Figure 1.
Post-translational modification of AIB1 occurs at the entry to mitosis.
(A) Western blot analysis of HeLa cells arrested at the different stages of the cell-cycle as indicated in the scheme. Anti-AIB1 antibodies reveal an electrophoretic mobility shift in mitotic cells (lane 6). Antibodies against indicated cyclins were used to confirm cell-cycle progression through the different stages. β-actin was used as loading control. Chemically-induced cell cycle arrest was produced using the following drugs at the doses and times described in materials and methods: lane 1: cyclosporin A; lane 2: wortmannin; lane 3: L-mimosine; lane 4: hydroxyurea; lane 5: etoposide, and lane 6: nocodazole. (B) Morphology of asynchronous culture of HeLa cells and cells treated with nocodazole to arrest them at the beginning of mitosis. Rounded up cells (light refracting) are cells that have entered mitosis. The increased number of rounded uping cells under nocodazole treatment visually confirms the enriched mitotic population. (C) Western blot analysis of asynchronous growing cells (A), nocodazole-treated cells and mitotic cells (M) isolated from an asynchronous culture by shake-off. Nocodazole-treated cells were divided into rounded up cells (R.up) and those that still remained attached (Adh.) to the surface of the culture dish. (D) Flow cytometry analysis was used to confirm the DNA content of cells treated as indicated in (C).
Figure 2.
Post-translational modification of AIB1 at mitosis does not correlate with ubiquitination.
(A) HeLa cells constitutively expressing 6xhistidine tagged-ubiquitin were grown asynchronously or arrested at mitosis with nocodazole and cell lysates were either directly analyzed (total extract) or subjected to purification through a Ni2+-agarose column and further analyzed by western blotting. Arrow indicates the AIB1 mitotic-specific band. (B) MCF-7 cell extracts (30 µg, 1∶100) from arrested cells with nocodazole were subjected to enzymatic assay with the 300 ng of deubiquitinating enzyme GST-USP2 in 50 µl deubiquitylation buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT). Samples were analyzed by western blot with anti-mono, poly-ubiquitin antibodies and anti-AIB1 antibodies. The altered electrophoretic mobility of the AIB1 band is indicated with an arrow, demonstrating insensitivity to USP2 activity. (C) HeLa cells were grown asynchronously, or treated with the proteasome inhibitor MG132, or with etoposide to arrest them at G2 or with nocodazole to arrest them at mitosis (M). Cell lysates were immunoprecipitated with AIB1 antibodies and immunocomplexes were analyzed by western blotting with anti-mono, poly-ubiquitin antibodies and anti-AIB1 antibodies. The arrow indicates the mitosis-specific AIB1 band that is not detected with ubiquitin antibodies.
Figure 3.
AIB1 is phosphorylated at entry to M phase by Cdk1/cyclin B.
(A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni2+-NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ-32P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).
Figure 4.
AIB1 is not a substrate of Aurora A, B, Plks or Nek2.
Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors [31], [32]. On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane [33]. Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.
Figure 5.
Desphosphorylation of AIB1 occurs at exit from mitosis.
(A) Rounded up HeLa cells arrested at mitosis with nocodazole were washed with PBS and allowed to progress through mitosis for the times indicated, in the presence or absence of 20 µM MG132 and 10 µg/ml cycloheximide (CHX). Western blot analysis was performed for AIB1 to monitor its phosphorylation status. β-actin were assessed as loading control. (B) Flow cytometry analysis of HeLa cells treated as above. (C) HeLa cells arrested at mitosis were allowed to progress through cell cycle and at 3 hours, the remaining rounded up cells (R.up) were separated from adherent cells (Adh.) and analyzed by western blotting. (D) Rounded up HeLa cells recovered after nocodazole treatment were washed with PBS and allowed to progress through mitosis for the indicated times in the absence or presence of 10 µg/ml cycloheximide (CHX).
Figure 6.
Desphosphorylation of AIB1 occurs at mitosis exit.
(A) Floating MCF7 cells arrested at mitosis with nocodazole were washed and liberated to progress through mitosis for the times indicated, in the presence or absence of 20 µM MG132 and 10 µg/ml cycloheximide (CHX). Western blot analysis was performed for AIB1 to monitor its phosphorylation status. Cyclin B1, protein phosphatase 1 (PP1), and the inhibitory phosphorylation of PP1 (P-PP1) were also assessed. β-actin was used as a loading control. (B) Flow cytometry analysis of MCF-7 cells treated as above was used to confirm cell-cycle progression as determined by DNA content.
Figure 7.
The dephosphorylation of AIB1 at the exit from mitosis is sensitive to okadaic acid and caliculin A inhibitors.
(A) Mitotic HeLa and MCF-7 cells were allowed to progress through mitosis for the indicated times, in the presence or absence of the indicated concentrations of okadaic acid (OA). Cell lysates were analyzed subsequently by western blotting with AIB1 antibodies to detect the mitotic specific band and β-actin as loading control. (B) Western blot analysis of mitotic HeLa cells allowed to progress through mitosis for the indicated times, in the presence or absence of 5 µM cyclosporin A. (C) Rounded up HeLa cells arrested in mitosis as in (A) were allowed to progress through cell cycle in the absence (−) or presence (+) of 0.2 µM okadaic acid (OA) or 20 nM caliculin A for 5 hours. Cell lysates were analyzed by western blotting as indicated. (D) Western blot analysis of AIB1 phosphorylation status and other indicated molecular species from nocodazole-arrested HeLa cells further treated (+) with 10 µM purvalanol A, 0.2 µM okadaic acid (OA) or 20 nM caliculin A.
Figure 8.
AIB1 is phosphorylated by Cdk1/cyclin B but not Cdk1/Cyclin A.
(A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ-32P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ-32P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.
Figure 9.
The band-shifted AIB1 is associated with the soluble fraction of cell lysates and is excluded from mitotic chromatin.
(A) Asynchonous culture of MCF-7 cells were used for an immunofluorescence assay with anti-AIB1 antibodies (red) and DAPI (blue) to stain the chromatin. Bottom panels represent merged images. Scale bar: 10 µm. Mitotic cells are indicated with an arrow demonstrate that AIB1 is excluded from chromatin. (B) Confocal microscopy analysis of same cells as in (A) stained with anti-AIB1 (Alexa Fluor 488) and anti-β-tubulin (Alexa Fluor 633) to reveal the mitotic spindle. (C) MCF-7 and HeLa cells were arrested at mitosis with nocodazole and adherent cells were further subjected to cellular fractionation based on the treatment with two subsequent lysis buffers. The first buffer with low ionic strength completely solubilized the phosphorylated AIB1 (upper band, lane S). The second buffer containing high ionic strength extracted only the lower AIB1 band (lane I), together with the nuclear proteins lamin A/C, revealing non cross-contamination with the soluble fraction S.
Figure 10.
Phospho-specific antibodies (pAIB1) confirm that AIB1 is phosphorylated on Ser728 exclusively during mitosis.
(A) Confocal microscopy analysis of an asynchonous culture of MCF-7 cells stained with DAPI (blue), anti-pAIB1(S728) (Alexa Fluor 488) and anti-β-tubulin (Alexa Fluor 633) to reveal the mitotic spindle. Scale bar: 10 µm. Only mitotic cells stain positively with pAIB1(S728) antibodies and suggest that pAIB1(S728) is excluded from chromatin. (B) Western blot analysis of MCF-7 cells arrested at mitosis with nocodazole. Cell lysates were probed with the indicated antibodies, revealing that anti-pAIB1(S728) antibodies detect exclusively the slower electrophoretic mobility band.
Figure 11.
AIB1 phosphorylation at Serine 728 regulates its coactivation capacity.
(A) To measure AIB1-dependent transcriptional activity, COS-1 cells were transfected with 500 ng of reporter gene (5×UAS-luciferase), 20 ng of tk-lacZ and 25 ng of empty vector pCMX-Gal4-DBD (control, C) or 46.5 ng of pCMX-Gal4-DBD-AIB1 wild type or mutant. 30 hours after the transfection cells were arrested at mitosis with nocodazole (150 ng/ml) for 17 hours. Finally, cells were lysed with passive lysis buffer (Promega) and tested for luciferase activity and normalized against β-gal activities in a luminometer (Berthold Detection Systems). Results are presented as relative units to the control cells as mean values ± S.E. (B) To measure AIB1-dependent coactivation, COS-1 cells were transfected with the luciferase reporter ERE2-tk-luciferasa and vectors expressing ERα, AIB1 or the mutated AIB1 (S728A) or (S728E). Cells were arrested at mitosis similarly to (A) and treated with 50 nM 17-β-estraxdiol during the last 5 hours.