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N S Duesbery

Publications and source records attributed to N S Duesbery.

10 recordsLinked to original sources
PubMed2005

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes.

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.

Animals↗
PubMed2001

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways.

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

3T3 Cells↗
PubMed1999

Anthrax toxins.

Though its lethal effects were ascribed to an exotoxin almost half a century ago, the pathogenesis of anthrax has yet to be satisfactorily explained. Subsequent work has led to the molecular identification and enzymatic characterization of three proteins that constitute two anthrax toxins. Protective antigen binds an as yet unknown cell receptor and mediates the entry of the other two components to the cytoplasm via the endosomal pathway. Edema factor, so named for its ability to induce edema, is a Ca2+/calmodulin-dependent adenylate cyclase. Lethal factor, the dominant virulence factor associated with the toxin, proteolytically inactivates mitogen-activated protein kinase kinases, key players in signal transduction. We describe the fascinating work that has led to these discoveries and discuss their relevance to our understanding of the pathogenesis of anthrax.

Animals↗
PubMed1999

Anthrax lethal factor causes proteolytic inactivation of mitogen-activated protein kinase kinase.

A search of the National Cancer Institute's Anti-Neoplastic Drug Screen for compounds with an inhibitory profile similar to that of the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059 yielded anthrax lethal toxin. Anthrax lethal factor was found to inhibit progesterone-induced meiotic maturation of frog oocytes by preventing the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Similarly, lethal toxin prevented the activation of MAPK in serum stimulated, ras-transformed NIH3T3 cells. In vitro analyses using recombinant proteins indicated that lethal factor proteolytically modified the NH2-terminus of both MAPKK1 and 2, rendering them inactive and hence incapable of activating MAPK. The consequences of this inactivation upon meiosis and transformed cells are also discussed.

3T3 Cells↗
PubMed1998

Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

Animals↗
PubMed1998

Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.

The title of this article is taken from a 1971 publication by Yoshio Masui and Clement Markert in which they describe the discoveries of the meiotic regulatory activities maturation promoting factor and cytostatic factor. Here we review the experiments that led to these discoveries and discuss their relation to our current knowledge of the biochemistry of oocyte maturation.

Animals↗
PubMed1997

CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II.

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from mos-deficient (MOS-/-) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.

Animals↗
PubMed1996

The role of microtubules and inositol triphosphate induced Ca2+ release in the tyrosine phosphorylation of mitogen-activated protein kinase in extracts of Xenopus laevis oocytes.

Microsomal fractions of Xenopus oocytes release preloaded 45 Ca2+ when treated with inositol triphosphate (InsP3). The effective concentration of InsP3 required for half-maximal release (EC50) is 59 nM and maximal release occurs at approximately 2 microM InsP3. Uptake and release of 45 Ca2+ are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50 for InsP3-induced Ca2+ release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+ release causes the tyrosine phosphorylation of a 42,000 (M(r) 42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+ release, thereby inhibiting phosphorylation of MAP kinase.

Animals↗
PubMed1993

Changes in protein association with intracellular membranes of Xenopus laevis oocytes during maturation and activation.

Intracellular membranes isolated from fully grown immature oocytes, mature oocytes (eggs) and activated eggs of Xenopus laevis were fractionated through a discontinuous sucrose density gradient into light, intermediate and heavy fractions. Electron microscopy showed that the light and intermediate fractions consisted mainly of smooth membranes, while the heavy fraction consisted mainly of rough membranes and mitochondria. Variations in the proteins associate with samples taken at different stages were observed by SDS-PAGE. The following differences were consistently observed: a 200 kDa protein was present only in the intermediate fraction of activated eggs, 29 and 44 kDa proteins were present only in the intermediate fractions of immature oocytes and activated eggs, and 120 and 145 kDa proteins were present only in the heavy fractions of mature oocytes and activated eggs. Examination of Western blots showed that cyclins A and B2 did not associate with membrane fractions at any stage of meiosis. Instead, cyclin A was present in the cytosols of mature oocytes and cyclin B2 was present in the cytosols of immature and mature oocytes. c-mos protein was detected in the cytosols and occasionally in the light fractions of mature oocytes and activated eggs. While alpha- and beta-tubulins were detected in the light and intermediate fractions at all the stages of meiosis examined, only beta-tubulin was present in the heavy fraction. beta-tubulin present in the heavy fraction was detected only at interphase, i.e. in immature oocytes and activated eggs, and not in mature oocytes. Using immunogold labelling we confirmed these results and found evidence to suggest that beta-tubulin associates with the rough endoplasmic reticulum of interphase cells by a linking protein.

Animals↗