KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification

Marjorie Fournier, Meritxell Orpinell, Cédric Grauffel, Elisabeth Scheer, Jean-Marie Garnier, Tao Ye, Virginie Chavant, Mathilde Joint, Fumiko Esashi, Annick Dejaegere, Pierre Gönczy, László Tora, Marjorie Fournier, Meritxell Orpinell, Cédric Grauffel, Elisabeth Scheer, Jean-Marie Garnier, Tao Ye, Virginie Chavant, Mathilde Joint, Fumiko Esashi, Annick Dejaegere, Pierre Gönczy, László Tora

Abstract

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.

Figures

Figure 1. Identification of the KAT2A- and…
Figure 1. Identification of the KAT2A- and KAT2B-dependent acetylome.
(a) KAT2A/2B knockdown efficiency. Tetracycline (Tet) inducible stable HeLa cell lines were used in which shRNAs either not targeting any endogenous transcript (Tet-shCtrl) or targeting KAT2A (Tet-shKAT2A) were expressed under doxycycline (Dox) induction. In the Tet-shKAT2A cell line, a siRNA against KAT2B (K2B) was also transfected. The knockdown efficiency of KAT2A, KAT2B and the acetylation of histone H3K9 was tested by western blot analyses. (b) In all, 398 potential KAT2A/2B acetylated protein targets were identified as being present in control samples (3/5), but absent in all KAT2A/2B KD-depleted cell lysates. (c) Analysis of the frequency of amino acids surrounding the acetylated lysines targeted by KAT2A and KAT2B, as compared with that of amino acids surrounding non-acetylated lysines. In all, 1,569 distinct ‘Ks' from the list of 398 proteins were used in this analysis, with 10 aa upstream (−10 on the x axis) and 10 aa downstream (+10 on the x axis). The K itself (at position 0) is not shown in these logos. A random selection of 5,243 sequences (K in the middle, 21 aa length, 0.05% of the total K) from the total human proteins was used for the comparison. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. Note that the y axes on the upper and lower graphs are different. (d) Gene ontology (GO) term enrichment analysis for biological processes using Manteia of the 398 identified proteins. Pathways at a false discovery rate below 1% are represented. The x axis represents the reverse P value obtained after GO term enrichment analysis in Manteia. Txn,transcription. (e) Examples of proteins acetylated by KAT2A/2B belonging to the same biological pathways targeted by KAT2A/2B (see GO term analysis in d).
Figure 2. KAT2A and KAT2B acetylate the…
Figure 2. KAT2A and KAT2B acetylate the kinase domain of PLK4.
(a) Alignment of the first 55 amino acids of human PLK4 with the corresponding sequences from several other species. Danre, Danio rerio; Drome, Drosophila melanogaster; Human, H. sapiens; Mouse, Mus musculus. The well-conserved acetylated K45 and K46 residues are highlighted with red dots. (b) In vitro AT assay on recombinant PLK4. Molecular weight markers are indicated on the left in kDa. (c) PLK4 kinase domain (KinDo) was acetylated by KAT2A, KAT2Amut or KAT2B, and the acetylation sites determined after LC–MS/MS analysis. Acetylation abundance factor (see Methods) values are depicted using the indicated colour code. (d) HEK293 cells were transfected with expression vectors coding for only the Flag tag, Flag-tagged KAT2A or Flag-KAT2B. Western blots were probed with the indicated antibodies. (e) PLK4ac (green) and Centrin-2 localization (red) were probed by immunofluorescence. Scale bars represent 5 μm for the high-magnification view, 1 μm for the inset.
Figure 3. KAT2A/2B and PLK4 co-localize at…
Figure 3. KAT2A/2B and PLK4 co-localize at centrosomes.
(a) The co-localization of KAT2A (red) or KAT2B (green) with several centriolar proteins, HsSAS-6, Centrin-2, CP110 and PLK4ac was tested by immunofluorescence in U2OS cells. Scale bars represent 5 μm, and insets are high-magnification views of the regions indicated in the low-magnification images. (b) PLK4 and KAT2A localization probed by immunofluorescence at different cell cycle stages following synchronization. U2OS cells were tested by immunofluorescence with antibodies against the kinase domain of PLK4 or KAT2A. (c,d) The histograms report the average frequency of cells displaying detectable PLK4 and KAT2A (c), or PLK4ac and KAT2A (d) Signal at centrosomes in different cell cycle stages. Data were collected from three experiments. n>100 cells per cell cycle stage. Error bars: s.e.m. (e) Cell cycle-staged HeLa cell extracts were prepared. The cell cycle distribution was verified by western blot. (f) Using these cell cycle-staged protein extracts IP experiments were carried out with either anti-KAT2A antibodies (KAT2A IP) or control antibodies (mock IP). The presence of KAT2A and PLK4 in the IPs was tested by western blot analysis (WB). M, molecular weight marker in kDa.
Figure 4. KAT2A and the ATAC complex…
Figure 4. KAT2A and the ATAC complex regulate centrosome duplication.
(a) U2OS cells were transfected with vectors either to overexpress (OE) or to knockdown the indicated proteins using shRNA-mediated depletion. Centrosomes were visualized by immunofluorescence. Cen-2, Centrin-2; γ-Tub, γ-tubulin. Note that all cells were analysed, without knowledge of their transfection status thus, the values reported here are probably underestimates. (b) The histograms report the average frequency of interphase cells displaying aberrant Centrin-2 (>4) and γ-tubulin (>2) numbers. Data from three experiments, n=50 cells per condition. Error bars: s.e.m.
Figure 5. Molecular modelling of PLK4 kinase…
Figure 5. Molecular modelling of PLK4 kinase domain suggests that acetylation shifts the dynamics of the acetylated PLK4 to the inactive conformation.
(a) Active (PDB ID 3COK) and (b) inactive (PDB ID 4JXF) conformations of the kinase domain (KinDo) of PLK4 are shown in the middle of the panels. The region where the structure was reconstructed is shown in grey. In both conformations, zoomed-in views have been generated to highlight the changes caused by K45/K46 acetylation. Zooms on the left show the non-modified WT KinDo structures, zooms on the right the KinDO structures with K45/K46 acetylations (ac). The zooms are rotated with the indicated angles when compared with the structures in the middle. On the right, the atoms of the backbone are included. Dotted lines are plotted from nitrogen atoms to oxygen atoms. The left side zooms give an overview of the most stable H-bonds of the WT KinDo, while the right side zoom only shows the H-bonds that differ in the acetylated forms. The thicker lines in a show the local H-bonds that are lost on acetylation: K45-E80 (red) and K46-D44 (yellow), whereas weakened H-bonds are shown in green. If an H-bond is not reported from left to right, its stability does not vary. In b, a distant H-bond newly formed in the acetylated structure (D154-L157) is highlighted in blue. The ATP and residues of the DFG motif are shown in sticks, whereas the αC helix and other structural motifs are in ribbons. (c–f) Ramachandran representation of the backbone dihedral angles of D154 (of the DFG motif), in the WT (c,e) and acetylated (d,f) forms of the active and inactive conformations. (d) In the active acetylated form, D154 explores conformations characteristic of the inactive structure, whereas this is not observed for the WT (c).
Figure 6. Acetylation of PLK4 kinase domain…
Figure 6. Acetylation of PLK4 kinase domain by KAT2A/2B inhibits its kinase activity.
(a,b) Kinase activity of the PLK4 kinase domain (KinDo) in the presence of 32Pγ-ATP after being acetylated by Flag-KAT2A (a) or Flag-KAT2B (b). KinDo acetylation was tested by western blot analyses (WB). Kinase activity was measured by the autophosphorylation reaction (32P) of the KinDo. M, molecular weight marker in kDa. (c,d) Kinase activity normalized to protein amount was measured in three independent experiments. Mean values from the three replicate experiments and the corresponding s.d.'s are shown. (e) Kinase activity of the KinDo was tested on His-CPB of PLK4. KinDo was first acetylated by Flag-KAT2A, Flag-KAT2Amut, Flag-KAT2B or Flag-KAT2Bmut and then 32Pγ-ATP was added to the reactions. The level of acetylated KinDo was tested by WB. Kinase activity was measured by phosphorylation (32P) of His-CPB by the KinDo. M, molecular weight marker in kDa. (f) Kinase activity of the recombinant purified WT or mutated (K45R/K46R) KinDos. Protein levels and autophosphorylation activities were analysed as in a,b. (e) The kinase activities of WT and mutated K45R/K46R KinDos were normalized and measured from three independent experiments as in c,d. In g, kinase activity of the K45/K46R KinDo was calculated relative to that of the WT KinDo, which was normalized to 100% in all replicates. In a,b,e,f, the reaction mixes were analysed by Coomassie blue (CB) staining. In a,b,e,f, a dotted line has been inserted to indicate where unnecessary lanes were cut out from the gels.
Figure 7. KAT2A/2B-mediated acetylation of PLK4 prevents…
Figure 7. KAT2A/2B-mediated acetylation of PLK4 prevents abnormal centrosome amplification in human cells.
(a) Distribution of PLK4 phosphorylated at Ser305 (pSer305) upon KAT2A/2B knockdown. U2OS cells were transfected with control siRNAs (control), or siRNAs targeting PLK4, KAT2A or KAT2B to knock down (KD) these factors. Cells were stained with anti-PLK4-pS305 antibodies (green) and γ-tubulin (red). Scale bar represents 5 μm, and insets are high-magnification views of the regions indicated in the low-magnification images. (b) Quantification of total centrosomal pS305 signal in cells of the indicated conditions (reflected in the images shown in a). n=10 cells for each condition. Student's two-tailed paired T-test (comparing with WT): KAT2A depletion <0.001; KAT2B depletion 0.038. Note that we found no statistically significant increase on KAT2A or KAT2B depletion if each focus of PLK4-pS305 was considered instead of the total centrosomal signal per cell. (c) U2OS cells were transfected with control siRNAs (upper row), or siRNAs targeting the 3′-untranslated region (UTR) of PLK4 (PLK4 3′-UTR siRNA, lower row). Twenty-four hours later, cells were transfected again with control-empty vectors, a vector encoding the full-length PLK4-GFP, or a vector encoding the full-length PLK4-K45R/K46R-GFP double point mutant. Cells were stained for GFP (green) and Centrin-2 (Cen-2). (d) The histogram reports the average frequency of mitotic cells displaying >4 centrioles in the siRNA control condition. (e) The histogram reports the average frequency of mitotic cells displaying <3 centrioles in the siPLK4 3'-UTR condition. Data from three experiments, n=50 cells per condition. Error bars: s.e.m.

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