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Abstract
We demonstrated previously that v-Src activates a phospholipase D (PLD) activity (Song, J., Pfeffer, L.M., and Foster, D.A. (1991) Mol. Cell. Biol. 11, 4903-4908) and that this activation is dependent upon a G protein(s) (Jiang H., Alexandropoulos, K., Song, J., and Foster, D.A. (1994) Mol. Cell. Biol. 14, 3676-3682). An in vitro PLD assay was developed to study G protein involvement in v-Src-induced PLD activity. Maximal PLD activity in membranes isolated from v-Src-transformed cells was dependent upon both GTP and cytosol. In this report, we present three lines of evidence demonstrating that v-Src-induced PLD activity is mediated by Ras. First, a neutralizing Ras monoclonal antibody (Y13-259) inhibits PLD activity in membranes isolated from v-Src-transformed Balb/c 3T3 cells. Second, immobilized Ras protein depleted cytosol of the ability to stimulate PLD activity. This effect was dependent upon preloading immobilized Ras with GTP. Last, expression of a dominant negative Ras mutant in v-Src-transformed cells reduced PLD activity to the level observed in the nontransformed parental cells. These data establish a novel role for Ras in the regulation of PLD activity.
Highlights
There is a strong correlation between the activation of phospholipase D (PLD)1 and mitogenesis (Boarder, 1994; Foster, 1993)
We demonstrated previously that v-Src activates a PLD activity that can be distinguished from the PLD activity induced by phorbol esters that activate protein kinase C (Song and Foster, 1993) and that this PLD activity is dependent upon a G protein(s) (Jiang et al, 1994a)
To examine the mechanism of PLD activation by v-Src, we developed an in vitro PLD assay to examine PLD activity in isolated membranes where greater than 90% of the increased PLD activity in v-Src-transformed cells fractionated
Summary
There is a strong correlation between the activation of phospholipase D (PLD)1 and mitogenesis (Boarder, 1994; Foster, 1993). PLD activity in membranes isolated from v-Src-transformed (Src) and parental Balb/c 3T3 (Balb) cells was determined in the presence and absence of cytosol and the nonhydrolyzable GTP analog GTPyS (10 J.LM). The synergistic effect of cytosol and GTPyS and the increased PLD activity in membranes from v-Src-transformed cells were not observed when the cells were prelabeled with [3Hlarachidonate, which is incorporated into phopspholipids (including phosphatidylcholine) not utilized by the PLD activated by v-Src (Song and Foster, 1993).
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