Regulation of the meiotic cell cycle in oocytes.

The mitotic and meiotic cell cycle share many regulators, but there are also important differences between the two processes. The meiotic maturation of Xenopus oocytes has proved useful for understanding the regulation of Cdc2-cyclin-B, a key activator of G2/M progression. New insights have been made recently into the signalling mechanisms that induce G2-arrested oocytes to resume and complete the meiotic cell cycle.

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides.

We designed a microplate-based assay method for mitogen-activated protein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in heretofore used assays. The micro-trap phosphorylation assay has advantages over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.

PAF-induced MAPK activation is inhibited by wortmannin in neutrophils and macrophages.

In the present study we examined the mechanism by which PAF activates MAPK in native cells such as guinea-pig neutrophils and P388D1 macrophage-like cells. We found that PAF activates MAPK through two distinct pathways. One calcium-dependent pathway that likely involves cPKC, and another calcium-independent but wortmannin-sensitive pathway. Using molecular biological methods we are presently examining whether hetrodimeric (p85/p110) type PI 3-kinase is the actual target of wortmannin involved in PAF mediated activation of MAPK.

On the mechanism of cytosolic phospholipase A2 activation in CHO cells carrying somatostatin receptor: wortmannin-sensitive pathway to activate mitogen-activated protein kinase.

We examined the mechanism of arachidonate release induced by somatostatin-14 (SS14) in CHO-K1 cells overexpressing rat hippocampal somatostatin receptor SSTR4. SSTR4 couples to pertussis toxin (PTX)-sensitive G-protein in CHO cells and does not lead to phosphoinositides breakdown or intracellular calcium ([Ca2+]i) mobilization (Bito et al.: J. Biol. Chem. 269, 12722-12730, 1994). SSTR4 activated mitogen-activated protein (MAP) kinase and induced the phosphorylation of 85kDa cytosolic phospholipase A2 (cPLA2), in a PTX-sensitive manner. Furthermore, activations of both MAP kinase and cPLA2 were inhibited by treatment with wortmannin, at almost identical IC50 values. Thus, SSTR4 appears to stimulate MAP kinase and cPLA2 in a Gi-dependent, and through a wortmannin-sensitive pathway. We also showed that stimulation with SS14, in combination with calcium-ionophore, strongly enhanced arachidonate release from these cells.

Wortmannin inhibits mitogen-activated protein kinase activation induced by platelet-activating factor in guinea pig neutrophils.

Stimulation of guinea pig neutrophils with platelet-activating factor (PAF) caused a rapid and transient activation of mitogen-activated protein kinase (MAPK). Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially (approximately 50%) inhibited PAF-induced MAPK activation. Half-maximal inhibition was observed with 200-300 nM wortmannin, while it did not inhibit phorbol ester-induced MAPK activation. Neutrophils preloaded with 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM) failed to raise cytosolic Ca2+ concentrations toward PAF, while they still responded to PAF with a 40-50% activation of MAPK. However, when cells were treated with BAPTA/AM and wortmannin in combination, the MAPK activation was completely inhibited. These results suggest that PAF activates MAPK through two distinct pathways in guinea pig neutrophils, one Ca(2+)-dependent, and the other Ca(2+)-independent but wortmannin-sensitive.

Two distinct signal transduction pathways for the activation of guinea-pig macrophages and neutrophils by endotoxin.

Lipopolysaccharide (LPS, endotoxin) is a major component of the outer membrane of gram-negative bacteria. Although it interacts with many types of cells and is linked to numerous events associated with sepsis and endotoxic shock, the mechanisms underlying these actions are poorly understood. We found that Ca-signaling induced by endotoxin in guinea-pig neutrophils and macrophages is caused by cross-recognition of LPS with platelet activating factor (PAF) receptors. However, the synthesis of tumor necrosis factor-alpha or the priming effect of O2- production was not affected by PAF antagonists. Thus, at least two distinct pathways are involved in the actions of LPS, one via the PAF receptor, while the other is independent of a PAF receptor and Ca-signaling.

A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.

The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.

Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activating factor through a mechanism independent of p85/p110-type phosphatidylinositol 3-kinase.

We have shown previously that wortmannin partially inhibits mitogen-activated protein kinase (MAPK) activated by platelet-activating factor (PAF) in guinea pig neutrophils (Ferby, M. I., Waga, I., Sakanaka, C., Kume, K., and Shimizu, T. (1994) J. Biol. Chem. 269, 30485-30488). To identify whether p85-dependent phosphatidylinositol 3-kinase is a target molecule of wortmannin in this inhibitory process, we established a murine macrophage cell line (P388D1), inducibly expressing a dominant-negative p85, delta p85. Upon induction of delta p85 by isopropyl-beta-D-thiogalactopyranoside, PAF still induced unaltered activation of MAPK, which was inhibited completely by wortmannin and 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester in an additive manner. Thus, PAF activates MAPK in P388D1 cells via two distinct pathways, one calcium-dependent and another calcium-independent, but wortmannin-sensitive. The inhibition of calcium-independent activation of MAPK by wortmannin does not involve p85-dependent phosphatidylinositol 3-kinase.

RINGO efficiently triggers meiosis resumption in mouse oocytes and induces cell cycle arrest in embryos.

RINGO was identified as a Cdc2-binding and activating protein which is necessary and sufficient to trigger G2/M progression in Xenopus oocytes. We have investigated whether the function of RINGO is conserved in mouse oocytes. We show that RINGO induces Germinal Vesicle BreakDown (GBVD) in mouse oocytes. Mos is known to induce GVBD in mouse oocytes, and is also involved in the metaphase II arrest, which is due to the CSF (CytoStatic Factor) activity. We found that RINGO also has CSF activity and induces cleavage arrest after injection into one blastomere of a late two-cell mouse embryo, like Mos. However, RINGO also inhibits polar body extrusion of wild type mouse oocytes. The same effect of RINGO on first and second polar body extrusion was observed in Mos -/- mouse oocytes. The injection of RINGO mimics Mos effects: GVBD induction and efficient cleavage arrest. However, our results in mouse oocytes suggest that RINGO may have additional functions in meiosis regulation.

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.

Xkid, a chromokinesin required for chromosome alignment on the metaphase plate.

Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces exerted at kinetochores and on chromosome arms drive chromosome movements that culminate in their alignment on the metaphase plate. In this paper, we show that Xkid, a kinesin-like protein localized on chromosome arms, plays an essential role in metaphase chromosome alignment and in its maintenance. We propose that Xkid is responsible for the polar ejection forces acting on chromosome arms. Our results show that these forces are essential to ensure that kinetochores and chromosome arms align on a narrow equatorial plate during metaphase, a prerequisite for proper chromosome segregation.