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2002 Scientific Report

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Developmental Cell

Developmental Cell Biology Laboratory Nicholas S. Duesbery, Ph.D. Dr. Duesbery received both his M.S. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada. Before his appointment at VARI, he served as a Postdoctoral Fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland (1996–1999). Dr. Duesbery joined VARI as a Scientific Investigator in April 1999. Staff Xudong Liang, M.D. Arun Prasad Chopra, Ph.D. Sherri Boone, B.S. Laboratory Members Visiting Scientist Jean-François Bodart, Ph.D. Students Jonathon Douglas Marie Graves Jeanine Myles Research Projects Our research group focuses on cellular aspects of oogenesis, meiosis, and mitosis in a variety of vertebrate model organisms. Using biochemical and molecular approaches, we seek to identify regulatory mechanisms involved in egg cell formation, fertilization, and early embryonic development, as well as to ascertain the roles of these mechanisms in human health and disease. Our current understanding of meiotic maturation in amphibian oocytes is largely based on extensive analyses of cell cycle regulation in Xenopus laevis. Immature amphibian oocytes, arrested at prophase of meiosis I, resume meiosis in response to hormonal stimulation. The resumption of meiosis is followed by germinal vesicle breakdown (GVBD) and the outward appearance of a white spot. Oocytes subsequently arrest at metaphase II as mature oocytes or eggs in anticipation of fertilization. In 1971, Masui and Markert found that cytoplasm from an egg, when injected into oocytes, was capable of inducing meiotic maturation. They thus concluded that eggs contain an activity, which they called maturation promoting factor (MPF), that was sufficient to induce maturation. Subsequent purification showed that MPF is a complex of two subunits: a catalytic subunit, p34 cdc2 , and a regulatory subunit, cyclin B. Since that initial report, MPF has been shown to be a universal regulator of entry into meiotic and mitotic metaphase. MPF in X. laevis oocytes possesses the ability to activate itself; injection of MPF from eggs can induce recipient oocytes to undergo GVBD in the presence of cycloheximide. The preexistence of cyclin B2 in immature oocytes may explain why amplification of MPF is observed even in the absence of protein synthesis. It has been proposed that X. laevis oocytes contain a precursor of MPF, called pre-MPF, that is activated by the injection of MPF. However, the mechanisms of autoamplification of MPF remain poorly understood. It has been shown that MPF can phosphorylate and activate its positive regulator Cdc25 in vitro and that this mechanism may play a role in the autoamplification of MPF. Still, even if Cdc25 phosphorylation by p34 cdc2 is required for the autoamplifying activity of MPF, it is not sufficient. Numerous kinases have also been implicated in the MPF autocatalytic loop; among them, members of the polokinase family have been involved in p34 cdc2 activation by phosphorylating Cdc25 and have been characterized in X. laevis. Mitogen-activated protein kinase (MAPK) is activated during meiotic maturation of X. laevis oocytes at the same time that maturation promoting factor is. Though it remains unclear whether MAPK is activated before MPF, interconnections exist between the MPF and MAPK pathways. MAPK kinase (MEK1) injection or constitutively active thiophosphorylated-MAPK injection into X. laevis oocytes can induce resumption of meiosis. The mechanisms by which MAPK activation induces meiotic maturation seem to involve Myt1 inhibition by p90rsk. Indeed, it has been shown that p90rsk, which is phosphorylated and activated by MAPK, is able 21

to bind to Myt1, to phosphorylate it, and thus to inactivate the protein. However, GVBD can occur even in the absence of MAPK activity, a fact that leads us to conclude that MAPK activity might not be required for meiosis I in X. laevis oocytes. Nevertheless, MAPK activity is required for reactivation of MPF and suppression of DNA replication between meiosis I and II. Despite the advantages of X. laevis as a model system, its amenability to genetic approaches is limited as a consequence of its pseudotetraploidy. The use of Xenopus tropicalis, a diploid member of the same genus, has been proposed as a way of circumventing this problem. In addition to its promise as a genetic model, X. tropicalis may also be useful as a comparative model to complement studies in X. laevis. Although X. tropicalis development superficially resembles that of X. laevis, it may not be assumed that this similarity holds at all levels, because these species evolutionarily diverged 30–100 million years ago. Consequently, we have compared the biochemical regulation of oocyte maturation in the two species, focusing on the regulation of MPF activation and MAPK activation in X. tropicalis. The time required for progesterone-induced maturation of X. tropicalis oocytes was shorter (GVBD50 = 148.8 ± 44 min) than that of X. laevis oocytes. The maturation of X. tropicalis oocytes was marked by the appearance of a white dot and then the formation of a dark ring coincident, respectively, with entry into meiosis I and the onset of anaphase I. As with X. laevis, X. tropicalis maturation required protein synthesis but not transcription. The activity of MPF during maturation first peaked at 0.67 GVBD50, transiently declined, and remained stable thereafter. Crude lysates and cytoplasmic extracts of mature X. tropicalis oocytes could induce immature oocytes to mature. X. tropicalis oocytes, however, appeared to lack stores of pre-MPF, because these extracts could not induce GVBD in the presence of protein synthesis inhibitors. MAPK activity increased in parallel with that of MPF but remained elevated after the first meiotic division. Whereas injection of constitutively active MEK2 triggered GVBD, MAPK appeared not to be required for GVBD in X. tropicalis oocytes. However, maturation in the absence of MAPK activation was delayed, and meiotic spindles failed to form. Our results indicate that the biochemical regulation of oocyte maturation in both of these species is similar in most respects, with the notable exception that X. tropicalis oocytes do not mature when injected with MPF in the presence of protein synthesis inhibitors. We are currently using a comparative approach to characterize the proteins present in MPF complexes isolated from X. laevis and X. tropicalis oocytes in order to identify elements required for MPF autoamplification. In the course of our studies, we serendipitously identified anthrax lethal factor (LF), a component of the toxin produced by Bacillus anthracis, as a proteolytic inhibitor of the MAPK pathway. Specifically, LF was found to remove seven amino acids from the amino terminus of MAPK kinase 1 (MEK1), the loss of which resulted in its inactivation. Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin on tumor cells. LF was very effective in inhibiting MAPK activation in V12 H-ras–transformed NIH 3T3 cells. Treatment of transformed cells in vitro with LF caused them to revert to a nontransformed morphology and also inhibited their ability to form colonies in soft agar and to invade Matrigel, without markedly affecting cell proliferation. In vivo, LF inhibited the growth of ras-transformed cells implanted in athymic nude mice—in some cases causing tumor regression—at concentrations that produced no apparent animal toxicity. Unexpectedly, LF also greatly decreased tumor neovascularization. These results demonstrate that LF potently inhibits ras-mediated tumor growth and is a novel, potential antitumor therapeutic. Current research efforts in our lab are designed to characterize the protein regions necessary for LF-MEK interaction, to identify regions of LF that are important for cleaving MEK, and to determine how this cleavage results in inactivation of MEK. Such information may allow us to develop drugs that would interfere with this interaction and ultimately block anthrax toxin activity. 22

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