Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6.
Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 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.
Xu
Lizhong
L
Department of Biological Sciences, Hunter College of The City University of New York, New York, New York 10021, USA.
Frankel
Paul
P
Jackson
Desmond
D
Rotunda
Thuy
T
Boshans
Rita L
RL
D'Souza-Schorey
Crislyn
C
Foster
David A
DA
D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D005799:Genes, Dominant; D011905:Genes, ras / Q000235:genetics*; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020725:ral GTP-Binding Proteins
D000089982:ADP-Ribosylation Factor 6; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; C118120:Rala protein, mouse; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D020725:ral GTP-Binding Proteins