Inactivation of Rho GTPases with Clostridium difficile toxin B impairs centrosomal activation of Aurora-A in G2/M transition of HeLa cells

During G2 phase of cell cycle, centrosomes function as a scaffold for activation of mitotic kinases. Aurora-A is first activated at late G2 phase at the centrosome, facilitates centrosome maturation, and induces activation of cyclin B-Cdk1 at the centrosome for mitotic entry. Although several molecules including HEF1 and PAK are implicated in centrosomal activation of Aurora-A, signaling pathways leading to Aurora-A activation at the centrosome, and hence mitotic commitment in vertebrate cells remains largely unknown. Here, we have used Clostridium difficile toxin B and examined the role of Rho GTPases in G2/M transition of HeLa cells. Inactivation of Rho GTPases by the toxin B treatment delayed by 2 h histone H3 phosphorylation, Cdk1/cyclin B activation, and Aurora-A activation. Furthermore, PAK activation at the centrosome that was already present before the toxin addition was significantly attenuated for 2 h by the addition of toxin B, and HEF1 accumulation at the centrosome that occurred in late G2 phase was also delayed. These results suggest that Rho GTPases function in G2/M transition of mammalian cells by mediating multiple signaling pathways converging to centrosomal activation of Aurora-A.     

Cell cycle differences in DNA damage-induced BRCA1 phosphorylation affect its subcellular localization

Phosphorylation of BRCA1 tumor suppressor protein is regulated during the cell cycle and in response to DNA damage. Several Ser/Thr kinases have been implicated in BRCA1 phosphorylation, including ATM/ATR, cdk2, and hChk2 kinases. In this study, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were employed to study BRCA1 phosphorylation during the S and G2/M phases under conditions of DNA damage. We observed that IR (ionizing radiation) treatment induced phosphorylation of Ser-988/Ser-1524 during the S phase and of Ser-988/Ser-1423 during the G2/M phase. UV treatment induced phosphorylation of Ser-988 during the S phase and of Ser-1423 during the G2/M phase. Phosphorylation of serines 1423 and -1524 was not induced in HCC1937 breast cancer cells, which contain mutant BRCA1 protein. Confocal microscopy revealed that unphosphorylated BRCA1 localizes on chromosomes from metaphase through telophase, whereas Ser-988-phosphorylated BRCA1 resides in the inner chromosomal structure, centrosome, and the cleavage furrow during prophase through telophase. We also found that Ser-988-phosphorylated BRCA1 relocalizes to the perinuclear region when cells are subjected to IR or UV radiation in the S phase. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.     

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.     

CDC25B Phosphorylation by Aurora A Occurs at the G2/M Transition and is Inhibited by DNA Damage

CDC25B is one of the three human dual-specificity phosphatases involved in the activation ofcyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsiblefor the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-Akinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/Mtransition. Here we have investigated this phosphorylation event during the cell cycle, and inresponse to activation of the G2 DNA damage checkpoint. We show that accumulation of theS353-phosphorylated form of CDC25B at the centrosome correlates with the relocalisation ofcyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation ofthe G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated andconsequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-Aresults in a bypass of the checkpoint that partially overcome by a S353A mutant of CDC25B.Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These resultsstrongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is animportant step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclinB1 activation.     

Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.     

Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes [corrected] of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.     

Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.     

Phosphorylation of maskin by Aurora-A participates in the control of sequential protein synthesis during Xenopus laevis oocyte maturation

At the end of oogenesis, Xenopus laevis stage VI oocytes are arrested at the G2/M transition (prophase) waiting for progesterone to release the block and begin maturation. Progesterone triggers a cascade of phosphorylation events such as a decrease of pK(a) and an increase of maturating-promoting factor activity. Progression through meiosis was controlled by the sequential synthesis of several proteins. For instance, the MAPK kinase kinase c-Mos is the very first protein to be produced, whereas cyclin B1 appears only after meiosis I. After the meiotic cycles, the oocyte arrests at metaphase of meiosis II with an elevated c-Mos kinase activity (cytostatic factor). By using a two-hybrid screen, we have identified maskin, a protein involved in the control of mRNA sequential translation, as a binding partner of Aurora-A, a protein kinase necessary for oocyte maturation. Here we showed that, in vitro, Aurora-A directly binds to maskin and that both proteins can be co-immunoprecipitated from oocyte extracts, suggesting that they do associate in vivo. We also demonstrated that Aurora-A phosphorylates maskin on a Ser residue conserved in transforming acidic coiled coil proteins from Drosophila to human. When the phosphorylation of this Ser was inhibited in vivo by microinjection of synthetic peptides that mimic the maskin-phosphorylated sequence, we observed a premature maturation. Under these conditions, proteins such as cyclin B1 and Cdc6, which are normally detected only in meiosis II, were massively produced in meiosis I before the occurrence of the nuclear envelope breakdown. This result strongly suggests that phosphorylation of maskin by Aurora-A prevents meiosis II proteins from being produced during meiosis I.     

CP110, a cell cycle-dependent CDK substrate,regulates centrosome duplication in human cells

Centrosome duplication and separation are linked inextricably to certain cell cycle events, in particular activation of cyclin-dependent kinases (CDKs). However, relatively few CDK targets driving these events have been uncovered. Here, we have performed a screen for CDK substrates and have isolated a target, CP110, which is phosphorylated by CDKs in vitro and in vivo. Human CP110 localizes to centrosomes. Its expression is strongly induced at the G1-to-S phase transition, coincident with the initiation of centrosome duplication. RNAi-mediated depletion of CP110 indicates that this protein plays an essential role in centrosome duplication. Long-term disruption of CP110 phosphorylation leads to unscheduled centrosome separation and overt polyploidy. Our data suggest that CP110 is a physiological centrosomal CDK target that promotes centrosome duplication, and its deregulation may contribute to genomic instability.     

Two different protein kinases act on a different time schedule as glial filament kinases during mitosis.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non-phosphorylated-epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2-M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.     

Aurora-A controls cancer cell radio- and chemoresistance via ATM/Chk2-mediated DNA repair networks

High expression of Aurora kinase A (Aurora-A) has been found to confer cancer cell radio- and chemoresistance, however, the underlying mechanism is unclear. In this study, by using Aurora-A cDNA/shRNA or the specific inhibitor VX680, we show that Aurora-A upregulates cell proliferation, cell cycle progression, and anchorage-independent growth to enhance cell resistance to cisplatin and X-ray irradiation through dysregulation of DNA damage repair networks. Mechanistic studies showed that Aurora-A promoted the expression of ATM/Chk2, but suppressed the expression of BRCA1/2, ATR/Chk1, p53, pp53 (Ser15), H2AX, γH2AX (Ser319), and RAD51. Aurora-A inhibited the focus formation of γH2AX in response to ionizing irradiation. Treatment of cells overexpressing Aurora-A and ATM/Chk2 with the ATM specific inhibitor KU-55933 increased the cell sensitivity to cisplatin and irradiation through increasing the phosphorylation of p53 at Ser15 and inhibiting the expression of Chk2, γH2AX (Ser319), and RAD51. Further study revealed that BRCA1/2 counteracted the function of Aurora-A to suppress the expression of ATM/Chk2, but to activate the expression of ATR/Chk1, pp53, γH2AX, and RAD51, leading to the enhanced cell sensitivity to irradiation and cisplatin, which was also supported by the results from animal assays. Thus, our data provide strong evidences that Aurora-A and BRCA1/2 inversely control the sensitivity of cancer cells to radio- and chemotherapy through the ATM/Chk2-mediated DNA repair networks, indicating that the DNA repair molecules including ATM/Chk2 may be considered for the targeted therapy against cancers with overexpression of Aurora-A.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation

Hec1 (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 mutant changing Ser(201) to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.     

Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser(149), Ser(229), and Ser(321) of Cdc25B, and explored the role of Ser(149) in G(2)/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr(15), resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser(149) was phosphorylated at G(1) and S phases, whereas dephosphorylated at G(2) and M phases, and the phosphorylation of Cdc25B-Ser(149) was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G(1) and S phases and translocated to the nucleus at the G(2) phase. Collectively, our findings provide evidence that Ser(149) may be another potential PKA phosphorylation target of Cdc25B in G(2)/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.     

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.     

ATM activation by ionizing radiation requires BRCA1-associated BAAT1

ATM (ataxia telangiectasia mutated) is required for the early response to DNA-damaging agents such as ionizing radiation (IR) that induce DNA double-strand breaks. Cells deficient in ATM are extremely sensitive to IR. It has been shown that IR induces immediate phosphorylation of ATM at Ser(1981), leading to catalytic activation of the protein. We recently isolated a novel BRCA1-associated protein, BAAT1 (BRCA1-associated protein required for ATM activation-1), by yeast two-hybrid screening and found that BAAT1 also binds to ATM, localizes to double-strand breaks, and is required for Ser(1981) phosphorylation of ATM. Small interfering RNA-mediated stable or transient reduction of BAAT1 resulted in decreased phosphorylation of both ATM at Ser(1981) and CHK2 at Thr(68). Treatment of BAAT1-depleted cells with okadaic acid greatly restored phosphorylation of ATM at Ser(1981), suggesting that BAAT1 is involved in the regulation of ATM phosphatase. Protein phosphatase 2A-mediated dephosphorylation of ATM was partially blocked by purified BAAT1 in vitro. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.     

BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M

Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G(2)/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G(2)/M phase of the cell cycle. G(2)/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G(2)/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G(2)/M.     

M phase-specific phosphorylation of BRCA2 by Polo-like kinase 1 correlates with the dissociation of the BRCA2-P/CAF complex

BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. We found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain "phosphorylation consensus motifs" for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulated the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolished Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression.