Claude Prigent

Phosphorylation of serine 10 in histone H3, what for?

Eukaryotic cells must possess mechanisms for condensing and decondensing chromatin. Chromatin condensation is particularly evident during mitosis and cell death induced by apoptosis, whereas chromatin decondensation is necessary for replication, repair, recombination and transcription. Histones are among the numerous DNA-binding proteins that control the level of DNA condensation, and post-translational modification of histone tails plays a critical role in the dynamic condensation/decondensation that occurs during the cell cycle. Phosphorylation of Ser10 in the tails of histone H3 has been extensively studied in many organisms. Interestingly, this modification is involved in both transcription and cell division, two events requiring opposite alterations in the degree of chromatin compaction. How does one and the same modification of histone H3 fulfil such roles? For instance, in interphase, phosphorylation of H3 correlates with chromatin relaxation and gene expression, whereas in mitosis it correlates with chromosome condensation. What is the kinase and under what circumstances does Ser10 becomes phosphorylated? Most importantly, what are the consequences of phosphorylation of this residue?

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (∼5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-α and -β. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-α and -β, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP–TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2–M transition

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.

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.

The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5.

We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from thebimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395–3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Dorée, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557–572). Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that inXenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interactin vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylatedin vivo first confirmed that XlEg5 was phosphorylated by p34 cdc2 and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.

APC/Fizzy-Related targets Aurora-A kinase for proteolysis.

Aurora‐A kinase is a mitotic spindle‐pole‐associated protein that has been implicated in duplication and separation of centrosomes and in spindle assembly. The proper timing and amplitude of Aurora‐A expression seems to be important, as elevated levels of this protein have been associated with centrosome abnormalities and aneuploidy in mammalian cells. We show that Aurora‐A increases at the G2–M transistion and disappears completely at G1 in XL2 cells. Using Xenopus oocyte extracts, we demonstrate that degradation of Aurora‐A is mediated by the anaphase‐promoting complex (APC) and is regulated by Fizzy‐Related but not by Fizzy. Degradation of Aurora‐A depends on a D‐Box, but not on its KEN‐Box motif, as mutation of its C‐terminal D‐Box sequence induces stabilization of the protein. Accordingly, addition into the extracts of a cyclin B‐type D‐Box‐motif‐containing peptide completely suppresses its degradation. Furthermore, APC/Fizzy‐Related ubiquitylates the wild type but not a D‐Box mutant form of Aurora‐A in vitro. Consistent with these data, ectopic expression of Fizzy‐Related in Xenopus oocytes induces complete degradation of endogenous Aurora‐A. Aurora‐A is thus the first protein, at least in our assay system, that undergoes a D‐Box‐dependent degradation mediated by APC/Fizzy‐Related but not by APC/Fizzy.

The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation

The CDK11 (cyclin‐dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110‐kDa isoform is expressed at constant levels during the cell cycle and the shorter 58‐kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. γ‐Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11p58 isoform, but not the CDK11p110 isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11p58 in centrosome maturation and bipolar spindle morphogenesis.

CDC25B phosphorylation by aurora-a occurs at the G2/M transition and is inhibited by DNA damage

CDC25B is one of the three human dual-specificity phosphatases involved in the activation ofcyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsiblefor the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-Akinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/Mtransition. Here we have investigated this phosphorylation event during the cell cycle, and inresponse to activation of the G2 DNA damage checkpoint. We show that accumulation of theS353-phosphorylated form of CDC25B at the centrosome correlates with the relocalisation ofcyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation ofthe G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated andconsequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-Aresults in a bypass of the checkpoint that partially overcome by a S353A mutant of CDC25B.Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These resultsstrongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is animportant step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclinB1 activation.