Fig 1: Acetylation of K100 in p27 induces its degradation via proteasome. (A) HCT116 cells were transfected with GFPp27WT or GFPp27K100R and then made quiescent by serum starvation for 48 h. Then, they were released from quiescence by serum addition. Cell samples were collected at Time 0 and at 10 h after re-entry into cell cycle. The levels of these two forms of p27 were detected by WB using anti-GFP. Cyclin D1 was detected by WB to monitor cell-cycle progression and WB with anti-actin was performed as a loading control. (B) HCT116 cells were transfected with GFPp27WT or GFPp27?91–120 and then made quiescent by serum starvation for 48 h. Then, they were released from quiescence by serum addition. Cell samples were collected at Time 0 and at 10 h after re-entry into cell cycle. The levels of these two forms of p27 were detected by WB using anti-GFP. Cyclin D1 was detected by WB to monitor cell-cycle progression and WB with anti-actin was performed as a loading control. (C) HCT116 cells were transfected with Flag–PCAF or with the empty vector as a control. At 8 h post-transfection, cells were additionally incubated for 16 h in the presence or in the absence of the proteasome inhibitor ALLN. Then, the levels of PCAF and p27 in the total cell extracts were examined by WB. WB with anti-actin was performed as a loading control. (D) HCT116 cells were transfected with Flag–PCAF or with the empty vector as a control. At 8 h post-transfection, cells were additionally incubated for 16 h in the presence or in the absence of the proteasome inhibitor MG132. Then, the levels of PCAF and p27 in the total cell extracts were examined by WB. WB with anti-actin was performed as a loading control. (E) PCAF stimulates the in vitro ubiquitylation of p27. Purified recombinant GSTp27WT or GSTp27-K100R proteins were incubated with radioactive acetyl-CoA and GSTHAT protein for in vitro acetylation reaction in the presence of reticulocyte lysates. After incubation, the proteins from the reticulocyte lysates were eliminated by purifying the GST-containing proteins with a glutathione column. The 50% of the material was analyzed for ubiquitylation by WB with anti-ubiquitin antibody (right panel). The other 50% of the material was used for the analysis of the acetylation of p27 by autoradiography (left panel). (F) The levels of endogenous Skp2 were down-regulated by incubating HCT116 cells with siRNA for Skp2 (siSkp2) during 48 h. Incubation with siRNA for GFP (siGFP) was used as a control. Then, cells were transfected with FlagPCAF expression vector. At 20 h post-transfection, the levels of Flag PCAF, Skp2, p27 and actin were determined in the cell extracts by WB using specific antibodies.
Fig 2: PCAF acetylates p27. (A) In vitro acetylation assays were performed by incubating purified GST–HAT with the indicated concentrations of purified GST–p27 in the presence of radioactive acetyl-CoA. Control assays were performed with GST–p27 in the absence of GST–HAT (C-), with GST–HAT in the absence of any substrate (-) or in the presence of GST. Acetylation was examined by autoradiography (left panel). A WB using anti-p27 is also showed (right panel). (B) Purified GST–HAT was incubated with GST–p27, GST–p21 or GST in the presence of radioactive acetyl-CoA. A similar assay was performed in the absence of any substrate (-). Acetylation was examined by autoradiography (left panel). Red Ponceau stain of the membrane is showed in the right panel. (C) Purified GST–CBP was incubated with GST–p27, GST or histones in the presence of radioactive acetyl-CoA. A similar assay was performed in the absence of any substrate (-). Acetylation was examined by autoradiography (left panel). Red Ponceau stain of the membrane is shown in the right panel. (D) HEK293T cells were cotransfected with PCAF plus p27WT or p27K13R, or transfected with an empty vector (ø). Then, cell extracts were subjected to IP with anti-p27. The levels of total or acetylated p27 were examined by WB with anti-p27 or anti-acetyl-lysine antibodies (Ac-K), respectively (upper left panel). A similar experiment was performed using p21 instead of p27 (upper right panel). Expression levels of p27 and p21 were examined by WB (bottom panels). (E) Cell extracts were subjected to IP with anti-p27 or anti-Flag (used as a control) and then the immunoprecipitates were analyzed by anti-p27 and anti-acetyl-Lysine antibodies (Ac-K).
Fig 3: 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.
Fig 4: 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.
Fig 5: PCAF acetylates Lys100 of p27. (A) Purified GST–HAT was incubated with GST, full-length GST–p27, GST–p27(1–110) or GST–p27(110–198), in the presence of radioactive acetyl-CoA. A similar assay was performed in the absence of any substrate (-). Acetylation was examined by autoradiography (left panel). Amido black stain of the membrane is showed in the right panel. (B) Amino acid sequences of peptides used for the identification of the in vitro acetylation sites of p27 by spot mapping analysis. Eight peptides, each one containing one of the lysines present in the N-moiety of p27, were spotted on a nitrocellulose membrane (peptides 2–9). A peptide from histone H3 was also spotted and used as a positive control (peptide 1). (C) The membrane containing the spotted peptides was subjected to an in vitro acetylation assay using GST–HAT as acetylase. Acetylation was examined by autoradiography. (D) Purified GST–HAT was incubated with GST, GST–p27WT, GST–p27–K96R, GST–p27–K100R, GSTp27–K96R,K100R (GST–p27-2R) or GST–p27–K25R,K96R,K100R (GST–p27–3R) in the presence of radioactive acetyl-CoA. A similar assay was performed in the absence of any substrate (-). Acetylation was examined by autoradiography (left panel). Red Pounceau staining of the membrane is showed in the right panel. (E) HEK293T cells were transfected with an empty vector (ø) or cotransfected with p27WT, p27-K13R, p27-K100R, p27-K3R or p27-K5R plus PCAF. Cell extracts were then subjected to IP with anti-p27 and the total levels of p27 or acetylated p27 were analyzed by WB with anti-p27 or anti acetyl-Lysine (Ac-K) antibodies (left panel). Expression levels of the different forms of p27 were determined by WB with anti-p27 (right panel). (F) Cells were transfected with an empty vector or cotransfected with p27WT or p27(?91–120) plus PCAF. Cell extracts were then subjected to IP with anti-p27 and the levels of p27 or acetylated p27 were analyzed by WB with anti-p27 or anti-acetyl-Lysine (Ac-K) antibodies (left panel). Expression levels of the different forms of p27 were determined by WB with anti-p27 (right panel).
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