Regulation of phospholipase D activity by small GTPases.
Item
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Title
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Regulation of phospholipase D activity by small GTPases.
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Identifier
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AAI9807963
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identifier
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9807963
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Creator
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Luo, Jingqing.
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Contributor
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Adviser: David A. Foster
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular
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Abstract
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The hydrolysis of phosphatidylcholine by phospholipase D (PLD) may generate several lipid second messengers or mediators including phosphatidic acid, lysophosphatidic acid and diacylglycerol. We investigated the activation of PLD by v-Src oncogene and demonstrated a requirement for the GTPase cascade of Ras and Ral. Another small GTPase Arf, previously implicated in vesicle transport, was also found to be involved in the activation of PLD by v-Src.;The data indicate that PLD activity associates with RalA in v-Src-transformed cells in vivo and in vitro. Overexpression of RalA potentiated PLD activation by v-Src, and expression of dominant negative RalA mutants inhibited both v-Src-and v-Ras-induced PLD activity. These data indicate that RalA is involved in the tyrosine kinase activation of PLD and that PLD is a downstream target of a Ras/Ral GTPase cascade. However, the association between RalA and PLD is not sufficient for PLD activation, another factor is required. We found the presence of Arf in RalA precipitates from v-Src transformed cell membrane lysates. Arf increased the RalA-associated PLD activity and contributed to differential activity of PLD associated with RalA mutants. The data also suggested that the N-terminal end of RalA is important for the binding of Arf and for PLD activation. Since the interaction between RalA and Arf is very weak, other molecules may be required. We also characterized the RalA associated PLD activity in v-Src transformed cells by examining the association of RalA with a purified PLD1 preparation and with a human PLD1 that was overexpressed in sf9 insect cells infected with baculovirus expressing a cloned hPLD1. Several lines of evidence demonstrate that the PLD associated with RalA in v-Src transformed cells is the Arf-responsive, PIP{dollar}\sb2{dollar}-dependent PLD1 and that this interaction is direct. Since Arf and PLD have been implicated in vesicle transport, the apparent involvement of Arf and PLD in the intracellular signals activated by v-Src suggest a role for vesicle transport in signal transduction and Arf may serve as a mediator of these two cellular functions.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
