Localization and regulation of phospholipase D in mitogenic signaling.
Item
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Title
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Localization and regulation of phospholipase D in mitogenic signaling.
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Identifier
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AAI3083721
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identifier
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3083721
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Creator
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Xu, Lizhong.
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Contributor
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Adviser: David A. Foster
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Date
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2003
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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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Chemistry, Biochemistry | Biology, Molecular | Biology, Cell
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Abstract
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Phospholipase D (PLD), a cell membrane enzyme that catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline, is activated by agonists that initiate cell signaling and by oncogenes that transform cells. But the mechanism is not clearly defined and the membrane localization of the mitogenic PLD is still debatable.;We found that the elevated PLD activity in NIH 3T3 cells transformed by activated oncogenic forms of Src, Ras, and Raf or in cells stimulated by epidermal growth factor (EGF) is largely restricted to the caveolin-enriched membrane microdomains (CEMMs). The apparent PLD substrate specificity in those cells for PC lacking polyunsaturated acyl groups is explained by the localization of PLD activity in the CEMMs where PC labeled with polyunsaturated fatty acids was excluded. Although both PLD1 and PLD2 were found in CEMMs, neither was particularly enriched in the CEMMs of the transformed relative to the parental cells. We suggest that PLD1 and PLD2 may work together in response to mitogenic signals initiated in the CEMMs where many signaling molecules co-localize.;We also found that PLD activity is elevated by the oncogenic H-Ras, but not K-Ras. Consistent with the previous report that Ral is required for PLD activity in H-Ras-transformed cells, we found that RalA was activated in H-Ras- but not in K-Ras-transformed cells. In cells transformed by H-Ras, increased levels of ARF6 interacted with RalA. ARF6 but not ARF1 co-localized with RalA in CEMMs. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited mitogenic PLD activity. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity; however, expression of both activated RalA and activated ARF6 in NIH3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.;This work has underscored the mechanism of the regulation of PLD in the Ras pathway. New models of the dynamic regulation of PLD in signaling cascades and the localization of Ras and PLD on the membrane are provided.
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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.
