Stéphanie Dutertre

On the role of aurora-A in centrosome function.

Mammalian aurora-A belongs to a multigenic family of mitotic serine/threonine kinases comprising two other members: aurora-B and aurora-C. In this review we will focus on aurora-A that starts to localize to centrosomes only in S phase as soon as centrioles have been duplicated, the protein is then degraded in early G1. Works in various organisms have revealed that the kinase is involved in centrosome separation, duplication and maturation as well as in bipolar spindle assembly and stability. Aurora kinases are found in all organisms in which their function has been conserved throughout evolution, namely the control of chromosome segregation. In human, aurora-A has focused a lot of attention, since its overexpression has been found to be correlated with the grade of various solid tumours. Ectopic kinase overexpression in any culture cell line leads to polyploidy and centrosome amplification. However, overexpression of aurora-A in particular cell lines such as NIH3T3 is sufficient to induce growth on soft agar. Those transformed cells form tumours when implanted in immunodeficient mice, indicating that the kinase is an oncogene.

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

Blooms syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3′–5′ DNA helicase identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modifications in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process.

ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation.

Blooms syndrome (BS), a rare genetic disease, arises through mutations in both alleles of the BLM gene which encodes a 3′–5′ DNA helicase identified as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer affecting the general population and BLM-deficient cells display a strong genetic instability. We recently showed that BLM protein expression is regulated during the cell cycle, accumulating to high levels in S phase, persisting in G2/M and sharply declining in G1, suggesting a possible implication of BLM in a replication (S phase) and/or post-replication (G2 phase) process. Here we show that, in response to ionizing radiation, BLM-deficient cells exhibit a normal p53 response as well as an intact G1/S cell cycle checkpoint, which indicates that ATM and p53 pathways are functional in BS cells. We also show that the BLM defect is associated with a partial escape of cells from the γ-irradiation-induced G2/M cell cycle checkpoint. Finally, we present data demonstrating that, in response to ionizing radiation, BLM protein is phosphorylated and accumulates through an ATM-dependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream effector.

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.

The absence of p53 aggravates polyploidy and centrosome number abnormality induced by Aurora-C overexpression.

Aurora-C is the third member of the aurora serine/threonine kinase family and was found onlyin mammals. Because Aurora-C is overexpressed in many different types of cancer cells wedecided to analyze the consequences of Aurora-C overexpression in human cells. We firstinvestigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis andinterphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimicsAurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to thecentrosome, like Aurora-A. We then examined the phenotype generated by Aurora-Coverexpression. Basically it looked similar to the phenotypes observed after overexpression ofthe other Aurora kinases. We observed an augmentation of polyploid cells containing morethan two centrosomes. More interestingly this phenotype was aggravated in the absence of afunctional p53. Although the physiological function of Aurora-C in somatic cells remains tobe clarified, our results, just like for the two other Aurora kinases, raised the question of a roleof Aurora-C in the development and progression of cancer especially in the presence ofmutated p53.

The Bloom syndrome helicase is a substrate of the mitotic Cdc2 kinase.

ABSTRACTBloom syndrome (BS) is a rare human autosomal recessive disorder characterized by marked genetic instability associated with greatly increased predisposition to a wide range of cancers affecting the general population. BS arises through mutations in both copies of the BLM gene which encodes a 3’-5’ DNA helicase identified as a member of the RecQ family. Several studies support a major role for BLM in the cellular response to DNA damage and stalled replication forks. However, the specific function(s) of BLM remain(s) unclear. The BLM protein is strongly expressed and phosphorylated during mitosis, but very little information is available about the origin and the significance of this phosphorylation. We show here that ATM kinase provides only a limited contribution to the mitotic phosphorylation of BLM. We also demonstrate that BLM is directly phosphorylated at multiple sites in vitro by the mitotic cdc2 kinase, and identify two new sites of mitotic BLM phosphorylation: Ser-714 and Thr-766. Our results identify BLM helicase as a new substrate for cdc2, which may have potential physiological implications for the role of BLM in mitosis.

Mnk1 kinase activity is required for abscission.

Summary MNK1 is a serine/threonine kinase identified as a target for MAP kinase pathways. Using chemical drug, kinase-dead expression or knockdown by RNA interference, we show that inhibition of MNK1 induces the formation of multinucleated cells, which can be rescued by expressing a form of MNK1 that is resistant to RNA interference. We found that the active human form of MNK1 localises to centrosomes, spindle microtubules and the midbody. Time-lapse recording of MNK1-depleted cells displays cytokinesis defects, as daughter cells fuse back together. When MNK1 activity was inhibited, no microtubule defect at the midbody was detected, however, anchorage of the membrane vesicle at the midbody was impaired as lumenal GFP-positive vesicles did not accumulate at the midbody. At the molecular level, we found that centriolin localisation was impaired at the midbody in MNK1-depleted cells. As a consequence, endobrevin – a v-SNARE protein implicated in the abscission step – was not properly localised to the midbody. Altogether, our data show that MNK1 activity is required for abscission.