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1.
Figure 9.

Figure 9. From: The transcriptional co-activator PCAF regulates cdk2 activity.

An outline representing the two different mechanisms, by which PCAF inhibits the activity of cyclin A/cdk2 complexes is shown.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
2.
Figure 7.

Figure 7. From: The transcriptional co-activator PCAF regulates cdk2 activity.

Cdk2 is acetylated by PCAF and GCN5 in vivo. (A) Cells were transfected with Flag-cdk2 alone or together with three different acetylases (p300, PCAF or GCN5). Cell extracts were subjected to IP with anti-Flag and WBs were performed with anti-Flag and anti-Acetyl-K. (B) Cells were transfected with Flag-cdk2 or Flag-cdk2K33R alone or together with HA-GCN5. Then, cell extracts were subjected to IP with anti-Flag followed by WB with anti-Flag and anti-Acetyl-K. (C) C2C12 cell extracts were subjected to IP with anti-PCAF. The obtained immunoprecipitates were used as a source of active PCAF and were used for in vitro cdk2 acetylation experiments. Autoradiography indicates cdk2 acetylation (top panel). A WB anti-cdk2 of the membrane used for autoradiography is shown on the bottom panel. Asterisks indicates non-specific bands. (D) The immunoprecipitates obtained in (C) were checked for the content of PCAF and SPT3 by WB.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
3.
Figure 4.

Figure 4. From: The transcriptional co-activator PCAF regulates cdk2 activity.

PCAF inhibition is not competitive with ATP nor with substrate histone H1 or cyclin A. Data from kinetic assays (see ‘Materials and Methods’ section) were analysed by double reciprocal plots. (A) Inhibition of cyclin A/cdk2 activity by PCAF at different ATP concentrations. The assay consisted in the incubation of 2 µg of substrate histone H1 with 400 nM cyclin A/cdk2 in the presence (square) or absence (triangle) of 800 nM PCAF. (B) Inhibition of cyclin A/cdk2 activity by PCAF at different histone H1 concentrations. The assay consisted in the incubation of 12.5 µM ATP with 400 nM cyclin A/cdk2 in the presence (square) or absence (triangle) of 800 nM PCAF. (C) Inhibition of cdk2 activity by PCAF at different cyclin A concentrations. The assay consisted in the incubation of 400 nM cdk2 (control) or 400 nM cdk2 plus 800 nM PCAF (+PCAF) with increasing concentrations of cyclin A in the presence of 12.5 µM ATP and 2 µg of substrate histone H1.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
4.
Figure 2.

Figure 2. From: The transcriptional co-activator PCAF regulates cdk2 activity.

PCAF inhibits cyclin/cdk2 complexes in vivo and in vitro. (A) Purified active cyclin A/cdk2, cyclin E/cdk2, cyclin B/cdk1 and cyclin D3/cdk6 complexes were incubated with increasing concentrations of purified recombinant PCAF and kinase assays were performed. (B) HeLa cells were transfected with YFP-PCAF or empty vector as a control. Cell extracts were subjected to IP with anti-cdk2. Kinase assays of the immunoprecipitated endogenous cdk2 were performed and phosphorylation of histone H1 was detected by PhosphorImager. Kinase activity was normalized to the amount of immunoprecipitated cdk2 and represented in the graph. Results shown are the mean ± SE of 3 independent experiments. *P-value <0.05.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
5.
Figure 3.

Figure 3. From: The transcriptional co-activator PCAF regulates cdk2 activity.

The C terminus of PCAF is responsible for the inhibition of cyclin A/cdk2 activity in vitro. (A) Schematic representation of the domains of PCAF used in the experiments. (B) Purified recombinant cyclin A/cdk2 complexes (400 nM) were incubated with increasing concentrations of Nterm, Cterm, Cterm ΔHAT and full-length PCAF and kinase assays were performed. (C) The same as in (B), but using different recombinant fragments of PCAF: HAT, ADA, Bromo and ADA-Bromo. (D) A typical kinase assay using purified recombinant cyclin A/cdk2 complex (400 nM) as a kinase and histone H1 (2 µg) as a substrate. Different reactions were performed by adding GST or different purified fragments of PCAF at a concentration of 800 nM. After incubation, reactions were stopped and samples were electrophoresed. Phosphorylation of histone H1 was detected by autoradiography using a PhosphorImager.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
6.
Figure 6.

Figure 6. From: The transcriptional co-activator PCAF regulates cdk2 activity.

Cdk2 is a substrate acetylated by PCAF in vitro. (A) Purified GST-cdk2 was subjected to in vitro acetylation assays using the catalytic domain of PCAF [GST-HAT(PCAF)], GST-CBP or GST-Tip60 in the presence of [14C]acetylCoA. Purified GST was used as a negative control substrate. In the assays with PCAF or Tip60 their autoacetylation was used as a positive control, whereas in the case of CBP, histones were used as a positive control substrate (see Supplementary Data Figure S4A). Acetylated proteins were visualized by autoradiography (top panel). A loading control gel was stained with coomassie blue (bottom panel). (B) Purified recombinant cdk2 fragments were subjected to in vitro acetylation assays as in (A) using GST-HAT (PCAF) as acetylase. Acetylated proteins were visualized by autoradiography (top panel). A loading control gel was stained with coomassie blue (bottom panel). (C) Fourteen peptides including one or two consecutive lysines from the cdk2 fragment including aa 1–106, were spotted on a membrane. As a positive control a peptide from histone H3 was added. The membrane was subjected to in vitro acetylation assays with GST-HAT(PCAF) and [14C]acetylCoA. Acetylation was visualized by autoradiography. (D) Purified GST-cdk2 WT and GST-cdk2 K33R were subjected to in vitro acetylation assays with GST-HAT(PCAF). Acetylation was visualized by autoradiography (top panel). A loading control gel was stained with red ponceau (bottom panel).

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
7.
Figure 8.

Figure 8. From: The transcriptional co-activator PCAF regulates cdk2 activity.

Acetylation of cdk2 impairs its kinase activity both in vivo and in vitro. (A) In vitro kinase assays were performed using 400 nM of purified recombinant GST-cdk2WT, K33R or K33Q together with 400 nM of cyclin A. Kinase activity ± SE was represented in the graph. (B) GST-cdk2 WT and K33R were expressed in bacteria co-expressing 6His-PCAF. After purification of GST-cdk2 WT and K33R, they were analysed by WB with anti-Acetyl-K (top panel). Red Ponceau staining of the proteins is shown in the bottom panel as a loading control. GST-cdk2 WT purified in the absence or presence (Ac-cdk2) of PCAF were tested for in vitro kinase activity. 400 nM of the proteins were incubated with 400 nM of purified GST-cyclin A in the presence of histone H1 as a substrate and [32P]ATP as a cofactor. Kinase activity ± SE was represented in the graph. (C) HeLa cells were transfected with Flag-cdk2 WT, K33R or K33Q. Cell extracts were subjected to IP with anti-Flag or IgG as a control. A WB performed with anti-Flag is shown in the top panel. Kinase assays were also performed with the immunoprecipitates and their kinase activity was quantitated with a PhosphorImager (bottom panel). Normalization of cdk activity with respect to the amount of immunoprecipitated cdk2 is shown in the graph. (D) 293-T cells were transfected with Flag-cdk2WT, K33R or K33Q. Cell extracts were subjected to IP with anti-Flag or IgG as a control. A WB performed with anti-Flag is shown in the upper panel. Interaction of the different cdk2 forms with cyclin A, p21 and p27 was analyzed by WB.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
8.
Figure 1.

Figure 1. From: The transcriptional co-activator PCAF regulates cdk2 activity.

Cdk2 interacts with the acetyltransferase PCAF. (A) C2C12 cells were fixed and stained with antibodies against PCAF and cdk2 and colocalization of both proteins was studied by fluorescence microscopy. (B) CNBr-sepharose beads coupled to GST, GST-HAT (PCAF) or GST-PCAF (full length) were incubated with HCT-116 cell extracts and pull-down experiments were performed. The presence of cdk2 in the precipitates was analysed by WB. NB, not bound; B, bound. (C) C2C12 cell extracts were subjected to IP with IgG as a control and anti-cdk2 in order to immunoprecipitate the endogenous protein. Then, WB was performed to detect endogenous cdk2, PCAF and SPT-3. A sample of cell lysate (input) is shown in the first lane. (D) HeLa cells were transfected with YFP-PCAF and Flag-cdk2. Cell extracts were subjected to IP using anti-Flag or IgG as a control followed by WB with antibodies against Flag, PCAF or cyclin A. A sample of cell lysate (input) was used as a control. (E) The putative direct interaction between PCAF and cdk2 was studied by Surface Plasmon Resonance as described in ‘Materials and Methods’ section. PCAF was fixed on the matrix and cdk2 was left to circulate on the chip. The interaction was represented in the sensorgram. (F) C2C12 cells were synchronized by a double-thymidine block or nocodazole as described in ‘Materials and Methods’ section. Then, the levels of endogenous PCAF, cyclin A and cdk2 were determined by WB. To confirm the time of mitosis a WB with antibodies against phosphorylated histone H3 was performed. (G) Cell extracts from synchronized cells described in (F) were subjected to IP with anti-cdk2 or IgG as a control and the amount of PCAF and cdk2 was analyzed by WB.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.
9.
Figure 5.

Figure 5. From: The transcriptional co-activator PCAF regulates cdk2 activity.

PCAF impairs cell proliferation by causing S and G2/M cell cycle arrest. (A) NIH3T3 cells were transfected with empty vector or Flag-PCAF, then counted, and equal amounts were seeded in triplicates in 6-well-plates. Cell proliferation was measured by counting the number of cells present in each well 24, 48 and 72 h after transfection. Results shown are the mean of three independent experiments ± SE. *P-value <0.05; **P-value <0.01; ***P-value <0.001. A WB showing the levels of transfected PCAF is shown in the top panel, and a WB with anti-actin was performed as a loading control (bottom panel). (B) Similar experiments as in (A) were performed using a NIH3T3 clone expressing CtΔHAT-PCAF under a Tet-off system generated as described in the ‘Materials and Methods’ section. Equal amounts of cells were seeded in triplicates in 6 well plates. Cell proliferation was measured by counting the number of cells present in each well 24, 48 and 72 h after seeding. CtΔHAT expressing cells were cultured in the absence of tetracyclin. As a control, the same clone was cultured in medium supplemented with tetracyclin. Results shown are the mean of three independent experiments ± SE. *P-value <0.05; **P-value <0.01; ***P-value <0.001. A WB showing the levels of CtΔHAT-PCAF is shown in the top panel, and a WB with anti-actin was performed as a loading control (bottom panel). (C) HCT-116 cells were transfected with YFP-PCAF, CFP-cdk2WT or both. At 48 h after transfection they were fixed, DNA was stained with TOPRO-3 and transfected cells were analysed by FACS. The percentage of cells in each phase of the cell cycle is represented in the graph. (D) The same as in (C), but in this case cells were transfected with YFP-PCAF, CFP-cdk2 K33R, or both.

Francesca Mateo, et al. Nucleic Acids Res. 2009 November;37(21):7072-7084.

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