Mitosis-specific MPM-2 phosphorylation of DNA topoisomerase IIalpha is regulated directly by protein phosphatase 2A

Abstract

Recent results suggest a role for topoIIalpha (topoisomerase IIalpha) in the fine-tuning of mitotic entry. Mitotic entry is accompanied by the formation of specific phosphoepitopes such as MPM-2 (mitotic protein monoclonal 2) that are believed to control mitotic processes. Surprisingly, the MPM-2 kinase of topoIIalpha was identified as protein kinase CK2, otherwise known as a constitutive interphase kinase. This suggested the existence of alternative pathways for the creation of mitotic phosphoepitopes, different from the classical pathway where the substrate is phosphorylated by a mitotic kinase. In the present paper, we report that topoIIalpha is co-localized with both CK2 and PP2A (protein phosphatase 2A) during interphase. Simultaneous incubation of purified topoIIalpha with CK2 and PP2A had minimal influence on the total phosphorylation levels of topoIIalpha, but resulted in complete disappearance of the MPM-2 phosphoepitope owing to opposite sequence preferences of CK2 and PP2A. Accordingly, short-term exposure of interphase cells to okadaic acid, a selective PP2A inhibitor, was accompanied by the specific appearance of the MPM-2 phosphoepitope on topoIIalpha. During early mitosis, PP2A was translocated from the nucleus, while CK2 remained in the nucleus until pro-metaphase thus permitting the formation of the MPM-2 phosphoepitope. These results underline the importance of protein phosphatases as an alternative way of creating cell-cycle-specific phosphoepitopes.

Figure 1. Interaction between CK2 and topoIIα in interphase cells

(A) Immunofluorescence microscopy reveals partial co-localization (indicated by yellow in panel d) between enhanced GFP–topoIIα (indicated in green in panels a and d) and the catalytic CK2α subunit (indicated in red in panels b and d) during interphase. DNA counterstaining (indicated in blue in panels c and d) shows that the co-localization of the two enzymes occurs preferentially in low-condensed chromatin domains. (B) In vitro phosphorylation assays reveal that mitotic and non-mitotic forms of CK2 are equally able to phosphorylate topoIIα. Left-hand panel, total topoIIα phosphorylation; right-hand panel, MPM-2 phosphorylation of topoIIα. Control lanes 1 show the absence of topoIIα phosphorylation in an immunoprecipitate obtained with a non-immune antibody. Lanes 2 show topoIIα phosphorylation by CK2 immunoprecipitated from nocodazole-blocked HeLa cells (M-phase). Lanes 3 and 4 show topoIIα phosphorylation by CK2 immunoprecipitated from HeLa cells blocked by aphidicolin and released in fresh medium for 1 or 4 h (S-phase) respectively. (C) The MPM-2 epitope on topoIIα (lane 1) disappears in the presence of HeLa cell extracts (lane 2). Incubation with increasing amounts of okadaic acid (10, 20 and 50 nM in lanes 3, 4 and 5 respectively) completely inhibits the MPM-2 phosphatase activity in the cellular extracts, while incubation with increasing amounts of protein inhibitor-2 (50, 100 and 500 nM in lanes 6, 7 and 8 respectively) has no effect. (D) Immunofluorescence microscopy reveals partial co-localization (indicated by yellow in panel d) between enhanced GFP–topoIIα (indicated in green in panels a and d) and the catalytic PP2A subunit (indicated in red in panels b and d) during interphase. DNA counterstaining (indicated in blue in panels c and d) shows that the co-localization of the two enzymes preferentially occurs in low-condensed chromatin domains. (E) HeLa cells were synchronized by double thymidine block. After synchronization, cells were released for 1 h in fresh medium containing 0, 50 or 500 nM okadaic acid (lanes 1, 2 and 3 respectively). The upper panel shows MPM-2-positive topoIIα after specific immunoprecipitation, and the lower panel shows MPM-2-positive proteins in total cell extracts. (F) The relative intensities of MPM-2-positive topoIIα (left-hand panel) and MPM-2-positive proteins in total cell extracts (right-hand panel), obtained as described in (E), were quantified by densitometric analysis. Results are means±S.E.M. for three independent experiments. White columns, cells released in drug-free medium for 1 h; hatched columns, cells released for 1 h in medium with 50 nM okadaic acid; black columns, cells released for 1 h in medium with 500 nM okadaic acid. htopoIIα, human topoIIα.

Figure 2. PP2A shows high specificity towards the MPM-2 phosphoepitope on Ser-1469

(A) Comparison of the ability of PP2A to dephosphorylate ³²P-labelled topoIIα (left-hand panel) and the topoIIα MPM-2 epitope (right-hand panel) reveals a strong preference of PP2A towards the Ser-1469 residue. Left-hand panel, PP2A is able to dephosphorylate topoIIα (lane 1) only in the presence of Mn²⁺ (lane 3), but not in the absence of Mn²⁺ (lane 2). Right-hand panel, PP2A completely dephosphorylates the MPM-2 phosphoepitope (lane 1) even in the absence of Mn²⁺ (lane 2). (B) Comparison of the capacity of PP2A to dephosphorylate ³²P-labelled peptides confirms the strong sequence preference towards Ser-1469 over Ser-1525. The R20A sequence corresponds to the region surrounding the MPM-2 phosphoepitope on Ser-1469, whereas the K15F sequence corresponds to the region surrounding the major phosphorylation site on Ser-1525. (C) CK2-mediated phosphorylation of topoIIα in the absence (lane 1) or presence (lane 2) of PP2A. Left-hand panel, total phosphorylation levels of topoIIα, as revealed by ³²P-labelling, are not affected by the presence of PP2A. Right-hand panel, the formation of the MPM-2 epitope on topoIIα, as revealed by Western blot analysis, is strongly inhibited when PP2A is present. Similar results were obtained in the presence and absence of Mn²⁺. (D) PP2A shows a high preference for Ser-1469 over Ser-1525. Lanes 1, peptide phosphorylation in the absence of PP2A; lanes 2, peptide phosphorylation in the presence of PP2A. CK2 phosphorylation of Ser-1469 on R20A is strongly inhibited in the presence of PP2A (right-hand bar), whereas CK2 phosphorylation of Ser-1525 on K15F is less inhibited by PP2A (left-hand bar) even in the presence of Mn²⁺. hTopoIIα, human topoIIα.

Figure 3. PP2A is excluded from the nucleus in early prophase while CK2 remains nuclear until pro-metaphase

Immunofluorescence reveals that PP2A (left-hand panels, red) disappears quickly from early mitotic nuclei while CK2 (right panels, red) stays associated with a non-chromosomal fraction until complete chromosome condensation and nuclear membrane breakdown. Series 1–4 show a rapid loss of PP2A during early prophase. Series 5–8 show gradual changes of CK2 localization until pro-metaphase. DNA counterstaining (left- and right-hand panels, blue) shows that neither PP2A or CK2 is associated with pre-condensed or condensed chromatin and that CK2 tends to surround the chromosomes.

Figure 4. Localization of CK2 and PP2A in early mitosis

(A) Immunofluorescence reveals co-localization (yellow, panel d) between GFP–topoIIα (green, panels a and d) and the catalytic CK2α subunit (red, panels b and d) in early mitosis (upper panels). Co-localization was observed for the non-chromosomal fraction of GFP–topoIIα as revealed by DNA counterstaining (blue, panel c). In pro-metaphase (lower panels), topoIIα (green, panel e) is fully associated with the condensed chromosomes (blue, panel g), whereas CK2 (red, panel f) shows a diffused pattern surrounding condensed chromosomes. No yellow is seen after superimposition (panel g). (B) Model for the cell-cycle-dependent interaction of CK2 with topoIIα. The CK2-mediated MPM-2 phosphorylation of topoIIα is regulated directly by PP2A during interphase. The translocation of PP2A from the nuclear compartment during early mitosis allows CK2 to phosphorylate the Ser-1469 MPM2 site. Solid lines represent preferential residues targeted by either CK2 or PP2A. Broken lines represent weaker substrates targeted by either CK2 or PP2A.