Jean-Pierre Tassan

Human pEg3 kinase associates with and phosphorylates CDC25B phosphatase: a potential role for pEg3 in cell cycle regulation

The pEg3 protein is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics. In Xenopus, pEg3 has been shown to be a cell cycle dependent kinase whose activity increases to a maximum level during mitosis of the first embryonic cell division. CDC25B is one of the three CDC25 phosphatase genes identified in human. It is thought to regulate the G2/M progression by dephosphorylating and activating the CDK/cyclin complexes. In the present study we show that the human pEg3 kinase is able to specifically phosphorylate CDC25B in vitro. One phosphorylation site was identified and corresponded to serine 323. This residue is equivalent to serine 216 in human CDC25C which plays an important role in the regulation of phosphatase during the cell cycle and at the G2 checkpoint. pEg3 is also able to specifically associate with CDC25B in vitro and in vivo. We show that the ectopic expression of active pEg3 in human U2OS cells induces an accumulation of cells in G2. This effect is counteracted by overexpression of CDC25B. Taken together these results suggest that pEg3 is a potential regulator of the G2/M progression and may act antagonistically to the CDC25B phosphatase.

An overview of the KIN1/PAR-1/MARK kinase family

Summry— Members of the KIN1/PAR‐1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization. Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability. Here we present an overview of known roles of KIN1/PAR‐1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression. Some common modes of action can be deciphered for this protein kinase family.

CDC25B Phosphorylated by pEg3 Localizes to the Centrosome and the Spindle Poles at Mitosis

The phosphatase CDC25B is one of the key regulators that control entry into mitosis throughthe dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here westudy the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of theKIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate thatCDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the Bdomain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 isphosphorylated in vivo, that this phosphorylated form of CDC25B accumulates duringmitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3expression is repressed by RNA interference. Taken together, these results support a model inwhich pEg3 contributes to the control of progression through mitosis by phosphorylation ofthe CDC25 phosphatases.

A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development

MELK is a serine/threonine kinase involved in several cell processes, including the cell cycle, proliferation, apoptosis and mRNA processing. However, its function remains elusive. Here, we explored its role in the Xenopus early embryo and show by knockdown that xMELK (Xenopus MELK) is necessary for completion of cell division. Consistent with a role in cell division, endogenous xMELK accumulates at the equatorial cortex of anaphase blastomeres. Its relocalization is highly dynamic and correlates with a conformational rearrangement in xMELK. Overexpression of xMELK leads to failure of cytokinesis and impairs accumulation at the division furrow of activated RhoA – a pivotal regulator of cytokinesis. Furthermore, endogenous xMELK associates and colocalizes with the cytokinesis organizer anillin. Unexpectedly, our study reveals a transition in the mode of cytokinesis correlated to cell size and that implicates xMELK. Collectively, our findings disclose the importance of xMELK in cytokinesis during early development and show that the mechanism of cytokinesis changes during Xenopus early development.

Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit

MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

M-phase MELK activity is regulated by MPF and MAPK

Caroline Badouel, Roman Körner, Marie Frank-Vaillant, Anne Couturier, Erich A. Nigg and Jean-Pierre TassanThe protein kinase MELK is implicated in the control of cell proliferation, cell cycleand mRNA splicing. We previously showed that MELK activity is correlated with itsphosphorylation level, is cell cycle dependent, and maximal during mitosis. Here we report onthe identification of T414, T449, T451, T481 and S498 as residues phosphorylated inXenopus MELK (xMELK) in M-phase egg extract. Phosphorylations of T449, T451, T481are specifically detected during mitosis. Results obtained in vivo showed that MPF andMAPK pathways are involved in xMELK phosphorylation. In vitro, MPF and MAPK directlyphosphorylate xMELK and MPF phosphorylates xMELK on T481. In addition,phosphorylation by MPF and MAPK enhances MELK activity in vitro. Taken together ourresults indicate that MELK phosphorylation by MPF and MAPK enhance its activity duringM-phase.

Cell‐cycle‐dependent cortical localization of pEg3 protein kinase in Xenopus and human cells

Background information. Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR‐1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell‐cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell‐cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells.

Cortical localization of Maternal Embryonic Leucine zipper Kinase (MELK) implicated in cytokinesis in early Xenopus embryos

MELK has been implicated in a large variety of functions. Because its level is elevated in cancer tissues and it is involved in cell proliferation, MELK is considered as a potential therapeutic target for cancers. In a recent, study we have shown that MELK is involved in cytokinesis in early Xenopus laevis embryos. MELK dynamically accumulates at the cell cortex including a narrow band corresponding to the presumptive division furrow shortly before cytokinesis onset. MELK co-localizes and interacts with anillin an important regulator of cytokinesis. In addition, MELK overexpression interferes with accumulation at the cleavage furrow of activated Rho GTPase another crucial regulator of cytokinesis. Interestingly, our study also revealed that a transition implying a change in the direction of asymmetric furrow ingression occurs during early development. After this transition, MELK, as well as other proteins involved in cytokinesis, do not localize anymore as a band at the equatorial cortex but still localizes at the cell cortex. Our results indicate that cortical localization is an important feature of MELK in X. laevis embryos.

Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos

Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cell-cell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells.

Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo.

Summary Maternal Embryonic Leucine zipper Kinase (MELK) was recently shown to be involved in cell division of Xenopus embryo epithelial cells. The cytokinetic furrow of these cells ingresses asymmetrically and is developmentally regulated. Two subpopulations of xMELK, the mMELK (for “mitotic” xMELK) and iMELK (“interphase” xMELK), which differ in their spatial and temporal regulation, are detected in Xenopus embryo. How cells regulate these two xMELK populations is unknown. In this study we show that, in epithelial cells, xMELK is present at a higher concentration at the apical junctional complex, in contrast to mesenchyme-like cells, which have uniform distribution of cortical MELK. Interestingly, mMELK and iMELK also differ by their requirements towards cell–cell contacts to establish their proper cortical localization both in epithelial and mesenchyme-like cells. Receptor for Activated protein Kinase C (RACK1), which we identified as an xMELK partner, co-localizes with xMELK at the tight junction. Moreover, a truncated RACK1 construct interferes with iMELK localization at cell–cell contacts. Collectively, our results suggest that iMELK and RACK1 are present in the same complex and that RACK1 is involved in the specific recruitment of iMELK at the apical junctional complex in epithelial cells of Xenopus embryos.