Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

BACKGROUND:Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS:We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE:These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein

New biomarkers for early diagnosis of Lesch-Nyhan disease revealed by metabolic analysis on a large cohort of patients

International audienceBackground: Lesch-Nyhan disease is a rare X-linked neurodevelopemental metabolic disorder caused by a wide variety of mutations in the HPRT1 gene leading to a deficiency of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). The residual HGprt activity correlates with the various phenotypes of Lesch-Nyhan (LN) patients and in particular with the different degree of neurobehavioral disturbances. The prevalence of this disease is considered to be underestimated due to large heterogeneity of its clinical symptoms and the difficulty of diagnosing of the less severe forms of the disease. We therefore searched for metabolic changes that would facilitate an early diagnosis and give potential clues on the disease pathogenesis and potential therapeutic approaches

Rôle de la régulation d'Eg5 et de ses propriétés motrices lors de la formation du fuseau mitotique dans l'extrait d'oeuf de Xenopus laevis

In this study, we show that Eg2 phosphorylation of Eg5 is not important for its function in bipolar spindle formation in Xenopus egg extract. Conversely, Eg5 needs to be phosphorylated by Cdk1 to be targeted to spindle microtubules and hence assemble a bipolar spindle in Xenopus egg extract. These findings confirm previous studies and furthermore indicate that Cdk1 phosphorylation site is not only conserved among kinesin-5 members but also that its mechanism of regulation is conserved among this subfamily. Although further experiments are required to fully characterize Eg5 motor properties in by means of our microtubule gliding assay in Xenopus egg extract, Eg5 intrinsic motor properties are definitely crucial for bipolar spindle assembly as none of the Eg5 chimeras could rescue spindle formation in Xenopus egg extract. Moreover, these experiments provide the first experimental evidence that the classification of kinesins in different subfamilies, according to their conserved motor domain sequences, has also yielded to classify them according to their diverse function.Par cette étude, nous montrons que la phosphorylation d'Eg5 par Eg2 n'est pas importante pour sa fonction dans la formation du fuseau mitotique dans l'extrait d'oeuf de Xénope. Au contraire, la phosphorylation d'Eg5 par Cdk1 est nécessaire pour son attachement aux microtubules. Cet attachement permettra par la suite l'assemblage du fuseau mitotique. En plus de confirmer de précédentes études, ces résultats indiquent que le site de phosphorylation de Cdk1 n'est pas seulement conservé parmi les membres des Kinésines 5, mais également que son mécanisme de régulation est conservé. Bien que des expériences plus approfondies soient nécessaires afin de caractériser les propriétés motrices d'Eg5 par l'intermédiaire de notre expérience de "microtubule-gliding" dans l'extrait de Xénope, nos expériences réalisées avec des chimères d'Eg5 ont souligné l'importance des propriétés motrices intrinsèques à Eg5 qui sont cruciales pour la formation du fuseau mitotique. En effet, aucune de ces chimères n'a pu rétablir la formation du fuseau mitotique. De plus, ces expériences ont fourni la première preuve expérimentale que la classification des Kinésines en différentes sous-familles selon la conservation de séquence de leur domaine moteur a également abouti à les classer selon leurs différentes fonctions

Role of Eg5 regulation and motor properties in bipolar spindle assembly in Xenopus laevis egg extract

Par cette étude, nous montrons que la phosphorylation d Eg5 par Eg2 n est pas importante pour sa fonction dans la formation du fuseau mitotique dans l extrait d oeuf de Xénope. Au contraire, la phosphorylation d Eg5 par Cdk1 est nécessaire pour son attachement aux microtubules. Cet attachement permettra par la suite l assemblage du fuseau mitotique. En plus de confirmer de précédentes études, ces résultats indiquent que le site de phosphorylation de Cdk1 n est pas seulement conservé parmi les membres des Kinésines 5, mais également que son mécanisme de régulation est conservé. Bien que des expériences plus approfondies soient nécessaires afin de caractériser les propriétés motrices d Eg5 par l intermédiaire de notre expérience de microtubule-gliding dans l extrait de Xénope, nos expériences réalisées avec des chimères d Eg5 ont souligné l importance des propriétés motrices intrinsèques à Eg5 qui sont cruciales pour la formation du fuseau mitotique. En effet, aucune de ces chimères n a pu rétablir la formation du fuseau mitotique. De plus, ces expériences ont fourni la première preuve expérimentale que la classification des Kinésines en différentes sous-familles selon la conservation de séquence de leur domaine moteur a également abouti à les classer selon leurs différentes fonctions.In this study, we show that Eg2 phosphorylation of Eg5 is not important for its function in bipolar spindle formation in Xenopus egg extract. Conversely, Eg5 needs to be phosphorylated by Cdk1 to be targeted to spindle microtubules and hence assemble a bipolar spindle in Xenopus egg extract. These findings confirm previous studies and furthermore indicate that Cdk1 phosphorylation site is not only conserved among kinesin-5 members but also that its mechanism of regulation is conserved among this subfamily. Although further experiments are required to fully characterize Eg5 motor properties in by means of our microtubule gliding assay in Xenopus egg extract, Eg5 intrinsic motor properties are definitely crucial for bipolar spindle assembly as none of the Eg5 chimeras could rescue spindle formation in Xenopus egg extract. Moreover, these experiments provide the first experimental evidence that the classification of kinesins in different subfamilies, according to their conserved motor domain sequences, has also yielded to classify them according to their diverse function.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Heat shock factor 1 is a potent therapeutic target for enhancing the efficacy of treatments for multiple myeloma with adverse prognosis

International audienceAbstractDeregulated expression of heat shock proteins (HSPs) encoding genes is frequent in multiple myeloma. HSPs, which are molecular chaperones involved in protein homeostasis pathways, have emerged recently as promising therapeutic targets. Using human myeloma cell lines and primary myeloma cells belonging to various molecular groups, we tested the efficacy of HSP90, HSP70, and heat shock factor 1 (HSF1) inhibitors alone or associated with current antimyeloma drugs. We report here that KNK-437 (an inhibitor of HSF1) and bortezomib have additive effects on apoptosis induction in cells belonging to groups with bad prognosis

HIF-1α and rapamycin act as gerosuppressant in multiple myeloma cells upon genotoxic stress

<p>Multiple myeloma (MM) is still an incurable hematological malignancy. Despite recent progress due to new anti-myeloma agents, the pathology is characterized by a high frequency of <i>de novo</i> or acquired resistance. Delineating the mechanisms of MM resistance is essential for therapeutic advances. We previously showed that long-term genotoxic stress induces the establishment of a senescence-associated secretory phenotype, a pro-inflammatory response that favors the emergence of cells with cancer stem-like properties. Here, we studied the short-term response of MM cells following treatment with various DNA damaging agents such as the energetic C-ion irradiation. MM cells are highly resistant to all treatments and do not enter apoptosis after they arrest cycling at the G2 phase. Although the DNA damage response pathway was activated, DNA breaks remained chronically in damaged MM cells. We found, using a transcriptomic approach that <i>RAD50</i>, a major DNA repair gene was downregulated early after genotoxic stress. In two gerosuppression situations: induction of hypoxia and inhibition of the mammalian target of rapamycin (mTOR) pathway, we observed, after the treatment with a DNA damaging agent, a normalization of RAD50 expression concomitant with the absence of cell cycle arrest. We propose that combining inhibitors of mTOR with genotoxic agents could avoid MM cells to senesce and secrete pro-inflammatory factors responsible for cancer stem-like cell emergence and, in turn, relapse of MM patients.</p