Regulation of Greatwall kinase during Xenopus oocyte maturation.

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase.

Identification of a polo-like kinase 4-dependent pathway for de novo centriole formation.

Supernumerary centrosomes are a key cause of genomic instability in cancer cells. New centrioles can be generated by duplication with a mother centriole as a platform or, in the absence of preexisting centrioles, by formation de novo. Polo-like kinase 4 (Plk4) regulates both modes of centriole biogenesis, and Plk4 deregulation has been linked to tumor development.

Centrosomal localization of cyclins E and A: structural similarities and functional differences.

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin.

A novel role for greatwall kinase in recovery from DNA damage.

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression. The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood.

The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication.

Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication.

Centrosomal localization of cyclin E-Cdk2 is required for initiation of DNA synthesis.

Cyclin E-Cdk2 is known to regulate both DNA replication and centrosome duplication during the G1-S transition in the cell cycle, and disruption of centrosomes results in a G1 arrest in some cell types. Localization of cyclin E on centrosomes is mediated by a 20 amino acid domain termed the centrosomal localization sequence (CLS), and expression of the GFP-tagged CLS displaces both cyclin E and cyclin A from the centrosome. In asynchronous cells, CLS expression inhibits the incorporation of bromodeoxyuridine (BrdU) into DNA, an effect proposed to reflect a G1 arrest.

Repo-man controls a protein phosphatase 1-dependent threshold for DNA damage checkpoint activation.

Background: In response to DNA damage, cells activate checkpoints to halt cell-cycle progression and prevent genomic instability. Checkpoint activation induced by DNA double-strand breaks (DSB) is dependent on the ATM kinase, a master regulator of the DNA damage response (DDR) that is activated through autophosphorylation and monomerization.

Results: Here we show that either protein phosphatase 1 or 2A is sufficient to suppress activation of the DDR and that simultaneous inhibition of both phosphatases fully activates the response.

Discovery of a distinct domain in cyclin A sufficient for centrosomal localization independently of Cdk binding.

Centrosomes have recently emerged as key regulators of the cell cycle. The G1/S transition requires a functional centrosome, and centrosomal localization of numerous proteins, including cyclin/Cdk complexes, is important for the G2/M transition. Here we identify a modular centrosomal localization signal (CLS) localizing cyclin A to centrosomes independently of Cdk binding.

Phosphorylation of TPX2 by Plx1 enhances activation of Aurora A.

Entry into mitosis requires the activation of mitotic kinases, including Aurora A and Polo-like kinase 1 (Plk1). Increased levels of these kinases are frequently found associated with human cancers, and therefore it is imperative to understand the processes leading to their activation. We demonstrate that TPX2, but neither Ajuba nor Inhibitor-2, can activate Aurora A directly.

Phosphorylation of p53 is regulated by TPX2-Aurora A in xenopus oocytes.

p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A.

Kicking off the polo game.

Polo-like kinase 1 (Plk1) is essential for checkpoint recovery and the activation of key mitotic enzymes; however, its own activation mechanism has remained elusive. Recent findings show that Bora, a G(2)-M expressed protein, facilitates Plk1 activation by the oncogenic kinase Aurora A in G(2). During mitosis, Plk1-dependent Bora degradation promotes Aurora A localization to the centrosome and/or spindle.

Cyclin E-dependent localization of MCM5 regulates centrosome duplication.

Centrosomes are the primary microtubule-organizing centers in animal cells and are required for bipolar spindle assembly during mitosis. Amplification of centrosome number is commonly observed in human cancer cells and might contribute to genomic instability. Cyclin E-Cdk2 has been implicated in regulating centrosome duplication both in Xenopus embryos and extracts and in mammalian cells.

Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis.

The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC.

Spindle pole regulation by a discrete Eg5-interacting domain in TPX2.

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis.

DNA damage signaling in early Xenopus embryos.

In Xenopus embryos, the first 12 cell division cycles before the midblastula transition (MBT) feature rapid and synchronous alternations between DNA replication and mitosis. Moreover, embryos at this stage lack checkpoints that halt the cell cycle in response to DNA damage, whereas introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. In a search for the underlying mechanism, we recently discovered that the checkpoint signal initiated by damaged DNA is enhanced by the undamaged threshold DNA.

Undamaged DNA transmits and enhances DNA damage checkpoint signals in early embryos.

In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA.

The role of Xenopus membrane progesterone receptor beta in mediating the effect of progesterone on oocyte maturation.

Rapid, nongenomic membranal effects of progesterone were demonstrated in amphibian oocytes more than 30 y ago. Recently, a distinct family of membrane progestin receptors (mPRs) has been cloned in fish and other vertebrate species. In this study we explore the role of mPR in promoting oocyte maturation in Xenopus laevis.

Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1.

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest.

The anaphase-promoting complex/cyclosome inhibitor Emi2 is essential for meiotic but not mitotic cell cycles.

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown.

Calcium elevation at fertilization coordinates phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to release metaphase arrest by cytostatic factor.

Background: Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest.

The Aurora A and Aurora B protein kinases: a single amino acid difference controls intrinsic activity and activation by TPX2.

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. Here we identify G205 in Xenopus Aurora A as a key determinant of both intrinsic activity and regulation by TPX2.

Regulation of the G(2)/M transition in Xenopus oocytes by the cAMP-dependent protein kinase.

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc).

Xenopus Polo-like kinase Plx1: a multifunctional mitotic kinase.

The Xenopus Polo-like kinase Plx1 plays multiple roles in mitosis. Accumulating evidence shows that Plx1 is the trigger kinase for the G2/M transition that phosphorylates and activates the phosphatase Cdc25C, which subsequently dephosphorylates Cdc2/cyclin B and initiates a positive feedback loop between Cdc25C and Cdc2/cyclin B. Recent findings indicate that Plx1 itself is also in a positive feedback loop.

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells.

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF.