Plk1 Protein Phosphorylates Phosphatase and Tensin Homolog (PTEN) and Regulates Its Mitotic Activity during the Cell Cycle

PTEN is a well known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pulldown analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated Ser-380, Thr-382, and Thr-383, but not Ser-385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only Ser-380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with the accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.     

Ubiquitin-specific cysteine protease 2a (USP2a) regulates the stability of Aurora-A

The ubiquitin/proteasome pathway plays critical roles in virtually all aspects of cell biology. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases) ubiquitin tags to or from their target proteins in a selective fashion. USP2a is a member of a subfamily of deubiquitinases, called ubiquitin-specific cysteine proteases (USPs). Although USP2a has been reported to be a bona fide oncogene that regulates the stability of MDM2, MDMX, and FAS, it is likely that there are other unidentified substrates for USP2a. In this study, we show that USP2a mediates mitotic progression by regulating the stability of Aurora-A. Through cell-based screening of a USP siRNA library, we discovered that knockdown of USP2a reduced the protein levels of Aurora-A. USP2a interacts with Aurora-A directly in vitro and in vivo. In addition, Aurora-A is a substrate for USP2a in vitro and in vivo. Our study provides a novel mechanism for the role of USP2a in mediating the stability of Aurora-A.     

Analysis of mutations that dissociate G(2) and essential S phase functions of human ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase

ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.     

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G₂/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G₂/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G₂ phase, but not at S phase, were ∼2–3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G₂ phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G₂/M transition.     

The phosphorylation network for efficient activation of the DNA replication checkpoint in fission yeast

Protein phosphorylation is the hallmark of checkpoint activation. Hundreds of targets of checkpoint kinases have been identified recently by genome-wide investigations. However, the complete picture of a phosphorylation network required for activation of a checkpoint pathway has not been available. The DNA replication checkpoint in Schizosaccharomyces pombe contains two major protein kinases, the sensor kinase Rad3 and the effector kinase Cds1, with the latter mediating most of the checkpoint functions. We show here that when DNA replication is arrested, efficient activation of Cds1 requires five phosphorylations that cooperate in a parallel or a sequential manner. Phosphorylation of a threonine residue (Thr(11)) in Cds1 by Rad3 occurs at a basal level in the absence of three other parallel Rad3-dependent phosphorylations on the mediator Mrc1 and Rad9 in the checkpoint clamp complex. However, the three parallel Rad3-dependent phosphorylations are all required for efficient phosphorylation of Thr(11) in Cds1 by Rad3. Phosphorylation of Thr(11) has been shown previously to promote autophosphorylation of Thr(328) in the kinase domain of Cds1, which directly activates the enzyme, leading to full activation of the checkpoint pathway. Interestingly, phosphorylation of Mrc1 by Rad3 does not require the phosphorylation of Rad9, suggesting that activation of the sensor kinase Rad3 in the replication checkpoint of fission yeast may involve a different mechanism.     

Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.     

Aurora-A kinase-inactive mutants disrupt the interaction with Ajuba and cause defects in mitotic spindle formation and G2/M phase arrest in HeLa cells

Aurora-A is a centrosome-localized serine/threonine kinase that is overexpressed in multiple human cancers. We previously reported an intramolecular inhibitory regulation of Aurora-A between its N-terminal regulatory domain (Nt, amino acids [aa] 1-128) and the C-terminal catalytic domain (Cd, aa 129-403). Here, we demonstrate that although both Aurora-A mutants (AurA-K250G and AurA-D294G/Y295G) lacked interactions between the Nt and Cd, they also failed to interact with Ajuba, an essential activator of Aurora-A, leading to loss of kinase activity. Additionally, overexpression of either of the mutants resulted in centrosome amplification and mitotic spindle formation defects. Both mutants were also able to cause G2/M arrest and apoptosis. These results indicate that both K250 and D294/Y295 are critical for direct interaction between Aurora-A and Ajuba and the function of the Aurora-A complex in cell cycle progression. [BMB Reports 2014; 47(11): 631-636]     

RED, a spindle pole-associated protein, is required for kinetochore localization of MAD1, mitotic progression, and activation of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.     

Aurora-A regulates MCRS1 function during mitosis

The mitotic spindle is made of microtubules (MTs) nucleated through different pathways involving the centrosomes, the chromosomes or the walls of pre-existing MTs. MCRS1 is a RanGTP target that specifically associates with the chromosome-driven MTs protecting them from MT depolymerases. MCRS1 is also needed for the control of kinetochore fiber (K-fiber) MT minus-ends dynamics in metaphase. Here, we investigated the regulation of MCRS1 activity in M-phase. We show that MCRS1 is phosphorylated by the Aurora-A kinase in mitosis on Ser35/36. Although this phosphorylation has no role on MCRS1 localization to chromosomal MTs and K-fiber minus-ends, we show that it regulates MCRS1 activity in mitosis. We conclude that Aurora-A activity is particularly important in the tuning of K-fiber minus-ends dynamics in mitosis.     

Mammalian Polo-like Kinase 1 (Plk1) Promotes Proper Chromosome Segregation by Phosphorylating and Delocalizing the PBIP1·CENP-Q Complex from Kinetochores

Mammalian Plk1 is critically required for proper M phase progression. Plk1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating Plk1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. Plk1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine Plk1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although Plk1 did not alter the level of PBIP1 and CENP-Q ubiquitination, Plk1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that Plk1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting Plk1-dependent mitotic progression, but also for resetting the timing of Plk1 recruitment to prekinetochores in the next cell cycle.     

Aurora-A phosphorylates Augmin complex component Hice1 protein at an N-terminal serine/threonine cluster to modulate its microtubule binding activity during spindle assembly

Proper assembly of mitotic spindles requires Hice1, a spindle-associated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.     

Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.     

Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

TopBP1 (topoisomerase IIβ-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.     

Phosphorylation Regulates the p31Comet-Mitotic Arrest-deficient 2 (Mad2) Interaction to Promote Spindle Assembly Checkpoint (SAC) Activity

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31^Comet is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31^Comet interacts with Mad2 throughout the cell cycle. We show that p31^Comet binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31^Comet for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31^Comet-Mad2 binding and enhances p31^Comet-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31^Comet and demonstrate a previously unknown event controlling SAC activity.     

Cell Cycle-dependent Subcellular Translocation of the Human DNA Licensing Inhibitor Geminin

Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G₁. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G₁ phase and at the transition from G₀ to G₁. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.