The Biochemistry of Mitosis

Downloaded from http://cshperspectives.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press The Biochemistry of Mitosis Samuel Wieser and Jonathon Pines The Gurdon Institute, Cambridge CB2 1QN, United Kingdom Correspondence: [email protected] In this article, we will discuss the biochemistry of mitosis in eukaryotic cells. We will focus on conserved principles that, importantly,are adapted to the biology of the organism. It is vital to bear in mind that the structural requirements for division in a rapidly dividing syncytial Drosophila embryo, for example, are markedly different from those in a unicellular yeast cell. Nevertheless, division in both systems is driven by conserved modules of antagonistic protein kinases and phosphatases, underpinned by ubiquitin-mediated proteolysis, which create molecular switches to drive each stage of division forward. These conserved control modules combine with the self-organizing properties of the subcellular architecture to meet the specific needs of the cell. Our discussion will draw on discoveries in several model systems that have been important in the long history of research on mitosis, and we will try to point out those principles that appear to apply to all cells, compared with those in which the biochemistry has been specifically adapted in a particular organism. he aim of mitosis is to separate the genome spatial organization of the mitotic cell has ram- Tand ensure that the two daughter cells inher- ifications for the machinery controlling mitosis. it an equal and identical complement of chro- In particular, the breakdown of the nuclear mosomes (Yanagida 2014). To achieve this, compartment disrupts the guanosine triphos- eukaryotic cells completely reorganize their mi- phate (GTP)–guanosine diphosphate (GDP) crotubules to build a mitotic spindle that pulls gradient of the small GTPase called Ran. Ran apart the sister chromatids after the cohesin usually controls nuclear-cytoplasmic transport complexes are cut (see Cheeseman 2014; West- through the importin chaperones; Ran-GDP in horpe and Straight 2014; Hirano 2015; Reber the cytoplasm promotes binding to nuclear and Hyman 2015) and, subsequently, use the transport substrates, whereas Ran-GTP in the actin cytoskeleton to divide the cell into two nucleus promotes their dissociation (Gu¨ttler (cytokinesis) (see D’Avino et al. 2015). In and Go¨rlich 2011). As a result of nuclear enve- some cells, such as in budding and fission yeasts, lope breakdown (NEBD), another Ran-GTP the spindle is built within the nucleus (closed gradient is generated around the chromosomes, mitosis), whereas in others, the nuclear enve- to which the RCC1 GTP-exchange factor binds lope breaks down and the condensed chromo- (Clarke 2008). This Ran-GTP gradient is impor- somes are captured by microtubules in the cy- tant for the interaction between microtubules toplasm (open mitosis). This difference in the and chromosomes because the high Ran-GTP Editors: Mitsuhiro Yanagida, Anthony A. Hyman, and Jonathon Pines Additional Perspectives on Mitosis available at www.cshperspectives.org Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a015776 1 Downloaded from http://cshperspectives.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press S. Wieser and J. Pines levels around chromosomes promote the disso- Lehner 1993; Jacobs et al. 1998].) The mitotic ciation between the importin b chaperone and cyclin–Cdk also binds a small protein cyclin- its binding partners, several of which help to kinase subunit (Cks). Cks has a conserved an- stabilize or nucleate microtubules (Carazo-Salas ion-binding site (Bourne et al. 1995), which et al. 1999; Kalab et al. 1999; Gruss et al. 2001; allows it to bind phosphoserine/phosphothreo- Wilde et al. 2001; Yokoyama et al. 2008). nine, and thereby retain interactions with sub- The dramatic reorganization of the cell at strates phosphorylated by the cyclin–Cdk. This mitosis must be coordinated in both time and allows the cyclin–Cdk to hyperphosphorylate space. There are several key temporal events: en- its substrate by phosphorylating even low affin- try to mitosis, sister chromatid separation, and ity sites, and this has been shown to be impor- mitotic exit, and these are effectively made uni- tant in the activation of substrates, such as directional by the biochemical machinery. We Xenopus Cdc25 and the anaphase-promoting will discuss the biochemistry behind each of complex or cyclosome (APC/C) (see below) these temporal events, in turn, but it is impor- (Patra et al. 1999) and in the degradation of tant to emphasize that the control mechanisms cyclin A (Wolthuis et al. 2008; Di Fiore and are also spatially organized. Our understanding Pines 2010). of this spatial organization has improved dra- The crystal structures of a number of differ- matically with advances in the technology to ent cyclins, Cdks, and cyclin–Cdk complexes detect gradients of activity in cells, and this have been solved (Brown et al. 1995, 1999; Jef- has revealed the importance of local gradients frey et al. 1995; Russo et al. 1996; Pavletich 1999). of antagonistic protein kinases and phospha- These show that the Cdk subunit is completely tases, GTP-binding protein regulators, and inactive as a monomer for two reasons. First, ubiquitin ligases and deubiquitylases, to name the structure of the ATP-binding pocket in the only a few of the more prominent examples (re- amino-terminal lobe coordinates ATP in the viewed in Pines and Hagan 2011). wrong conformation for b-g bond hydrolysis; second, the “T-loop,” which contains an activa- tory phosphorylation site, obscures the sub- ENTRY TO MITOSIS strate binding cleft. Cyclin binding rectifies both defects by altering the structure of the Cyclin B–Cyclin-Dependent Kinase (CDK): Cdk amino-terminal lobe to remodel the ATP- The Main Switch binding site and draw the T-loop down and Entry to mitosis appears to have switch-like away from the substrate-binding cleft. When properties in most organisms studied; once a the T-loop is phosphorylated by Cdk-activating cell is committed to mitosis, there is no going kinase (CAK), it interacts more strongly with a back (see Fig. 1). But how the machinery is basic patch on the carboxy-terminal lobe of the made switch-like does differ between systems. Cdk, and this, then, is the fully active state. Nevertheless, the core of the switch seems to be Cyclin binding and CAK phosphorylation the same in all cells: the activation of the cyclin can be used to control entry to mitosis directly B–Cdk1 mitotic protein kinase (Nurse 1990). A if a threshold of cyclin–Cdk activity must be combination of genetics, cell biology, and bio- reached to push cells into mitosis. The validity chemistry led to the identification of this pro- of this “threshold” hypothesis has been elegantly tein kinase as the key regulator of mitosis, which shown in genetically engineered fission yeast is conserved from yeast to man (Doree 2002). (Coudreuse and Nurse 2010), and the regulation The kinase is a heterodimer composed of a Cdk of Cdk activity by local cyclin levels alone ap- subunit and an activating cyclin subunit. Both pears to be used to control mitosis in systems, are members of multigene families, and, usual- such as rapidly dividing early Drosophila embry- ly, the B-type cyclins are essential to trigger mi- os (Su et al. 1998; McCleland et al. 2009). In tosis. (One exception is Drosophila, in which the other systems, however, the mitotic cyclin– A-type cyclin is most important [Knoblich and Cdk complex accumulates in an inactive form 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a015776 Downloaded from http://cshperspectives.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press The Biochemistry of Mitosis p21 14-3-3 Cdk1/2 14-3-3 Gwl Cyc Arrp1 ? A 9 ? Ensa ? NF-Y B-Myb CdK1 Wee1 Cdc25 /Myt1 B Cyc Cdc25 B C FoxM1 Cdc25 ? PP2A/ A ? B55 Cyclin B ? Plk1 ? Plk1 Cdc25 Aurora B Cdc25 transcription A PP1 Aur A Cyclin B/CDK1 substrates Activate Inhibit Nuclear envelope breakdown Transcription/translation Chromosome condensation Intracellular trafficking Cell rounding Golgi integrity APC/C activation ER organization? SAC signaling Endocytosis? Kinetochore assembly Cytokinesis Figure 1. Mitotic entry network. The core regulatory module is indicated as a green rectangle. Dark gray lines indicate the interactions during a nonperturbed mitotic cell cycle. Yellow lines indicate interaction on DNA damage or stress-activated G2 checkpoint signaling. Question marks indicate interactions that are contentious or not proven. Green and red colored text in the text box indicates processes that are stimulated and inhibited by cyclin B/Cdk1 during mitosis, respectively. Question marks indicate processes in which the direct involvement of cyclin B/Cdk1 is not clear or not proven. APC, Anaphase-promoting complex; CDK, cyclin-dependent kinase; ER, endoplasmic reticulum; SAC, spindle assembly checkpoint. through phosphorylation on a conserved tyro- pathwaystocontrolthe timing of mitosis. Which sine residue and, in some systems, the adjacent pathways converge on the Wee1 and Cdc25 reg- threonine residue, both in the ATP-binding site ulators depends on the biology of the organism. of the Cdk. Phosphorylation on these sites pre- For example, in budding yeast, instead of entry

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