Suzanne Vigneron

Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance.

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phos-phatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.

Mps1 Is a Kinetochore-Associated Kinase Essential for the Vertebrate Mitotic Checkpoint

The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.

Beyond the Greatwall Protein phosphorylation and dephosphorylation provide a central mechanism that controls the eukaryotic cell division cycle and entry of cells into mitosis. A form of protein phosphatase 2A (PP2A) has an important role inhibiting phosphorylation-dependent activation of cyclin-dependent kinase 1 (CDK1) itself and also dephosphorylating substrates of the active CDK1 that promote mitosis. PP2A activity is inhibited when another protein kinase, known as Greatwall, is activated (see the Perspective by Virshup and Kaldis). Mochida et al. (p. 1670) and Gharbi-Ayachi et al. (p. 1673) searched for substrates of Greatwall that might participate in the cell cycle regulatory machinery. When phosphorylated by Greatwall, a pair of small related proteins, Arpp19 and α-endosulfine, inhibited activity of PP2A. These effects were critical for regulation of mitosis in Xenopus egg extracts and in human cancer cells. Greatwall itself is phosphorylated and activated by CDK1—thus, apparently contributing to a feed-forward loop that contributes to the switchlike commitment of cells to mitosis. The protein kinase Greatwall controls cell division by phosphorylating and activating an inhibitor of protein phosphatase 2A. Initiation and maintenance of mitosis require the activation of protein kinase cyclin B–Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate–regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.

The anaphase-promoting complex: a key factor in the regulation of cell cycle.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC). In this review, we describe the present knowledge about the APC. We pay particular attention to the latest results concerning APC structure, APC regulation and substrate recognition, and we discuss the implication of these findings in the understanding the APC function.

Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts

The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X‐FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin‐dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase‐promoting factor (MPF)‐dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF‐dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level.

Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A‐dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW‐depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW.

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.

A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis

We have identified new members (X‐PAKs) of the Ste20/PAK family of protein kinases in Xenopus, and investigated their role in the process that maintains oocytes arrested in the cell cycle. Microinjection of a catalytically inactive mutant of X‐PAK1 with a K/R substitution in the ATP binding site, also deleted of its Nter‐half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3‐containing proteins, greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. Addition of the same X‐PAK1 mutant to cell cycle extracts from unfertilized eggs induced apoptosis, as shown by activation of caspases and cytological changes in in vitro‐assembled nuclei. This was suppressed by adding Bcl‐2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant‐negative mutant with its constitutively active X‐PAK1 counterpart. Such results indicate that X‐PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked. That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II‐arrested oocytes are less sensitive to apoptotic signals than those from activated eggs.

The D‐Box‐activating domain (DAD) is a new proteolysis signal that stimulates the silent D‐Box sequence of Aurora‐A

We have demonstrated previously that Xenopus Aurora‐A is degraded at late mitosis by the APC/Fizzy‐Related in a D‐Box‐dependent manner. Here we demonstrate that, although Aurora‐B possesses the same D‐Box as Aurora‐A, Aurora‐B is not degraded by this ubiquitin ligase. We have constructed a chimera Aurora‐A/B with the N‐terminus of Aurora‐A and the C‐terminus of Aurora‐B and we have examined its degradation by APC/Fizzy‐Related. We demonstrate that the N‐terminus of Aurora‐A confers degradation capacity on the C‐terminus of Aurora‐B and that this feature is blocked by mutation of the conserved D‐Box sequence. We characterize the minimal degradation signal at the N‐terminus of Aurora‐A and demonstrate that its deletion blocks the degradation of this protein by APC/Fizzy‐Related. Thus, we conclude that two different degradation signals are required for proteolysis of Aurora‐A. The first one, which we designated D‐Box‐activating domain, within the N‐terminal domain of Aurora‐A confers the functionality to the second, a silent D‐Box, present within the C‐terminus of the kinase.

Xkid is degraded in a D-Box, KEN-Box, and A-Box-independent pathway

ABSTRACT During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.