensity was measured by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition. Representative of at least four independent experiments. E: T11hT and T1 cells were counted and seeded at 2×10⁵cells/35 mm plate. At 24 hrs cells were collected and treated for analysis of cellular DNA content by flow cytometry. Percentages of cells in the SubG1, G0–G1, S and G2–M phases were quantified with Summit 4.1 software. Representative of three different experiments. The numerical ratios reported on the right highlight the different behaviour of T1 cells when grown in absence or presence of serum. Table 1 provides the mean values (and standard deviations in parentheses) relative to this analysis.

Figure 4. Silencing of TBP-1 determines an increase of the invading capability.

A: Cells from the T1 clone or control cells (wt T11hT) were plated in Boyden chambers and allowed to migrate on filters coated with Matrigel. The values are the mean ± SE of three experiments performed in triplicate. (*) p = 0.046 as determined by the Student's t test. B: Cells from the T1 clone or control cells (wt T11hT) were plated in Boyden chambers and allowed to migrate toward EGF on Matrigel filters. 100% values represent cell migration in the absence of chemoattractants. The values are the mean ± SE of three experiments performed in triplicate. (*) p = 0.027 as determined by the Student's t test. C: Cells transiently transfected with TBP1 si-RNA or with the control si-RNA (si-Luc) were plated in Boyden chambers and allowed to migrate toward EGF on filters coated with Matrigel. 100% values represent cell migration in the absence of chemoattractants. The values are the mean±SE of three experiments performed in triplicate. (*) p = 0.016 as determined by the Student's t test.

Figure 5. TBP-1 knockdown determines activation of the Akt/PKB kinase.

A: Cells from the T1, T10C and control cells (wt T11hT) were cultured in DMEM+10%FBS for 24 hrs. Activation of Akt/PKB was revealed by Western Blot with anti-Phospho-Akt Ser473 antibody. As control, extracts were also probed with anti-Akt, anti-Phospho-GSK-3β/pSer219, anti-pERK_1/2, anti-ERK_1/2 and anti-actin antibodies. Bands Intensity was measured by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point, arbitrarily set to 1. Representative of at least four independent experiments. B: Cells from the T1 clone or control cells (wt T11hT) were plated at the cell density of 2.5×10⁵ in DMEM+10%FBS in six wells. After 24 hrs, either DMSO (/) or with Wortmannin or LY294002, where indicated, were added to the cells at the concentrations indicated and left for either 1 hour (with Wortmannin) or 15′(with LY294002). Extracts were then probed in Western Blot with antibodies against Akt, Phospho-Akt Ser473 and actin. C, D: T11hT cells (C) or U2OS cells (D) were transfected with an siRNA directed against TBP-1 or Luciferase. Extracts were probed with antibodies against Phospho-Akt Ser473, Akt and actin. E: Cells from the T1 clone were transfected with empty vector (first lane) or increasing amounts of TBP-1 expression plasmid. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473 and, as control, with anti-Akt and anti-actin. F: U2OS cells were transfected with empty vector (lanes 1–4) or TBP-1 expression plasmid (lanes 5, 6). After 24 hrs cells were starved for 4 hrs and treated with 10 ng/ml insulin for 10′ where indicated. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473. Extracts were also probed with anti-Akt, anti-actin and anti-Xpress (to reveal transfected TBP-1).

Figure 6. TBP-1 is a downstream target of Akt activation.

A: U2OS cells or T11hT cells were starved for 4 hrs and then treated with 10 ng/ml insulin for the times indicated. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473 and anti-Akt. Levels of endogenous TBP-1 and of two proteasome components (C8 and Rpt-6) were analyzed where indicated. TBP-1 bands intensity was measured by ImageQuant analysis on two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point, (i.e. TBP-1 levels in starved cells) arbitrarily set to 1. Representative of three independent experiments. B: U2OS cells or T11hT cells were treated, 24 hrs after plating, either with DMSO (/) or with 200 nM Wortmannin or 50 mM LY294002 for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, anti-Akt, anti-TBP-1, anti-C8 and anti-Rpt-6. TBP-1 bands intensity was calculated as in A. Representative of three independent experiments. C: U2OS cells were transfected with empty vector (lane 1) or increasing amounts of the constitutive active mutant of the Akt kinase (CA-Akt). After 24 hrs cells were lysed and whole cell lysates probed with anti-Phospho-Akt Ser473, anti-Akt, anti-TBP-1, anti-Rpt-1, anti-Rpt-6, and anti-phospho-GSK3b. D: U2OS cells were transfected with a siRNA directed against MDM2 or Luciferase, as control, at the final concentration of 10 nM. After 24 hrs, cells were starved for 4 hrs and then treated with 10 ng/ml insulin for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, TBP-1, MDM2, Akt, and actin. E: U2OS cells were transfected with a siRNA directed against MDM2 or Luciferase, as control. After 48 hrs, either DMSO (/) or 200 nM Wortmannin was added to the cells and left for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, TBP-1, MDM2, Akt and actin.

Figure 7. TBP-1 is a downstream target of MDM2 activation.

A: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-TBP-1 antibody where indicated or, with anti-GFP antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecipitation (input). B: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-MDM2 antibody where indicated or, with anti-Flag antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecpitation (input). C: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs, cells were lysed and whole cell extracts probed with anti-TBP-1, anti-MDM2, and anti-actin, for loading control. D: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs cells were treated either with DMSO (first four lanes) or with 10 µM MG132 where indicated. Cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. E: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2_wt, MDM2_S166A or MDM2_S166A/S186A expression plasmids. After 24 hrs cells were lysed and whole cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. F: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2_wt, MDM2_1–441 or MDM2_Δ150–230 expression plasmids. After 24 hrs cells were lysed and whole cell lysates analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control.