Acetylation by GCN5 regulates CDC6 phosphorylation in the S-phase of the cell cycle Roberta Paolinelli 1,2, Ramiro Mendoza-Maldonado 2, Anna Cereseto 1 and Mauro Giacca 2 1 Molecular Biology Laboratory, Scuola Normale Superiore, AREA della Ricerca del CNR, Via Moruzzi 1, Pisa, Italy and 2 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, Italy
Supplementary Figure 1. CDC6 is acetylated by and binds GCN5 both in vitro and in vivo Mapping of the interacting regions. (a) Flag-tagged CDC6 is acetylated in transfected 293T cells treated with TSA. Upper blot: immunoprecipitation with anti-flag antibody and detection with anti-ac-lys antibody. Lower blot: detection with anti-flag antibody. (b) GST-CDC6 is acetylated by GCN5 acetyltransferase in vitro. HAT assay was performed incubating recombinant GCN5 and GST-CDC6 or GST proteins, as indicated. In addition to CDC6, GCN5 and its major degradation product were also positive for acetylation, due to the autocatalytic activity of the enzyme. (c) Schematic representation of the GST-CDC6 fragments used for the pull down assay shown in (d). (d) GCN5 binds the N-terminal domain of CDC6 in vitro. GST-CDC6 fragments or GST alone as a control were incubated with [ 35 S]-GCN5, extensively washed and then resolved by SDS-PAGE. The panel shows the gel exposed to a phosphoimager. The graphs show the amounts of bound proteins expressed as percentages of radiolabelled input. (e) Schematic representation of the [ 35 S]-CDC6 deletion mutants used for the pull down assay shown in (f).
(f) CDC6 binds the C-terminus of GCN5. GST-GCN5 fragments or GST were incubated with [ 35 S]- CDC6, extensively washed and then analyzed by SDS-PAGE. The panel shows the gel exposed to a phosphoimager. The graphs show the amounts of bound proteins as percentages of radiolabelled input. (g) CDC6 binds GCN5 in vivo. Whole cell lysate (WCL) was subjected to immunoprecipitation with an anti-gcn5 antibody and then analyzed by western blotting using a specific anti-cdc6 antibody. CDC6 was detected together with GCN5 in the bound fraction. (h) Endogenous CDC6 binds overexpressed HA-GCN5 in vivo. Extracts prepared from cells cotransfected with wt HA-GCN5 or HA-GCN5mut were immunoprecipitated with anti-cdc6 or anti-ha antibodies and immunoblotted with anti-ha or anti-cdc6 antibodies, respectively. CDC6 binds both wt GCN5 and its catalytically inactive mutant GCN5mut, which bears two amino acid substitutions (Y260A and F261A) at critical residues within the enzyme's catalytic site, further supporting the notion that the interaction between the two proteins does not involve the HAT domain of GCN5.
Supplementary Figure 2. Quantification of the levels of endogenous acetylated CDC6. To determine what is the fraction of acetylated CDC6 inside the cells, we first determined the sensitivity of the anti-lysine antibody used in this study relative to an antibody detecting both acetylated and nonacetylated (i.e. total) CDC6. Two peptides were synthesized, corresponding to the CDC6 acetylated region, one carrying unmodified lysines and the other acetylated lysines; the former peptide was used to raise a polyclonal antiserum recognizing the corresponding region of CDC6. The two peptides were diluted serially and tested in parallel with the newly developed antibody recognizing total CDC6 or the commercial antibodies against anti-acetylated lysines, by exposing the blots for exactly the same time. The intensity of each dot (measured by a densitometer) was plotted against the amount of input peptide and the statistical correlation between the two value sets was determined (correlation coefficients R 2 : 0.976 and 0.972 for the anti-total CDC6 and anti-acetylated lysine antibody, respectively). On the basis of the mathematical equations fitting the experimental points, it was concluded that the anti-acetylated lysine antibody was 1.43 times more efficient than the anti-total CDC6 antibody at detecting the peptide. Of note, the anti-acetylated lysine antibody did not react at all with the non-acetylated peptide. (Next, a whole cell lysate (WCE) of T98G cells was obtained, CDC6 was immunoprecipitated with an anti-total CDC6 antibody, and the immunoprecipitate was divided into two equal fractions, which were then used for western blotting experiments using the anti-total CDC6 antibody or the anti-acetylated lysine antibody. The ratio between the intensity of the two bands, determined densitometrically, after correction for the relative sensitivity of the antibodies, provides a quantitative estimate of the amount of acetylated CDC6 inside the cells (160.5/42.4*1.43=5.43). Thus, it can be concluded that ~18% of CDC6 is found acetylated in the cells. In evaluating this result, one should however take into account that the actual levels of acetylated CDC6 are expected to be strictly correlated with the phase of the cell cycle. (a) Peptides corresponding to the CDC6 acetylated region. ac: acetylated residues in peptide Ac-Pep. (b) Dot immunoblot on serially diluted Pep and ac-pep peptides using a polyclonal antiserum raised against peptide Pep (upper part) or an anti-acetylated lysine (Ac-Lys) antibody. (c) Quantification of the dot immunoblot experiment shown in (b). The equation of the lines fitting the experimental points are shown, along with the correlation coefficients. (d) Western blot experiment on whole cell lysates (WCE) of T98G cells after immunoprecipitation (IP) with the anti-cdc6 antibody and immunodetection with the same antibody (gel on the left side) or with the anti-ac-lys antibody (gel on the right side). IgH: immunoglobulin heavy chain; Ac-BSA: acetylated bovine serum albumin, used as control acetylated protein. Below the anti-cdc6 lanes, the quantification of the CDC6 band is reported (a.u.: arbitrary units).
(e) Expression of wt GCN5, but not of its catalytically inactive point mutant, promotes acetylation of endogenous CDC6. Immunoblots in the upper panel show the acetylation of endogenous CDC6 after immunoprecipitation of lysates from TSA-treated or GCN5/GCN5mut-transfected HeLa cells with an anti-cdc6 antibody and subsequent detection with anti-ac-lys or anti-cdc6 antibodies. Immunoblots in the lower panel show both HA-tagged GCN5 and α Tubulin levels in cell lysates (WCL).
Supplementary Figure 3. Anti-phospho-S106 antibody recognizes its phosphorylated epitope also in the context of the K3R mutation. Four GST-fusion proteins were obtained (GST alone, GST-CDC6, GST-CDC6(K3R) and GST- CDC6(111-561), the last carrying a deletion removing the N-terminal region up to aa 111. The four proteins were incubated with the immunoprecipitate obtained by incubating extracts from S-phase T98G cells (synchronization by serum starvation, 20 h after serum addition) with an antibody against Cyclin A. In this way, we wanted to force the in vitro phosphorylation of CDC6 and its mutants by the CDKs associating with Cyclin A. The proteins were then resolved by SDS-PAGE and immunoblotted with antibodies recognizing total CDC6 or specific for CDC6 phospho-s54 or CDC6 phospho-s106, as indicated. The results of this experiment show that both anti-phospho antibodies recognize the wt CDC6 and the K3R mutant, but not the mutant deleted in aa 1 110. Thus, the anti-phospho-s106 antibody is indeed able to recognize its phosphorylated epitope also in the context of the K3R mutation. The fact that S106 phosphorylation is lower in the K3R mutant compared to the wt protein might are most likely related to the fact that these recombinant proteins are not acetylated: while we force phosphorylation by this in vitro assay, we assume that this modification occurs less efficiently if CDC6 is not acetylated. In contrast, the K3R mutant is phosphorylated equally well on S54 and on the wt protein. In evaluating this experiment, it should be noted that the anti-cdc6 phospho-s106 antibody is less active than the anti-cdc6 phospho-s54 antibody when used in straight immunoblotting experiments.
Supplementary Figure 4. Levels of phosphorylation of CDC6 and other Cyclin A-CDK2 substrates upon downregulation of GCN5 or overexpression of Cyclin E or Cyclin A. (a) Western blot analysis of the levels of the indicated proteins in cells treated with anti-gcn5 (si- GCN5) or anti-luciferase (si-luc) sirnas. Down-regulation of GCN5 caused an accumulation of total CDC6 and S54-phophorylated-CDC6 together with a decrease of S106-phoshorylated-CDC6, while it left the levels of Rb phosphorylated on S807 and S811 (which are targets of CDK4/Cyclin D1) and on T821 (target of CDK2/Cyclin A) unaffected. (b) Overexpression of Cyclin A, but not that of Cyclin E, specifically increased phosphorylation of CDC6 on serine 106. The immunoblots show the levels of the indicated proteins in lysates from cells transfected with Cyclin A or Cyclin E.
Supplementary Figure 5. Interaction between GCN5 and Cyclin A-CDK2 (a) Cyclin A-CDK2 co-immunoprecipitates with CDC6 and GCN5 in early S phase. Immunoblots for the investigated proteins after immunoprecipitation with the specific anti-cyclin A antibody are shown at the indicated time points after serum addition. M: marker lane (b) Anti-GCN5 antibody immunoprecipitates CDK2 from lysates of T98G cells collected at 20 hours after serum addition.
Supplementary Figure 6. Ser106 CDC6 phosphorylation preferentially occurs on acetylated CDC6. (a) Phosphorylation of CDC6 on Ser106 preferentially occurs on acetylated CDC6. The immunoblots on the left side show the levels of endogenous acetylated CDC6 or total endogenous CDC6 after immunoprecipitation with antibodies against either total CDC6, or ps106-cdc6. The graphs on the right side shows the ratio acetylated CDC6/total CDC6 for the two immunoprecipitates. The levels of acetylated CDC6 were remarkably enriched in the phospho-s106 immunoprecipitate (ratio acetylated CDC6:total immunoprecipitated CDC6=0.45 using the anti-cdc6 antibody; =1.90 using the antiphospho-s106 antibody). IP Control: immunoprecipitation with an irrelevant antibody. Ac-BSA: acetylated BSA as a blotting control. (b) Selective enrichment of acetylated CDC6 after immunoprecipitation with anti-ps106-cdc6 antibody. The immunoblots on the left side show the levels of acetylated (upper) and total (lower) CDC6 after immunoprecipitation with phospho-specific antibodies in lysates from cells treated or not treated with TSA. The lower panel shows the total CDC6 protein level detected on the same lysates. Immunoprecipitation with an unrelated antibody was used as a control. The graph on the right side shows the ratio acetylated CDC6/total CDC6 after immunoprecipitation with the two phospho-specific antibodies. The anti-phospho-s106 antibody immunoprecipitated a remarkably higher amount of acetylated CDC6, compared to the anti-phospho-s54 antibody, even though the levels of total CDC6 immunoprecipitated by the anti-phospho-s106antibody were lower. Upon quantification, the ratio between acetylated CDC6 and total immunoprecipitated CDC6 was 2.10 for the anti-phospho-s106 antibody (similar to the experiments shown in panel a) and 0.35 for the anti-phospho-s54 antibody. The only detectable effect of TSA was to slightly increase the levels of total CDC6 in the cells.
Supplementary Figure 7. GCN5 overexpression drives cells into the S phase. (a) Overexpression of GCN5 increases the levels of endogenous Cyclin A. Extracts from HeLa cells transfected with HA-GCN5 and Cyclin A, as indicated, were immunoblotted with anti-cyclin A, anti- HA or anti-α-tubulin antibodies. (b) Flow cytometry profiles after transfection. The histogram on the right side shows the distribution of the cells in the different phases of the cell cycle; the increase and in the number of S-phase cells after GCN5 or Cyclin A overexpression is indicated by an arrow.
Supplementary Figure 8. Nuclear localization of CDC6(K3R) and CDC6(S106A) mutants is not modified by GCN5 overexpression. The picture shows HeLa cells transfected with HA-GCN5 together with Flag-tagged wt CDC6 or the two CDC6 mutants, followed by staining with anti-flag (green) and anti-ha (red) antibodies. The graphs show the percentage of nuclear (N) and cytoplasmic (C) subcellular distribution of HA-positive cells in wt CDC6, CDC6(K3R) and CDC6(S106A) transfected cells (mean±sem of at least three independent experiments).
Supplementary Figure 9. Phospho-Ser106 CDC6 is highly enriched in the cytoplasmic compartment. The picture shows immunoblots of cytoplasmic (Cyt), nucleoplasmic (Sol) and insoluble (Ins) fractions prepared from asynchronous HeLa cells. Endogenous proteins were revealed with specific antibodies, as indicated on the left side.