Protein phosphatase activities in vivo in Xenopus laevis oocyte: inhibition by okadaic acid

Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected beta casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50% of activity was recovered 15-30 minutes after microinjection.     

Timing of Plk1 and MPF activation during porcine oocyte maturation

A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40-44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes.     

Dissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes.

MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.     

Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.     

Membrane-anchored cyclin A2 triggers Cdc2 activation in Xenopus oocyte

In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2-Delta152 and a viral or cellular ER-targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M-phase promoting factor.     

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparent "activation" of protein kinases by okadaic acid class tumor promoters

A cytosolic fraction of mouse brain gave two peaks of protein kinase activity on DEAE-cellulose column chromatography. The first peak of protein kinase corresponded to protein kinase C. The second peak contained protein kinases that were "activated" dose-dependently by the okadaic acid class tumor promoters, okadaic acid and dinophysistoxin-1. This "activation" was not achieved by other tumor promoters, such as 12-0-tetradecanoyl-phorbol-13-acetate, teleocidin, aplysiatoxin, or palytoxin. In addition, the second peak contained phosphatases. The phosphate liberation from phosphorylated histone type III-S by incubation with the second peak was inhibited by okadaic acid or dinophysistoxin-1, dose-dependently. The resulting apparent "activation" of protein kinases by okadaic acid is indicated and would imply a new pathway of tumor promotion on mouse skin.     

Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Stimulation of Xenopus laevis oocyte maturation by methyl-beta-cyclodextrin

The cholesterol-depleting drug methyl-beta-cyclodextrin (Me-beta-CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5-50 mM Me-beta-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-beta-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [(3)H]cholesterol, 30-90 min of treatment with 5-50 mM Me-beta-CD removed 50%-70% of cell- associated label, and cholesterol depletion was not observed with alpha-cyclodextrin. After long-term (20-23 h) labeling of oocytes with [(3)H]cholesterol, Me-beta-CD treatment resulted in dose- dependent cholesterol depletion in the 5-50 mM range, and 50 mM Me-beta-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5-50 mM Me-beta-CD also decreased recovery of low-density membrane by detergent-free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.     

Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated membrane currents in the Xenopus laevis oocyte.

External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.     

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis.

L-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na+ dependent and presumably coupled to Na+ gradient. Our results indicate that progesterone (10(-6) M) Blocks abruptly, around the germinal vesicle breakdown, the saturable transport of L-leucine. p-Chloromercuribenzoate (10(-4) M) induces maturation and after a short lag of time strongly inhibits L-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.     

The protein-phosphatase inhibitor okadaic acid mimics MSH-induced and melatonin-reversible melanosome dispersion in Xenopus laevis melanophores.

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 microM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I-VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III-VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.