Mitosis Kinase Research Articles

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

Wnt and Hh are known signalling pathways involved in neural differentiation and recent work has shown the cell cycle regulator, Never in Mitosis Kinase 2 (Nek2) is able to regulate both pathways. Despite its known function in pathway regulation, few studies have explored Nek2 within embryonic development. The P19 embryonal carcinoma cell model was used to investigate Nek2 and neural differentiation through CRISPR knockout and overexpression studies. Loss of Nek2 reduced cell proliferation in the undifferentiated state and during directed differentiation, while overexpression increased cell proliferation. Despite these changes in proliferation rates, Nek2 deficient cells maintained pluripotency markers after neural induction while Nek2 overexpressing cells lost these markers in the undifferentiated state. Nek2 deficient cells lost the ability to differentiate into both neurons and astrocytes, although Nek2 overexpressing cells enhanced neuron differentiation at the expense of astrocytes. Hh and Wnt signalling were explored, however there was no clear connection between Nek2 and these pathways causing the observed changes to differentiation phenotypes. Mass spectrometry was also used during wildtype and Nek2 knockout cell differentiation and we identified reduced electron transport chain components in the knockout population. Immunoblotting confirmed the loss of these components and additional studies showed cells lacking Nek2 were exclusively glycolytic. Interestingly, hypoxia inducible factor 1α was stabilized in these Nek2 knockout cells despite culturing them under normoxic conditions. Since neural differentiation requires a metabolic switch from glycolysis to oxidative phosphorylation, we propose a mechanism where Nek2 prevents HIF1α stabilization, thereby allowing cells to use oxidative phosphorylation to facilitate neuron and astrocyte differentiation.

38P - Bub3 is phosphorylated by the ataxia-telangiectasia mutated kinase in mitosis and required for activation of the mitotic spindle checkpoint in breast cancer

BackgroundATM is an important cell cycle checkpoint kinase. This protein is an controller of cell cycle checkpoint that is required for cell response to DNA damage and for genome stability. The spindle assembly checkpoint (SAC) controls accurate chromosome segregation to maintain genome stability. All kinetochores correctly attach to the spindle, which allow mitosis to proceed. Bub3 is the mainly component of the SAC, in which its kinase activity can stabilize the correct kinetochore–microtubule attachments. Genomic stability is a hallmarks of breast cancer, and the level of Bub3 in breast cancer are higher than that in normal tussue. Understanding how the ATM-Bub3 signal contributes to kinetochore-based signaling may help us to recognize the occurrence of breast cancer. MethodsStable Isotope Labeling with Amino Acid in Cell Culture (SILAC) to identify ATM interation protein in breast cancer. Bioinformatics analyze the phosphorylation site of Bub3. Biacore assay and in vitro kinase assay to validate the phosphorylation site. Flow cytometry assay to check the mitotic index. Mutation of the phosphorylation site of bub3 and Co-IP were proformed to identify the interation of Bub3 and Bub1 or ZNF207, and western blot to be used to test the activity of Bub1. ResultsThrough Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC), we identified additional mitotic proteins that interact and can be phosphorylated by mitotically activated ATM kinase. We provide both in vitro and in vivo evidence that ATM phosphorylates Bub3 on Serine 135. This phosphorylation event promotes Bub1 activity in the mitotic spindle checkpoint. Further, we find that phosphorylation of Bub3 on Ser135 is required for formation of the Bub3-Bub1 complex, which is essential for Bub1 Ser314 phosphorylation. Mutation of Ser135 to alanine of Bub3 resulted in an enhanced interaction with BuGZ/ZNF207 and reduced interaction with Bub1, which lead to a spindle checkpoint defect. ConclusionsOur findings highlight the functional significance of ATM-mediated kinetochore protein phosphorylation and elucidate a detailed regulatory mechanism of the SAC in breast cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract P1-09-04: Proliferation and p21 refine risk of relapse in residual disease after HER2-directed therapies

Abstract Background: Patients (pts) with residual disease (RD) after neoadjuvant therapy are at higher risk of relapse. We investigated whether biomarkers assessed at surgery in patients pts with RD in the NeoSphere study were informative for risk of distant event free survival (DEFS) Methods: In NeoSphere 417 HER2+ pts were randomized to neoadjuvant TD, TPD, TP or PD (T=trastuzumab, P=pertuzumab, D=docetaxel), and received FAC/FEC and trastuzumab after surgery. 296 pts had RD. Affymetrix derived gene expression profiles (GEPs) were available at surgery for 201 pts (67.9%). 176 pts (60.1%) had paired samples before and after treatment with available GEPs. We investigated the prognostic value of proliferation evaluated by Mitosis Kinase Score (MKS) (Bianchini G Cancer Res 2010), and performed a gene discovery for association between gene expression at surgery and DEFS. Results: MKS as continuous marker was associated with significantly higher risk of relapse when assessed at surgery (HR 1.80 [1.23-2.65]; p=0.002), but not before treatment (HR 1.50 [0.80-2.78]; p=0.20). In paired samples, there was an average decrease (p=9.2E-11) of MKS after treatment, which was prominent in ER+ and chemotherapy-containing arms. In ER- and TP arm there were cases of increase and of decrease of MKS. In ER+ the 5 years DEFS was 94.3% in the Low/Int MKS tertiles group (pooled) vs 70.5% in the High MKS tertile group (HR 5.41 [1.87-15.6]; p=0.002). In ER-, the 5 years DEFS was 85.0% in the Low/Int vs 64.1% in the High group (HR 2.89 [1.08-7.76]; p=0.035). Notably, MKS at surgery after the two monoclonal alone was also prognostic. In the gene discovery approach only the expression of CDKN1A (p21)at surgery was associated with DEFS after correction for false discovery rate (FDR=0.01). Pre-treatment p21 was not associated with DEFS. Paired comparison showed significant upregulation of p21 in all patients, treatment arms and ER groups. The Int/High p21 tertiles group (pooled) had lower risk of recurrence than the low tertile in ER+ (HR 4.31 [1.60-11.6]; p=0.004) and in ER- (HR 5.81 [1.87-18.1]; p=0.002) groups. p21 in TP arm was also prognostic. MKS and p21 expression provided independent prognostic information and remained significant after correction for clinico-pathological variables (nodes and T stage) and tumor-infiltrating lymphocytes. Combining the two markers, there was a group at very low risk (Low/Int MKS and Int/High p21) and one at high risk (High MKS and Low p21). The other tertiles combinations had intermediate risk. In ER+, the 5 yrs DEFS was 94.9% in the low risk group and 52.9% in the high risk (p=1.9E-05). In ER-, the 5 yrs DEFS was 96.5% in the low and 45.5% in the high risk group (p=0.001).The markers' combination was also prognostic in the two monoclonal only arm. Conclusions: Proliferation (MKS) and p21 expression are modulated by trastuzumab and/or pertuzumab regimens. Tumors with high MKS and low p21 in RD after neoadjuvant therapy defined a group at very high risk of relapse. Tumors with low/int proliferation and int/high p21 had low risk of recurrence similar to that of patients achieving pCR. Whether the pharmacodynamic modulation of p21 could be used as surrogate marker of long term benefit in patients with RD deserves additional investigation. Citation Format: Bianchini G, Pienkowski T, Im Y-H, Bianchi GV, Tseng L-M, Liu M-C, Lluch A, de la Haba-Rodríguez J, Semiglazov V, Oh D-Y, Poirier B, Pedrini JL, Valagussa P, Gianni L. Proliferation and p21 refine risk of relapse in residual disease after HER2-directed therapies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-04.

Mechanisms for nonmitotic activation of Aurora-A at cilia.

Overexpression of the Aurora kinase A (AURKA) is oncogenic in many tumors. Many studies of AURKA have focused on activities of this kinase in mitosis, and elucidated the mechanisms by which AURKA activity is induced at the G2/M boundary through interactions with proteins such as TPX2 and NEDD9. These studies have informed the development of small molecule inhibitors of AURKA, of which a number are currently under preclinical and clinical assessment. While the first activities defined for AURKA were its control of centrosomal maturation and organization of the mitotic spindle, an increasing number of studies over the past decade have recognized a separate biological function of AURKA, in controlling disassembly of the primary cilium, a small organelle protruding from the cell surface that serves as a signaling platform. Importantly, these activities require activation of AURKA in early G1, and the mechanisms of activation are much less well defined than those in mitosis. A better understanding of the control of AURKA activity and the role of AURKA at cilia are both important in optimizing the efficacy and interpreting potential downstream consequences of AURKA inhibitors in the clinic. We here provide a current overview of proteins and mechanisms that have been defined as activating AURKA in G1, based on the study of ciliary disassembly.

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

Abstract 2115: CASZ1 suppresses neuroblastoma cell growth by restoring Rb activity in G1 and regulating kinases involved in G2/M progression

Abstract A hallmark of cancer is the disruption of cell cycle regulation. Frequently this occurs in the Rb signal transduction pathway and causes tumor cells to become insensitive to antigrowth signals. In neuroblastoma (NB), poor prognosis tumors have been found to over-express multiple cell cycle regulatory genes, including those that participate in Rb regulation. Specifically, while amplification of Cyclin D1 and Cdk4 and mutations in Cdk6 occur at low frequency in NB, more than 75% of tumors contain elevated cyclin D1 expression. Recently, we identified CASZ1 to be a chromosome 1p36 tumor suppressor gene that suppresses NB tumor growth. To determine how loss of the CASZ1 tumor suppressor might contribute to cell cycle deregulation in NB tumors, we investigated how CASZ1 reconstitution affects proteins required for transition of NB cells through the G1/S and G2/M phases of the cell cycle by western blot analysis. CASZ1 restoration causes a 2-fold increase in the time required for NB cells to progress through the cell cycle with no major changes in any particular phase of the cell cycle. CASZ1 decreases DNA synthesis by ∼30% using a Click-iT EdU assay. Within 24 hr of CASZ1 induction there is a 2-fold increase in the level of the Cdk2/4 inhibitor p21 and a 1.5-fold increase in p27 levels at 72 hr. Steady-state levels of p16 levels do not change. There is also a 50% decrease in the steady-state levels of Cdk2 and Cdk6 at 24 hr. Consistent with the increase of Cdk inhibitors and decrease in Cdks levels, there is a 60% decrease in phosphorylation of their target Rb at p-RbS780 and p-RbS807/S811. Thus, increased levels of CASZ1 restore Rb activity, which regulates the G1/S transition. CASZ1 causes a near 40% decrease of cyclin E at 24 hr, and Cyclin E:Cdk2 is required for G1/S transition in addition to Rb. Cyclin A decreases about 40% at 24 hr after CASZ1 induction and cyclin A:Cdk2 is required for the cell to progress through the S phase. Cyclin B:Cdk1 is required for G2/M progression, CASZ1 induces 70% decrease of cyclin B1 and 40% decrease of its up-stream activator phosphates Cdc25c although Cdk1 level does not change. CASZ1 causes a 70% decrease in the steady-state levels of Aurora A kinase, which a key regulatory kinase in mitosis. These decreases occur at 48 hr suggesting that they may be related to changes occurring in cells progressing through G1. Taken together, this study indicates that restoration of CASZ1 activates Rb in G1 and inhibits the G2/M regulators cyclin B1, Cdc25c and aurora kinase. The decreases in the key regulators controlling G1/S and G2/M transition leads to a lengthening of NB cell cycle progression and a subsequent decreased growth in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2115. doi:10.1158/1538-7445.AM2011-2115

The WW-HECT protein Smurf2 interacts with the Docking Protein NEDD9/HEF1 for Aurora A activation

The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.

Stem-like Cancer Cells Are Inducible by Increasing Genomic Instability in Cancer Cells

The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of tumor formation and recurrence. However, the origin of such cells remains controversial with two competing hypotheses: CSCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Compelling evidence has determined the chromosomal aneuploidy to be one of the hallmarks of cancer cells, indicating genome instability plays an important role in tumorigenesis, for which CSCs are believed to be the initiator. To gain direct evidence that genomic instability is involved in the induction of SLCCs, we utilized multiple approaches to enhance genomic instability and monitored the percentage of SLCC in cultured cancer cells. Using side population (SP) cells as a marker for SLCC in human nasopharyngeal carcinoma (NPC) and CD133 for human neuroblastoma cells, we found that DNA damage inducers, UV and mitomycin C were capable of increasing SP cells in NPC CNE-2 and neuroblastoma SKN-SH cells. Likewise, either overexpression of a key regulator of cell cycle, Mad2, or knock down of Aurora B, an important kinase in mitosis, or Cdh1, a key E3 ligase in cell cycle, resulted in a significant increase of SP cells in CNE-2. More interestingly, enrichment of SP cells was observed in recurrent tumor tissues as compared with the primary tumor in the same NPC patients. Our study thus suggested that, beside transformation of tissue stem cells leading to CSC generation, genomic instability could be another potential mechanism resulting in SLCC formation, especially at tumor recurrence stage.

Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.

Phosphorylation of threonine 204 of DEAD-box RNA helicase DDX3 by cyclin B/cdc2 in vitro

DDX3 is a DEAD-box RNA helicase involved in human immunodeficiency virus mRNA export and translation. Previously, we reported that DDX3 is required for cyclin A expression. To examine whether DDX3 is regulated at the post-transcriptional level, we determined the phosphorylation sites of hamster DDX3 in vitro. Threonine 204 (Thr204) is a conserved amino acid residue of DDX3 homologues in yeast, frog, hamster, and human that is located within motif Q of DEAD-box RNA helicases. A Thr204 to Glu204 DDX3 mutant protein lost its function, suggesting that phosphorylation at Thr204 affects DDX3 function. Thr204 was phosphorylated by cyclin B/cdc2. Thr323 in motif Ib was also phosphorylated by cyclin B/cdc2 kinase. We propose a novel function of cyclin B/cdc2 kinase in mitosis, which is to cause a loss of DDX3 function to repress cyclin A expression and to decrease ribosome biogenesis and translation during mitosis.

Immunolocalization of phospho-S6 kinases: a new way to detect mitosis in tissue sections and in cell culture

During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-fixed tissue mitotic cells appeared as bright spots in immunofluorescence using an antibody specific for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4× and 10×. Immunofluorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunofluorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid fixation, mitotic cells could be identified with the immunoperoxidase technique in paraffin sections of immersion-fixed tissue. This is the first report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with anti-phospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magnification.

Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer's disease pathology.

Nucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimer's disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration.

Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E.

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

Specificity and Determinants of Sam68 RNA Binding: IMPLICATIONS FOR THE BIOLOGICAL FUNCTION OF K HOMOLOGY DOMAINS

Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.