Regulation of phospholipase D activity by RalA and protein kinase C in cell transformation.
Item
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Title
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Regulation of phospholipase D activity by RalA and protein kinase C in cell transformation.
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Identifier
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AAI9917661
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identifier
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9917661
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Creator
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Hornia, Armand J.
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Contributor
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Adviser: David A. Foster
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Date
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1999
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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, Molecular | Biology, Cell
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Abstract
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We extend previous studies from cells overexpressing the non receptor c-src kinase to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor, 3Y1 EGFR. Unlike c-Src overexpressing cells, downregulation of PKC isoforms with TPA did not transform the EGFR cells. Treatment of EGFR cells with EGF transforms these cells. We examined the effects of PKC alpha- and PKC delta-specific inhibitors and the expression of dominant negative mutants of PKC alpha and delta. Both the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and inhibition of PKC delta. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity. The elevation of PLD activity in response to inhibiting PKC delta, like transformation, was dependent upon PKC alpha and restricted to the 3Y1 EGFR overexpressing cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects upon both transformation and PLD activity.;The GTPase RalA exists in a complex with PLD and is required for the activation of PLD activity by oncogenic Src and Ras. Upon EGF treatment, RalA is activated and its activation is dependent upon Ras. The activation of PLD by EGF was dependent upon both HaRas and RalA. The transformed phenotype induced by EGF was also dependent upon RalA; Overexpression of wild type RalA or an activated RalA mutant transformed the 3Y1 EGFR cells in the absence of EGF. The involvement of RalA suggested that elevated PLD activity might explain the RalA dependence for transformation of the EGFR cells in response to EGF. To establish a role for PLD in EGF signaling, 3Y1 EGFR cells were stably transfected with a PLD1 expression vector. EGFR cells not only tolerated expression of PLD but they were transformed even in the absence of EGF. These data indicate that PKC and the Ras/RalA/PLD signaling pathway is an essential component of the growth and transforming properties of EGF.
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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.
