FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton.

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.

RAD18 directs DNA double-strand break repair by homologous recombination to post-replicative chromatin.

RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

CDC25B is required for the metaphase I-metaphase II transition in mouse oocytes.

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment.

Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.

Many Ways to the Cell Cycle Exit after Inhibition of CDK4/6.

Cyclin-dependent kinases (CDKs) are master regulators of proliferation, and therefore they represent attractive targets for cancer therapy. Deve-lopment of selective CDK4/6 inhibitors including palbociclib revolutionized the treatment of advanced HR+/HER2- breast cancer. Inhibition of CDK4/6 leads to cell cycle arrest in G0/G1 phase and eventually to a permanent cell cycle exit called senescence. One of the main features of the senescence is an increased cell size. For many years, it was believed that the non-dividing cells simply continue to grow and as a result, they become excessively large. There is now emerging evidence that the increased cell size is a cause rather than consequence of the cell cycle arrest. This review aims to summarize recent advances in our understanding of senescence induction, in particular that resulting from treatment with CDK4/6 inhibitors.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex.

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration.

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

Nuclear localisation of 53BP1 is regulated by phosphorylation of the nuclear localisation signal.

BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin ß and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.

Bora and Aurora-A continue to activate Plk1 in mitosis.

Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora-Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora-Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.

Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression.

Activation of the DNA damage checkpoint causes a cell-cycle arrest through inhibition of cyclin-dependent kinases (cdks). To successfully recover from the arrest, a cell should somehow be maintained in its proper cell-cycle phase. This problem is particularly eminent when a cell arrests in G2, as cdk activity is important to establish a G2 state. Here, we identify the phosphatase Wip1 (PPM1D) as a factor that maintains a cell competent for cell-cycle re-entry during an ongoing DNA damage response in G2. We show that Wip1 function is required throughout the arrest, and that Wip1 acts by antagonizing p53-dependent repression of crucial mitotic inducers, such as Cyclin B and Plk1. Our data show that the primary function of Wip1 is to retain cellular competence to divide, rather than to silence the checkpoint to promote recovery. Our findings uncover Wip1 as a first in class recovery competence gene, and suggest that the principal function of Wip1 in cellular transformation is to retain proliferative capacity in the face of oncogene-induced stress.

Ppm1d truncating mutations promote the development of genotoxic stress-induced AML.

Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.

Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress.

ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.

A novel assay for screening WIP1 phosphatase substrates in nuclear extracts.

Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.

Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1.

Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.