BRCA1 downregulates the kinase activity of Polo-like kinase 1 in response to replication stress

Abstract

In response to DNA damage or replication stress, proliferating cells are arrested at different cell cycle stages for DNA repair by downregulating the activity of both the cyclin-dependent kinases (CDKs) and other important cell cycle kinases, including Polo-like kinase 1 (PLK1) . The signaling pathway to inhibit CDKs is relatively well understood, and breast cancer gene 1 (BRCA1) and other DNA damage response (DDR) factors play a key role in this process. However, the DNA damage-induced inhibition of PLK1 is still largely a mystery. Here we show that DNA damage and replication stress stimulate the association between BRCA1 and PLK1. Most importantly, we demonstrate that BRCA1 downregulates the kinase activity of PLK1 by modulating the dynamic interactions of Aurora A, hBora, and PLK1. Together with previous findings, we propose that in response to replication stress and DNA damage, BRCA1 plays a critical role in downregulating the kinase activity of both CDKs and PLK1.

Introduction

When encountering a variety of endogenous and exogenous genotoxic stresses, eukaryotic cells initiate a cascade of DNA damage responses (DDR). These include activating cell cycle checkpoints, modulating transcription, repairing DNA damage, or committing cells to undergo apoptosis.^1-3 Two groups of kinases play critical roles in orchestrating the complex DDR. Group I kinases include three upstream phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs): Ataxia telangiectasia mutated (ATM), ataxia telangiectasia, Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PKcs). Group II kinases include three downstream checkpoint kinases: checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and p38/MK2, all of which are serine/threonine kinases and function to transduce and amplify the DDR signal. In addition to protein kinases, a cohort of DNA damage mediators, including 53BP1, BRCA1, Claspin, MDC1, and TopBP1, function to facilitate the activation of checkpoint kinases by the PIKKs. Based on the temporal sequence, DDR can be sub-divided into three stages: sensing (or initiation), transduction, and maintenance. Because the nature of the DNA damage can be quite different, it is sensed, transduced, and executed by different pathways. For example, DDR can be classified into two major branches: the ATM-Chk2 pathway, which is activated primarily by double-strand breaks (DSBs), and the ATR-Chk1 pathway, which is activated mainly by replication stress. However, there can be extensive cross-talk between the two branches.

Among the many effectors that the six DDR kinases act on, several of them are key kinases that regulate cell cycle transition, including cyclin-dependent kinases (CDKs), Aurora A, and Polo-like kinase 1 (PLK1).^3,4 CDKs are essential for normal cell cycle progression. For example, CDK2/cyclin E and CDK2/cyclin A2 regulate events in S and G₂, while CDK1/cyclin B1 functions primarily in G₂/M transition as well as during mitosis. Although not required for normal cell cycle transition, PLK1 becomes essential for the G₂/M transition in cells attempting to recover from DNA damage.⁵ Aurora A was identified as the upstream activating kinase of PLK1 in late G₂ and during DNA damage recovery.^6,7 Because of their roles in promoting cell cycle transition under normal conditions or DNA damage recovery, it is essential to downregulate the activity of CDKs, PLK1, and Aurora A in cells experiencing DNA damage to maintain the G₂ checkpoint and allow time for accurate DNA repair. For example, during DDR, the activation of both ATM/Chk2 and ATR/Chk1 pathways induces the destruction of the Cdc25 family of phosphatases, one of the most important functions of which is to reverse the inhibitory phosphorylation of CDKs.^3,4,8 In addition, Chk1 stimulates the kinase activity of Wee1, which catalyzes the inhibitory phosphorylation of CDKs.⁹ Consequently, the combination of proteolytic destruction of CDK activating phosphatases (e.g., Cdc25A) and the activation of the inhibitory kinase, Wee1, DDR rapidly and effectively downregulates the kinase activity of CDKs. Similarly, to prevent premature recovery from cell cycle arrest after DNA damage, the activity of both PLK1 and Aurora A are also reduced.^10-12 However, the mechanism by which the activity of PLK1 and Aurora A is downregulated is less well understood.

BRCA1 is a well-recognized tumor suppressor gene and has been linked to both familial and sporadic breast and ovarian cancers.^13-16 BRCA1 functions in a variety of biological processes, including the centrosome cycle, cell-cell interactions, cell death, transcription, ubiquitination, X-chromosome silencing, oxidative stress, and DDR^17-20. We and others have shown that, with regards to its functions in DDR, BRCA1 exists in at least four distinct complexes in mammals: A complex (BRCA1/BARD1-Abraxas), B complex (BRCA1/BARD1-BACH1), C complex (BRCA1/BARD1-CtIP), and the BRCA2-containing P complex (BRCA1/BARD1-PALB2-BRCA2).^21-24 These four distinct BRCA1 complexes are thought to function in either different processes or different steps within the DDR. For example, the BRCA1 A complex is critical during the sensing/initiating stage of DDR. Immediately after the DSBs, RAP80 is recruited to the damage sites by associating with an unknown ubiquitinated factor(s) in the vicinity of the breaks through its two ubiquitin interacting motifs (UIM).^22,25,26 Abraxas then bridges the interaction between RAP80 and BRCA1 and further recruits the BRCA1 A complex to the DSBs.^22,27,28 Subsequently, BRCA1 promotes CDK inhibition by binding and activating Chk1.²⁹ In addition, BRCA1 has a well-established role in regulating the repair of DSB through the error-free homologous recombination (HR) repair pathway^30,31. The HR function of BRCA1 is through its collaboration with CtIP (part of the BRCA1 C complex) during the S and G₂ stages of the cell cycle to promote DNA end resection.^32-34 The HR function of BRCA1 is considered the reason why BRCA1-deficient cells are hypersensitive to DSB.

Here we identified a strong interaction between BRCA1 and PLK1 in response to a variety of DNA damaging agents, including replication stress. Most importantly, we demonstrate that, in response to DNA replication stress, BRCA1 inhibits the kinase activity of PLK1, likely through modulating the dynamic interactions of Aurora A-hBora-PLK1. Together with previous studies, we propose that BRCA1 regulates the checkpoint response by inhibiting both CDKs and PLK1.

Results

Replication stress strongly stimulates the interaction of BRCA1 and PLK1

BRCA1 is a key player during DDR, and one of its critical checkpoint functions is to downregulate the CDK activity.²⁹ However, no functional connection between BRCA1 and PLK1 has been established yet. To investigate the potential biological interplay between BRCA1 and PLK1 during DDR, we first examined whether the two proteins interact with each other. We chose to damage cells with hydroxyurea (HU), because it is a less lethal form of DNA damage and primarily induces reversible replication stress. We treated U2-OS cells with 4 mM HU for 24 h to achieve a tight and complete arrest. We then performed a co-immunoprecipitation (co-IP) assay using an antibody against either BRCA1 or PLK1. HU treatment has minimal effects on the protein level of PLK1 but has a pronounced effect on the mobility of BRCA1 (Fig. 1A–C), which is likely due to multiple phosphorylations on BRCA1.^35,36 As shown in Figure 1A–C, we detected a weak interaction between BRCA1 and PLK1 in non-stressed cells; however, their interaction becomes much stronger after HU treatment. To demonstrate the specificity of this interaction, we first depleted BRCA1 using two different small interfering (siRNA) and then performed IP using antibody against PLK1. As shown in Figure 1D, depletion of BRCA1 by siRNA reduced the band pulling-down with the anti-PLK1 antibody and migrating corresponding to the size of BRCA1, suggesting that pulling down BRCA1 with the anti-PLK1 antibody is very specific.

Figure 1. HU stimulates the interaction between PLK1 and BRCA1. (A–C) U2-OS cells were either left untreated (−) or treated with 4 mM HU for 24 h (+). Equal amount of cell lysate was used for immunoprecipitation (IP). The antibodies used for IP are indicated on the top. PLK1^m is a mouse monoclonal antibody. PLK^R is a rabbit polyclonal antibody. IgG was used as the negative control. Immunoblotting (IB) antibodies are indicated on the right. (D) Depletion of BRCA1 by siRNA abolishes the interaction between PLK1 and BRCA1. U2-OS cells were first transfected with either control siRNA, or two different siRNA against BRCA1 (BRCA1-B or BRCA1-C), and then treated with 4 mM HU for 24 h. Equal amount of cell lysate was used for IP. IB antibodies are indicated on the right. (E) The PLK1-BRCA1 interaction is independent of the kinase activity of PLK1 and Aurora A. U2-OS cells were first treated with 4 mM HU for 24 h. 100 nM BI2536 or 3 μM MLN8054 was added to the medium, and cells were incubated for another 24 h. Equal amount of cell lysate was used for IP. IB antibodies are indicated on the right. (F) GFP-tagged BRCA1 and Myc-tagged hBora were con-transfected into 293T cells. Cell lysates were used for IP with either control IgG or anti-Myc antibody. Antibodies used for immunoblotting are indicated on the right.

Aurora A was recently identified as an upstream kinase that phosphorylates PLK1 in late G₂ and during DNA damage recovery at threonine-210 (T210) within its T-loop.^6,7 PLK1 itself is also a kinase.^37-40 To examine if the kinase activity of either PLK1 or Aurora A is required for the BRCA1-PLK1 interaction, we inhibited their activity using two chemical inhibitors, BI2536 for PLK1,⁴¹ and MLN8054 for Aurora A.⁴² We first treated U2-OS cells with HU for 24 h, then incubated cells with BI2536 or MLN8054 for another 24 h. Cells were collected and an equal amount of cell lysate was used for IP with PLK1 antibody. As seen in Figure 1E, neither the kinase activity of PLK1 nor Aurora A is required for the interaction of BRCA1-PLK1.

Previously, Ouchi and colleagues reported an interaction between BRCA1 and Aurora A, which we validated here as well⁴³ (Fig. S1). Since PLK1 also interacts with hBora, a co-activator of Aurora A during G₂ and DNA damage recovery,^44,45 we then tested whether hBora can also associate with BRCA1. We co-transfected GFP-tagged BRCA1 with Myc-tagged hBora (Myc-hBora) and then performed IP analysis using antibody against Myc. Interestingly, we can also robustly IP GFP-BRCA1 with Myc-hBora (Fig. 1F), suggesting that BRCA1 also interacts with hBora. Consistent with these co-IP analyses, we found that BRCA1, Aurora A, hBora, and PLK1, also exist in overlapping fractionations in a continuous glycerol gradient assay and HU further stimulates their co-fractionation (Fig. S2). Taken together, these data demonstrate that BRCA1 forms complexes with Aurora A, hBora, and PLK1. The interaction between BRCA1 and PLK1 is strongly induced by replication stress, suggesting a potential role of BRCA1 in modulating the activity of PLK1 in response to replication stress.

PLK1 interacts with both the RING domain and the BRCT domain of BRCA1, while hBora binds primarily to the RING domain of BRCA1

To further characterize the BRCA1-PLK1 interaction, we mapped the region in BRCA1 that PLK1 binds. BRCA1 contains three major domains (Fig. 2A): a RING domain at its N-terminus (1–302 aa), a coiled-coil domain (CC, 1393–1476 aa), and two BRCT domains at its C-terminus (1528–1863 aa). The RING domain is commonly found in enzymes that catalyze the conjugation of ubiquitin. The BRCT domain is primarily involved in protein-protein interactions and binds phosphorylated peptides, such as phosphorylated Abraxas, BACH1, and CtIP^19,46. We first co-transfected HA-tagged PLK1 (HA-PLK1) with either GFP-tagged full-length BRCA1 or different BRCA1-truncated variants in 293T cells and then performed IP analysis using an antibody against HA. As seen in Figure 2B, deletion of either the RING domain or the BRCT domains of BRCA1 significantly weakened its interaction with PLK1. Consistently, HA-PLK1 can pull-down the RING or BRCT domain alone (Fig. 2B; Fig. S3A), suggesting that PLK1 binds both the RING domain and the BRCT domains of BRCA1.

Figure 2. PLK1 interacts with both the RING domain and the BRCT domain of BRCA1, while hBora primarily binds the RING domain of BRCA1. (A) Diagram of the domain structure of BRCA1 and the summary of the domain mapping. (B) HA-PLK1 and GFP-tagged full-length BRCA1 or different variants were co-transfected into 293T cells. Cell lysates were used for IP with anti-HA antibody. IB antibodies are indicated on the right. (C) Myc-hBora and GFP-tagged full-length BRCA1 or different variants were co-transfected into 293T cells. Cell lysates were used for IP with anti-Myc antibody. IB antibodies are indicated on the right. WCL, whole-cell lysate.

To map the interaction region between hBora and BRCA1, we co-transfected Myc-hBora with different GFP-tagged BRCA1-truncated variants in 293T cells and performed IP analysis using an antibody against Myc. As seen in Figure 2C, deletion of the RING domain completely abolishes the BRCA1-hBora interaction, while hBora still can bind BRCA1 when the BRCT domain is deleted, suggesting that hBora interacts primarily with the RING domain of BRCA1. Nonetheless, when the BRCT domain alone is overexpressed, we can detect its interaction with hBora, suggesting that hBora binds the BRCT domain either weakly or indirectly through another protein (Fig. S3B).

These domain mapping studies indicate that both the RING domain and the BRCT domain of BRCA1 are required for its association with PLK1, while the RING domain appears to be more important for its association with hBora. Interestingly, Ouchi and colleagues showed previously that Aurora A binds to BRCA1 amino acids 1314–1863.⁴³ Since the truncated BRCA1 used by Ouchi and colleagues contains both the coiled-coil domain and the BRCT domain, which domain of BRCA1 Aurora A specifically binds is still unknown. Nonetheless, our data indicate that BRCA1 is capable of associating with all three proteins involved in regulating PLK1 activity, suggesting that BRCA1 may regulate the activity of PLK1 by modulating the dynamic interactions of the Aurora A-hBora-PLK1 complex.

Replication stress regulates the dynamic interactions of the BRCA1-PLK1-hBora complex and inhibits the kinase activity of PLK1

To further investigate the dynamic interactions of BRCA1, PLK1, and hBora, U2-OS cells were first treated with HU for different amounts of time. Cell lysate was then used for IP analysis with anti-PLK1 antibody. HU treatment has no pronounced effects on the protein level of PLK1 (Fig. 3A and B); however, it dramatically increased the protein level and altered the mobility of hBora (Fig. 3B). A few groups showed that hBora is phosphorylated during G₂ and mitosis, and Cdk1/cyclin B1, GSK3β, PLK1, and Aurora A are the candidate kinases.^44,45,47 To examine whether the mobility change of hBora seen in Figure 3B is due to phosphorylation, we treated cell lysates with λ phosphatase. As shown in Figure 3C, treatment with λ phosphatase clasped the smeared hBora band into one major band, indicating that HU induced mobility shift of hBora is most likely due to phosphorylation. Because the kinase activities of Cdk1/cyclin B1, PLK1, and Aurora A are inhibited by DNA damage, they are less likely to be kinases responsible for hBora phosphorylation. As seen in Figure 3A, we detected an increased interaction of BRCA1-PLK1 as early as 4 h after the addition of HU. This interaction becomes stronger with time, peaks around 36 h and persists to 48 h (Fig. 3B). The dynamic interaction between PLK1 and hBora is also very intriguing. In non-stressed cells, the level of hBora is quite low, and the PLK1-hBora interaction is either weak or transient. In addition, the hBora that associates with PLK1 is mostly a phosphorylated form judging from the mobility shift. HU treatment first weakens the interaction between PLK1 and hBora (Fig. 3A). However, 24 h after HU treatment, the level of hBora starts to increase and the PLK1-hBora interaction becomes stronger again, peaking around 36 h. The peak of increased PLK1-hBora interaction also coincides with the period of the strongest interactions between PLK1-BRCA1, both persisting to at least 48 h (Fig. 3B). These time-course studies demonstrate that replication stress induces dynamic interactions among BRCA1-PLK1-hBora and that PLK1 interacts preferentially with the phosphorylated hBora. When treated with HU, the interaction between PLK1 and hBora is first weakened and later strengthened, suggesting that the phosphorylated hBora under non-stress condition is likely to be different from the phosphorylated hBora 24-h after HU treatment. The former is likely modified by CDKs, PLK1, and Aurora A, while the latter is likely modified by checkpoint kinases such as ATR and Chk1. Furthermore, at the early time points of HU treatment, there is a nice inverse correlation between PLK1-hBora and PLK1-BRCA1 (Fig. 3A), while at later time-points, the interaction of PLK1-BRCA1 tracks well with that of PLK1-hBora (Fig. 3B). These data strongly indicate that HU induces dynamic interactions among BRCA1, PLK1, and hBora.

Figure 3. HU inhibits the kinase activity of PLK1 and induces dynamic interactions of PLK1, hBora, and BRCA1. U2-OS cells were either left untreated (0 h) or treated with 4 mM HU for the indicated time. Equal amount of cell lysate was used for IP with antibody against either PLK1 (A and B) or phosphorylated T-210 of PLK1 (D and E) (pT210-PLK1). IB antibodies are indicated on the right. (C) U2-OS cells were either left untreated (0 h) or treated with 4 mM HU for the indicated time. Equal amount of cell lysate was incubated with either λ phosphatase or phosphatase inhibitor at 30 °C for 30 min. Cell lysates were then run on a SDS-PAGE and immunoblotted with antibody against hBora. WCL, whole-cell lysate.

To examine the changes of PLK1 activity in HU-treated cells, we took advantage of a phospho-specific antibody (pT210 Ab) that recognizes the phosphorylated Threonine-210 (pT210) within the T-loop of PLK1.¹² Phosphorylation of T210 is critical for PLK1 kinase activity both in vitro and in vivo.^48,49 The level of phosphorylation of T210 correlates very well with the activity of PLK1 during G₂, mitosis, and DNA damage recovery.^6,7 Because we were unable to detect the pT210-PLK1 using a whole-cell lysate, we opted to IP with the pT210 Ab first and then perform immunoblotting with an antibody that recognizes total PLK1. Using this strategy, a robust pT210-PLK1 signal was detected in unstressed cells (Fig. 3D and E). HU treatment significantly reduced the level of pT210-PLK1 as early as 4 h after treatment (Fig. 3D). Forty-eight hours after HU treatment, there is very little detectable pT210-PLK1 (Fig. 3E). This result is consistent with previous findings that treatment of cells with either UV (UV) or Adriamycin inhibits the PLK1 activity.^10,12 Intriguingly, neither hBora nor BRCA1 could be co-precipitated using the pT210 Ab (Fig. 5B and data not shown). Though it is possible that the pT210 Ab may not be optimal for IP hBora and BRCA1, it is more likely that the interaction between hBora and pT210-PLK1 is transient, while BRCA1 may only interact with the non-pT210-PLK1, which is the inactive form of PLK1.

Figure 5. Depletion of BRCA1 impairs the inhibition of PLK1 activity in HU treated cells. (A) Depletion of BRCA1 induces hyper-phosphorylation of hBora. U2-OS cells were first transfected with either control siRNA, or one of two different siRNAs against BRCA1 (BRCA1-B or BRCA1-C). Cells were then either left untreated (0 h) or treated with 4 mM HU for the indicated times. Equal amount of cell lysate was then run on a SDS-PAGE and immunoblotted with the antibody indicated on the right. (B) U2-OS cells were first transfected with either control siRNA, or one of two different siRNAs against BRCA1 (BRCA1-B or BRCA1-C). Cells were either left untreated or treated with 4 mM HU for 48 h. Equal amount of cell lysate was used for IP with antibody against pT210-PLK1. IB antibodies are indicated on the right. (C) U2-OS cells were first transfected with either control siRNA, or one of two different siRNAs against BRCA1 (BRCA1-B or BRCA1-C). Cells were then treated with 4 mM HU for 48 h. Equal amount of cell lysate was used for IP with either mouse IgG or an antibody against PLK1 (left panel). The immunoprecipitated PLK1 was then used for kinase assay using GST-PBIPtide as the substrate and in the presence of [γ-³²P]-ATP (right panel). Samples were then run on a 4–12% SDS-PAGE. Phosphorylated GST-PBIPtide was visualized by autoradiography. The amount of PLK1 that had been pulled down was determined by immunoblotting with an antibody against PLK1. The intensity of the ³²P-GST-PBIPtide was quantified on a Typhoon Phosphoimager and ImageQuant software.

Taken together, these interaction studies suggest that, soon after DNA damage, binding of BRCA1 to the Aurora A-hBora-PLK1 complex quickly inhibits the phosphorylation of PLK1 by either actively recruiting a phosphatase, or blocking the access of Aurora A to PLK1, or both. At later time points, BRCA1 either blocks the binding of Aurora A to PLK1, accelerates its dissociation from the BRCA1-PLK1-hBora complex, or both. Meanwhile, because the activity of PLK1 is dramatically reduced in response to HU treatment, hBora is protected from PLK1-induced proteasome-dependent degradation^44,45 and becomes trapped in the BRCA1-PLK1-hBora complex. Since by 36 and 48 h after HU treatment, the activity of PLK1 activity is quite low, PLK1 is unlikely the kinase that phosphorylates hBora. We propose that the function of the BRCA1-PLK1-hBora complex at later time points after HU treatment is to sequester PLK1 and block access of Aurora A, thus preventing the premature activation PLK1.

Replication stress-induced interaction between BRCA1 and PLK1 is regulated by ATR/ATM

Replication stress induces pronounced phosphorylation of BRCA1 (Fig. 1A and B).³⁶ Since ATM and ATR are the major kinases for DDR, we next tested whether the interaction between BRCA1 and PLK1 is regulated by ATM/ATR. U2-OS cells were first treated with HU for 12 h and then with the ATM/ATR inhibitor caffeine for another 12 h. Cells were then collected for IP analysis. As shown in Figure 4A, addition of caffeine significantly decreased the interaction between BRCA1 and PLK1. Intriguingly, addition of caffeine did not affect the interaction between PLK1 and hBora, while slightly enhancing the level of the phosphorylated forms of hBora.

Figure 4. Genotoxic stresses induce strong interaction between PLK1 and BRCA1. (A) Two sets of U2-OS cells were treated with 4 mM HU. Twelve hours later, 5 mM caffeine was added to one set of cells. Twelve hours after this addition, equal amount of cell lysate was used for IP with antibody against PLK1. IB antibodies are indicated on the right. Arrows indicate the IgG heavy chain. (B and C) U2-OS cells were either treated with vehicle (mock), or with 4 mM HU, 3 μM aphidicolin, 50 μM etoposide, 1 μM Daunorubicin, or 5 μM cis-platin (CDDP) for 24 h. Equal amount of cell lysate was used for IP with antibody against either PLK1 (B) or pT210-PLK1 (C). IB antibodies are indicated on the right. WCL, whole-cell lysate.

A variety of DNA damage agents also stimulate the BRCA1-PLK1 interaction and inhibit the kinase activity of PLK1

To examine whether the BRCA1-PLK1 interaction can also be simulated by other DNA damaging agents, we performed similar immunoprecipitation analysis as in Figure 3 using cell lysates from cells treated with 4 other DNA damaging agents for 24 h: aphidicolin, a DNA polymerase inhibitor that also blocks DNA replication; etoposide, a topoisomerase II inhibitor that induces DSBs; daunorubicin, a DNA strand intercalator that inhibits topoisomerase II and induces DSB; and cis-platin (also called cis-diamminedichloroplatinum [II], CDDP), a DNA strand cross-linker. Amazingly, they all induce a strong interaction between BRCA1 and PLK1, with HU being the most efficient (Fig. 4B). Intriguingly, only HU and aphidicolin stabilize hBora, suggesting that the way BRCA1 regulates PLK1 may vary for different genotoxic stresses. In addition, since HU and aphidicolin lead to higher levels of BRCA1 and hBora proteins, the stronger interactions of PLK1-BRCA1 and PLK1-hBora induced by them could be due to the protein increase. Similar to HU, aphidicolin, etoposide, daunorubicin, and CDDP also inhibit the phosphorylation of PLK1, indicating that suppressing the activity of PLK1 is a common response to DNA damages (Fig. 4C).

In response to replication stress, BRCA1 inhibits the kinase activity of PLK1 through regulating the phosphorylation and abundance of hBora

The dynamic interactions of BRCA1, PLK1, and hBora induced by genotoxic stress suggest that BRCA1 may play a critical role in regulating the kinase activity of PLK1. Since previous studies and data shown in Figures 3 and 4 demonstrate that DNA damage inhibits the kinase activity of PLK1,^10,12 the role of BRCA1 is likely to be inhibitory. To test this hypothesis, we first depleted BRCA1 using two different siRNA and then treated cells with HU for 24 or 48 h. Consistent with data shown in Figure 3B, HU treatment leads to an increase in hBora and induces its phosphorylation (Fig. 5A). Intriguingly, depletion of BRCA1 leads to further increases in hBora and stimulates more phosphorylation, as indicated by the increased slower migrating forms of hBora. Most importantly, in BRCA1-depleted cells we are able to pull-down more pT210-PLK1 than control siRNA-treated cells using the pT210 Ab in HU-treated cells but not nontreated cells, suggesting that BRCA1 only inhibits PLK1 activity in response to DNA damage (Fig. 5B). Next, we directly measured the PLK1 activity using an in vitro kinase assay. We previously showed that PLK1 efficiently phosphorylates a centromeric protein, PBIP, at Threonine-78 (T78).⁵⁰ Furthermore, PLK1, but not its two closely related kinases PLK2 and PLK3, phosphorylates GST-PBIPtide, which contains four repeats of the T78 motif.⁵¹ Therefore, we decided to use GST-PBIPtide, an endogenous PLK1 substrate, as the substrate in the kinase assay. U2-OS cells were first transfected with two different siRNAs against BRCA1 and then treated with HU for 48 h. Equal amounts of cell lysate were used for IP using the antibody against total PLK1. The immunoprecipitated PLK1 was then incubated with [γ-³²P]-ATP and GST-PBIPtide in a kinase reaction. The ³²P-labeled GST-PBIPtide was visualized, and the intensity of the band was quantified. IgG was used as the negative control during IP. Consistent with the results using the pT210 Ab (Fig. 5B), cells depleted of BRCA1 show increased PLK1 activity (Fig. 5C). Taken together, our data clearly demonstrate that in BRCA1-deficient cells, PLK1 activity cannot be efficiently inhibited by DNA damage, and this is likely due to the combination of increased level and phosphorylation of hBora (Fig. 5A).

Discussion

Following genotoxic stress, kinases involved in regulating cell cycle-related events, including CDKs, Aurora A, and PLK1 are all downregulated to arrest cells at different stages of the cell cycle for DNA repair and at the same time to prevent cells from prematurely entering into mitosis.^8,10-12,52 While the pathway that modulates the inhibition of CDKs is well-established,⁴ the mechanism of how the kinase activity of Aurora A and PLK1 is inhibited and kept low for the duration of cell cycle arrest and DNA repair is less well understood. Here we demonstrate a critical role for BRCA1 toward inhibiting the kinase activity of PLK1 in response to replication stress. We show that a variety of genotoxic stresses stimulate the interaction of BRCA1-PLK1 and concomitantly inhibit the kinase activity of PLK1. Our data suggest that BRCA1 likely participates in the early inhibition of PLK1 activity as well as the subsequent inhibition observed during the period of DNA damage-induced cell cycle arrest. This is based on the fact that the interaction between BRCA1 and PLK1 is enhanced as early as 4 h after HU treatment and persists as late as 48 h (Fig. 3A and B), a kinetic profile that corresponds with the reduced activity of PLK1 (Fig. 3D and E). Most importantly, PLK1 activity is increased in BRCA1-depleted cells compared with control siRNA-treated cells (Fig. 5), providing further evidence for the downstream effects of this interaction.

In co-ordination with other cell cycle kinases, PLK1 participates in the regulation of a variety of cell cycle-related processes including centrosome maturation, spindle assembly, chromosome segregation, cytokinesis, and recovery after DNA damage through interacting with a variety of proteins.^53,54 Because of its multifaceted functions, spatial and temporal control of PLK1 becomes crucial. Indeed, PLK1 localizes to distinctive subcellular compartments during G₂ and mitosis, including centrosome, kinetochore, and midbody.⁴⁰ In addition, PLK1 can also be regulated by activators, for example hBora. Both in non-stressed cells and during checkpoint recovery, hBora stimulates the activation of PLK1 by Aurora A.^6,7,55 In our studies, HU initially reduced the kinase activity of PLK1 (Fig. 3D) as well as the interaction between PLK and hBora (Fig. 3A), which is consistent with the role that hBora plays as an activator of PLK1. Surprisingly, the PLK1-hBora interaction becomes stronger 24 h after HU treatment (Fig. 3B), even though the kinase activity of PLK1 is actually very low (Fig. 3E). The stronger interaction between PLK1 and hBora at these later time points of HU treatment correlates nicely with the stronger interaction between PLK1 and BRCA1, suggesting that when in complex with both BRCA1 and PLK1, hBora is no longer an activator of PLK1. Intriguingly, this dual role of hBora was also observed by Seki and colleagues in unperturbed HeLa cells.⁶ They showed that in G₂ cells, hBora functions as an activator to facilitate the access of Threonine-210 of PLK1 by Aurora A while in mitosis, through direct binding to PLK1, hBora impedes the further activation of PLK1 by phosphorylated substrates. In addition, replication stress also increases both the protein level as well as the phosphorylation of hBora (Fig. 3C), and part of it is likely due to the reduced kinase activity of PLK1, since PLK1 also promotes the β-TCRP-dependent degradation of hBora.^44,45 Based on the data presented here, we propose that in the early phase of DDR, binding of BRCA1 to PLK1-hBora-Aurora A quickly inhibits the PLK1 activity by, for example, actively recruiting a phosphatase, or blocking the access of Aurora A to PLK1, or both (Fig. 6). Interestingly, BRCA1 is known to bind multiple serine/threonine phosphatases and their regulatory subunits, including phosphatase 1, 2A, and 6 (thebioGRID.org). During the maintenance phase of DDR, hBora-PLK1 is sequestered in the BRCA1-hBora-PLK1 complex away from Aurora A, thereby preventing the re-association of hBora-PLK1 with Aurora A. In light of our new findings, we propose that BRCA1 is critical for the inhibition of both CDKs and PLK1 and therefore is central to the DNA damage response.

Figure 6. A Model of how BRCA1 inhibits PLK1 activity during DDR.

We noted a striking similarity between the regulation of CDKs and PLK1 and Aurora A. In a cycling cell, the protein level of CDKs does not change much at different cell cycle stages. Among the many other regulatory mechanisms, the kinase activity of CDKs varies with the cell cycle because of the fluctuation of different cyclins. Interestingly, the protein level of neither PLK1 nor Aurora A showed pronounced changes in response to replication stress (Fig. 3A and B and data not shown). On the other hand, both the protein level as well as the phosphorylation status of hBora and BRCA1 manifested dynamic changes after HU treatment (Fig. 3A–C). This suggests that in response to replication stress, the cell modulates the kinase activity of PLK1 or Aurora A through regulating the protein level and phosphorylation of the regulators, such as hBora and BRCA1, to control when and where Aurora A can access its substrate, PLK1.

Previously, van Vugt and colleagues showed that the inhibition of PLK1 in both UV- and adriamycin-treated cells is dependent on both ATM and ATR.⁵² In response to a variety of DNA damages, including IR, UV, and HU, BRCA1 can be phosphorylated by ATM and ATR.^35,36 These phosphorylation events could potentially regulate the dynamic interactions of BRCA1, Aurora A, hBora, and PLK1. Indeed, when we inhibited ATM and ATR with caffeine (Fig. 4A), the interaction between BRCA1 and PLK1 is dramatically reduced. Interestingly, caffeine treatment does not affect the interaction between PLK1 and hBora, suggesting that the inhibition of PLK1 in response to DNA damage is most likely through the dynamic binding of BRCA1 to the PLK1-hBora-Aurora A complex.

As shown in Figure 5B, siRNA depletion of BRCA1 only partially relieved the inhibition of PLK1, suggesting that other DDR proteins may also be involved in inhibiting PLK1 after DNA damage. Specifically, 53BP1, another DNA damage mediator, has been reported to interact with PLK1 during mitosis and is required for the DNA damage recovery after IR.⁵⁶ In certain model organisms, such as budding yeast and frog, after a prolonged arrest, cells can enter mitosis even with unrepaired DNA lesions. This process is called DNA damage adaptation,⁵⁷ and the DNA damage mediator Claspin has been shown to regulate the DNA damage adaptation process.⁵⁸ It is worth noting that Claspin also interacts with PLK1. PLK1 phosphorylates and promotes the degradation of Claspin as a way to turn off the checkpoint. 53BP1 plays an important role in response to DSBs, whereas Claspin is mainly involved in replication stress. Future work within our lab will investigate whether 53BP1 and Claspin will modulate the PLK1 activity during the induction and maintenance phase of DDR.

What is the biological consequence of downregulating PLK1 activity by BRCA1 in response to replication stress? Previously van Vugt and colleagues have shown that PLK1 promotes checkpoint recovery after DSB response.^5,56 We thus first examined whether BRCA1-depleted cells recovered prematurely when treated with HU. When cells were treated with different concentrations of HU and analyzed at different times after HU treatment, BRCA1-depleted cells yielded a similar percentage of mitotic cells when compared with control siRNA-transfected cells (data not shown). This finding indicates that the de-repression of PLK1 seen in BRCA1-depleted cells (Fig. 5) does not induce cells prematurely entering mitosis. These findings are not unexpected, because HU tends to arrest cells at early S-phase, making it difficult for HU-treated cells to overcome both S phase and G₂ phase arrest in order to prematurely enter mitosis. Thus, the biological implication of BRCA1-dependent downregulation of PLK1 in response to replication stress warrants further investigation.

We and others have shown that cellular BRCA1 exists in at least four different DDR complexes.^21,22,25,26 Among the BRCA1-associated DDR proteins, Abraxas, BRCA2, CtIP, PALB2, and RAP80 have all demonstrated an ability to promote the G₂/M checkpoint in response to IR, though the mechanism is still unknown.^{21,22,25-28,59,60} Our future work will be toward identifying the complex that is involved in regulating the kinase activity of PLK1.

Three groups showed that the abundance and phosphorylation of hBora is highly regulated throughout the cell cycle.^44,45,47 Both the protein level and the phosphorylation of hBora peak in late S and G₂ and then decrease during mitosis. CDK1/cyclin B1, GSK3β, Aurora A, and PLK1 can all phosphorylate hBora. One consequence of the phosphorylation of hBora is to promote its binding to the E3 ligase, β-TRCP, which then targets it for proteasome-dependent degradation.^44,45 Here we found that both HU and aphidicolin increase the abundance of hBora and induce its phosphorylation (Figs. 3 and 4B). Two intriguing questions are raised from this observation: (1) which kinase(s) is responsible for the phosphorylation of hBora under replication stress? (2) What is the functional significance of these phosphorylations? Because the kinase activity of CDK1/cyclin B1, Aurora A, and PLK1 are all downregulated by DNA damage (Fig. 4C),^4,57 they are less likely to be the contributing kinases. Therefore, checkpoint kinases such as ATR and Chk1 could be the potential candidates. Possible functions of the replication stress induced phosphorylation of hBora include: (1) switching hBora from an activator of PLK1 to an inhibitor, or (2) together with BRCA1, blocking the access of Aurora A to PLK1, thereby preventing the activation of PLK1 by Aurora A.

BRCA1 is a well-known tumor suppressor. At the early stage of tumorigenesis, due to dysregulation of many oncogenes, such as c-Myc or E2F, cells are under constant replication stress.^61,62 In wild-type cells, downregulation of PLK1 will prevent them from prematurely exiting cell cycle arrest. However, under replication stress, BRCA1-deficient cells will likely have a higher PLK1 activity and consequently will have a better chance to escape the cell cycle arrest. Therefore, we speculate that the inhibitory role of BRCA1 toward PLK1 is very important for its tumor suppression functions. Moreover, overexpression of PLK1 is found in many cancers, including breast cancers.⁶³ PLK1 is considered to be a potential anticancer target, and many PLK1 inhibitors are now in different stages of clinical trial.^37,38 In this study, we found that BRCA1 deficiency causes inefficient inhibition of PLK1 in response to DNA damage. It will be interesting to investigate whether breast cancer patients with mutant BRCA1 or reduced expression of BRCA1 are more sensitive to PLK1 inhibitors alone or in combination with DNA damaging agents. These studies will be very informative to determine whether treatment of patients with BRCA1 deficient breast cancer with PLK1 inhibitors will be beneficial or detrimental.

Materials and Methods

Plasmids, siRNA, and chemicals

The construction HA-PLK1 and Myc-hBora were described previously.⁴⁴ Plasmids expressing GFP-tagged full-length BRCA1 and different truncation mutants of BRCA1 were generously provided by Dr Natsuko Chiba.⁶⁴

The following siRNAs used in this paper were purchased from DHARMACON: ON-TARGETplus siCONTROL non-targeting pool (D-001810-10-20) was used as a negative control for all siRNA transfections. BRCA1 was depleted with ON-TARGETplus SMARTpool siRNA against BRCA1, BRCA1-B (CCAAAGCGAGCAAGAGAAU), and BRCA1-C (UGAUAAAGCUCCAGCAGGA). Human cells were transfected with 50 nM siRNA twice using RNAiMAX (Invitrogen).

Following chemicals were purchased from Sigma: aphidicolin (A0781), cis-platin (CDDP, P4394), daunorubicin (30450), etoposide (E1383), and hydroxyurea (HU, H8627). BI2536 (S1109) and MLN8054 (S1100) were purchased from Selleck.

Antibodies

PCNA (Santa Cruz, sc-56). Actin (Santa Cruz, sc-1616). GAPDH (Bethyl, A300-641A). BRCA1: Ab-1 (Calbiochem, OP92). A rabbit polycolonal antibody against BRCA1 was generously provided by Dr Jeffery Parvin. Antibody against human hBora was generously provided by Dr Erich Nigg. GFP (Clontech, clone JL-8), HA (Convance, MMS 101P), Myc (Santa Cruz, sc-40), PLK1^m (Millipore, 05-844), PLK1^R (bethyl, A300-251A), Aurora A (BD, 610938), and (Calbiocem, PC742), pT210-PLK1 (Cell Signaling, 5472S). Phospho-Histone H3 (Cell Signaling, 9701S).

Cell lines and cell culture

293T and U2-OS cells were purchased from ATCC. 293T cells were grown in D-MEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. U2-OS cells were grown in McCoy’s 5A supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. All cells were cultivated at 37 °C in a humidified incubator with 5% CO₂.

Cell lysis and immunoprecipitation

For whole-cell lysates (WCL), cells were lysed in NETN-150 lysis buffer (20 mM TRIS-HCl, pH 8.0; 150 mM NaCl; 1 mM EDTA; 0.5% NP-40) containing a cocktail of phosphatase and protease inhibitors (Sigma). For immunoprecipitation, equal amount of cell lysate were incubated with primary antibody and protein A Sepharose CL-4B beads (GE Healthcare, 17-078-01) with rotation at 4 °C overnight. The beads were then washed twice with NETN-150 buffer.

PLK1 kinase assay done as previously described.⁵¹ Briefly, PLK1 were immunoprecipitated using anti-PLK1 antibody (Millipore, 05-844) and incubated in a kinase reaction mixture containing: 50 mM Tris-Cl (pH 7.5), 10 mM Mg₂Cl, 2 mM DTT, 2 mM EGTA, 0.5 mM NaV₃O in the presence of 10 μM ATP (10 μCi of [ γ-³²P]ATP; 1 Ci = 37 Gbq) and 1 μg of GST-PBIPtide. The mixture was incubated at 30 °C for 30 min and terminated by the addition of 2× SDS-PAGE sample buffer and resolved on a 4–12% SDS-PAGE. γ-³²P labeled GST-PBIPtide was visualized with Typhoon Phosphoimager. The intensity of the γ-³²P labeled GST-PBIPtide band is quantitated by the ImageQuant software.

Glossary

Abbreviations:

BRCA1

breast cancer gene 1

PLK1

Polo-like kinase 1

CDK

cyclin-dependent kinase

DDR

DNA damage response

ATM

ataxia telangiectasia mutated

ATR

ataxia telangiectasia Rad3-related

DSB

double-strand breaks

UIM

ubiquitin interacting motif

HR

homologous recombination

CDDP

cis-diamminedichloroplatinum [II], also called cis-platin

HU

hydroxyurea

UV

ultra violet

WCL

whole-cell lysate

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Author Contributions

DZ supervised the experimental design and wrote the paper. JZ, FT, and DZ performed the experiments. HW, KR, and KL provided crucial reagents. All the authors understand their responsibilities connected to authorship.

Supplemental Materials

Supplemental materials may be found here: 
http://www.landesbioscience.com/journals/cc/article/25349