«To cite this version: Patrick Sala¨n, Yoann Rannou, Claude Prigent. Cdk1, Plks, Auroras, and Neks: the mitotic u bodyguards. Advances in ...»
Cdk1, Plks, Auroras, and Neks: the mitotic bodyguards.
Patrick Sala¨n, Yoann Rannou, Claude Prigent
To cite this version:
Patrick Sala¨n, Yoann Rannou, Claude Prigent. Cdk1, Plks, Auroras, and Neks: the mitotic
bodyguards.. Advances in Experimental Medicine and Biology, Kluwer, 2008, Advances in
experimental medicine and biology - Vol 617, 617, pp.41-56. hal-00277808
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e Cdk1, Plks, Auroras and Nrks … the mitotic bodyguards.
Patrick Salaun, Yoann Rannou and Claude Prigent* CNRS UMR6061 Génétique et Développement, Université de Rennes 1, IFR140, Faculté de médecine, 2 Av du Pr léon Bernard, 35043 Rennes cedex *Corresponding Author: firstname.lastname@example.org • Introduction “omnis cellula e cellula”, in 1858, an important dogma in cell biology was born, when Rudolf Virchow established that every cell must derives from a pre-existing cell. And indeed cell division is the only way for life to expend, it is also the way for immortalization, and unfortunately when uncontrolled also the way for cancer. But unrevealing mechanisms leading to cell division took quite a while. How does a mother cell divide to give two daughters? This is known as the cell cycle, which describes a series of events that insures faithfully transition of the genetic information from one cell generation to the next. These dividing mechanisms have been conserved throughout evolution; they underlie growth and development in all living organisms and are central to their heredity and evolution.
In eukaryotic cells, the cell cycle was first described as two distinct phases: interphase and mitosis that just precedes cell division. The interphase was later on divided into three phases, S-phase standing for DNA synthesis surrounded by two G-phases G1 and G2 standing for Gap-phases. Fully described by Walter Flemming in 1882, mitosis remains the most spectacular and sophisticated part of the cell cycle. In less than an hour the mother cell organizes a complex machine aim to separate its genetic information and all its subcellular components into two identical sets that will be inherited by the two daughter cells. If mitosis proceeds without any error it eventually ends up with cytokinesis corresponding to the physical separation of the two daughter cells. Theodor Boveri predicted errors during mitosis to be at the origin of cancer in 1902. Hundred years later the scientific community is still debating on whether or not this might be true. The coordination of progression through mitosis is mainly orchestrated by protein phosphorylation insured by several serine/threonine kinases. In this short review we will focus on the four main mitotic kinase families: the cyclin-dependent kinase: Cdks, the polo-like kinases: Plks, the Aurora kinases and the NIMArelated kinases: Nerks.
“Cyclin Dependant Kinases” Cdks that must associate to a cyclin to become active kinases are key regulators of cell cycle progression. There are now about twelve Cdks; the fisrt one Cdk1 (or cdc2) has long been considered as THE cell cycle master kinase, thought to be responsible for all cell cycle transitions (1). This is true in yeast where Cdk1 kinase activity is required for the G1/S and the G2/M transition (2). In mammalian cell however, Cdk1 activity is only required for the G2/M transition (3). Cdk1 binds to cyclin A, cyclin B or Ringo to become an active kinase (4, 5).
The “Polo-Like Kinases” Plks form a family of four different proteins that regulates many aspects of the cell cycle progression. They all share small conserved domains named polo-box required fort protein localization. Only Plk1 that is the most extensively studied, is a true mitotic kinase homolog to the Drosophila polo kinase (6). Plk2, Plk3 and Plk4 are more likely involved only in interphase. However, Plk4 activity is required for centriole duplication, an event that must be achieved before entering mitosis, and necessary to assemble the bipolar mitotic spindle (7).
Aurora kinases were first identified in S.cerevisae and Drosophila (8, 9). Yeast cells possess only one Aurora-related kinase, invertebrates such Drosophila and C. elegans have two (A and B type) and mammals have three, named Aurora A, B and C (10). From an evolution point of view the A and B types have evolved from a common ancestor while C type have evolved from the B type (11). Consequently, Aurora A has distinct functions while Aurora B and C share same functions, though all three kinases are involved in the control of many processes required for mitosis.
“NIMA related kinases” Nrks belong to a very large family of protein kinases with 13 different Nek proteins in human, from Nek1 to Nek11 (Nek2A and Nek2B and Nek11L and Nek11S) (12). The belonging to the Nrk family is defined by the sequence homology with the kinase NIMA (Nerver In Mitosis A), a true Aspergillus nidulans mitotic kinase (13). However not all of the Nek kinases are involved in mitosis (14). Nek2 is the most studied of all; its activity is required for centrosome behavior and for cytokinesis (15, 16).
Mitosis (figure 1) To get ready for mitosis Mitosis comprises many complex events that must be accomplished in less than an hour. The length of a human full cell cycle is approximately 24h during which a dividing cell is preparing itself to enter mitosis. First of all the cell must have replicated its DNA (S phase) and possess two full copies of its genome (G2 phase). Secondary the cell must also have duplicated its centrosome and possess two centrosomes (four centrioles). These centrosomes then need to go through a maturation process, meaning that proteins involved in mitotic microtubule nucleation, such as γ-tubulin for instance, must have been recruited to the centrosome before cells may enter mitosis.
Prophase: leaving the starting blocks During the prophase stage, the chromatin start to condense to form well-defined chromosomes, each chromosome consists of two sister chromatids connected at the level of their centromeres. While centrosome maturation is continuing during prophase, duplicated centrosomes must have separated and started to migrate around the nucleus to reach opposite position (the two centrosomes are now separated by the nucleus). By the end of prophase the nuclear membrane starts to breakdown.
Prometaphase: a cell without nucleus At this the stage, the nuclear membrane has been dissolved, the chromosomes have become thicker. Centrosomes nucleate asters of microtubule that search for chromosomes to attach to.
Other microtubules nucleated by the chromosomes will help to assemble the bipolar spindle.
The chromosome centromeres where the kinetochores are assembled are an important attachment point for the microtubules. This attachment is controlled by the metaphase spindle checkpoint..
Metaphase: being under surveillance The chromosomes have reached their maximum condensation state. One pair of sister chromatids linked together by cohesins forms each chromosome. Each pair of chromatid kinetochores must have one kinetochore attached to microtubules nucleated by a centrosome and the opposite kinetochore attached to microtubules emanating from the opposite centrosome. During all this process the spindle formation is controlled by the dynamic instability of the microtubules. At the end of metaphase, the spindle must be under tension with all the chromosome kinetochores attached to both centrosomes and aligned at the metaphase plate. Cell will remain in metaphase until all the above conditions are fulfilled leading to the spindle checkpoint switch off.
Anaphase: chromosome segregation This stage is triggered once the cell has controlled the spindle is under tension and all the kinetochores have been captured by microtubules. When the spindle seems the most stable, the cohesins that maintain the sister chromatids are degraded and each sister chromatid is pulled towards each centrosome forming two identical set of chromosomes.
Telophase: get ready for cell division During anaphase while chromosomes are moving, many kinetochore proteins detached from chromosomes to remain at the center of the cell where a central spindle is assembled. A contractile actin ring forms under the surface of the plasma membrane, around the central spindle. All these events lead to a contraction of the plasma membrane at the middle of the cell that will form two cells attached by the midbody.
Cytokinesis and abscission: daughter cells separation This is the less understood event of mitosis: the two daughter cells must separate. To do so, the midbody must be broken and one of the cells will inherit a flemming body (remaining of the midbody). But more importantly the cell must repair the plasma membrane to avoid leaking of cell contain. This is achieved by recruiting membrane vesicles from the previously dissolved Golgi. These vesicles also carry proteins required for cytokinesis. The very last step of cytokenesis called abscission is the physical separation of the two daughter cells.
Control of mitosis by phosphorylation The protein kinases described here are all involved in the regulation of multiple events during mitotic progression. Analyzing the function of a mitotic kinase is not easy since knock down of the protein expression by RNA interference usually generates a phenotype that corresponds to the first event controlled by the enzyme. For instance, eliminating CDK1 leads to a cell cycle arrest in G2 phase. The cell doesn’t enter mitosis because CDK1 is required for the G2/M transition. But CDK1 is also required for progression through mitosis.
The function of each kinase is also tightly linked to their localizations during progression through mitosis, “being at the right place at the right time” (figure 2). One can for instance rescue Aurora B knock down by an Aurora A kinase chimera containing Aurora B localization sequences (17).
CDK1/cyclinB activity delimits mitosis Cdk1/cyclin B activity appears in late G2 and peaks at metaphase (the middle of M phase) and is inactivated upon exit from mitosis by cyclin B destruction, degraded first on the spindle at the chromosome level together with cohesins (18). Cdk1 kinase plays important roles in early stages that contribute to the G2/M transition. Cdk1 phosphorylates motor proteins involved in centrosomes separation required for bipolar spindle assembly (19). Cdk1 phosphorylates lamina inducing a destabilization of the nuclear structure leading to nuclear envelope breaks down (20). It also phosphorylates condensin contributing to chromosome condensation (21). When Cdk1 activity is maximum, it participates to the activation of the APC/C that insure the ubiquitination of the proteins targeted to be degraded at the metaphase/anaphase transition, including cyclin B and securin (22).
Plk1: a very busy kinase Plk1 kinase activity peaks in mitosis. The kinase is composed of a catalytic domain and a PBD (polo Box Domain) that must bind to a docking protein previously phosphorylated by a priming kinase to allow Plk1 activation (23). Also, Plk1 is activated by phosphorylation of its T-loop by an activated kinase (24). Plk1 localizes to the centrosomes, the kinetochores and the midbody during mitosis. The kinase plays multiple roles during mitosis; it participates to the G2/M transition, its inhibition delays entry in mitosis. Among the Plk1 substrates one finds all the major players involved in the G2/M transition, CDC25, Myt1 and cyclin B1 (25Plk1 would be involved in the feed back loop that controls the activation of Cdk1/cyclin B.
Plk1 activity is also required for centrosome maturation by recruiting protein necessary to nucleate the microtubules that will participate to bipolar spindle assembly; the kinase also interacts with and phosphorylates many proteins involved in microtubules dynamic (28, 29).
In addition to be localized and active at the centrosome level, Plk1 also localizes to the chromosome kinetochores (30) where its activity participates to the localization of spindle checkpoint proteins. The exact function of Plk1 at the kinetochores and its participation to the spindle checkpoint remains to be clarified.
Plk1 is also required to activate the E3 Ubiquitine ligase APC/C required to trigger mitotic protein degradation. But although Plk1 directly phosphorylates APC/C subunits the effect of this phosphorylation on APC/C activity is minor (31) compare to the phosphorylation by Cdk1/cyclin B1 (32). However Plk1 contributes indirectly to APC/C activation by phosphorylating the APC/C-cdc20 inhibitor Emi1 in somatic cells. Phosphorylation of Emi1 by Plk1 triggers Emi1 degradation and APC/C-cdc20 activation (33, 34). This contributes to metaphase-anaphase transition controlled by APC/C-cdc20 and M/G1 transition controlled by APC/C-cdh1.
Finally evidence for a function of Plk1 in cytokinesis has been found in different organisms.
Septum formation is impaired in the fission yeast kinase defective mutants while ectopic septum are formed when the kinase is overexpressed (35, 36). In Drosophila polo kinase mutant also shows cytokinesis defects at various stages of spermatogenesis (37). In vertebrate cells the kinase localizes at the midbody (38). Plk1 also interact with and phophorylates kinesin proteins required in cytokinesis such as MKLP1 (39).