α1‐Adrenergic Activation of ERK by Phosholipase D is Ras Dependant

Abstract

Phospholipase D (PLD) is considered an important signaling intermediate in many growth factor pathways. PLD converts phosphatidylcholine into choline and phosphatidic acid, (PA). The PA generated by PLD is thought to recruit Raf to the lipid rafts of cell membranes leading to stimulation of growth factor signaling complexes. Previous experiments in our laboratory have shown that the addition of primary butanol inhibits ERK activation by blocking PA production. While this work suggests that PLD is involved in the activation of the ERK signaling pathway if doesn’t explain its mechanism. To investigate the role of PLD in ERK activation, two short-chain, cell-permeable phosphatidic acids (1,2-Dihexanoyl-sn-Glycero-3-Phosphate and 1,2-Dilauroyl-sn-Glycero-3-Phosphate) were incubated at several times and concentrations with CCL39 Chinese hamster lung fibroblasts. Both short chain phosphatidic acids (scPA) are able to act as endogenously added PLD product. Both the 6 and 12 acyl scPA stimulated ERK activation in a dose and time dependent manner with maximum ERK activation observed with the 6 acyl scPA. Ras activation pull down assays conducted with phenylephrine (PE) stimulating cells showed Ras activation. When a primary butanol was added prior to agonist stimulation, PE did not activate Ras. Additional evidence for a Ras dependent PLD-ERK activation was determined with the use of dominant negative Ras (D/N Ras). Expression of D/N Ras blocked activation of ERK by PE. The ability of D/N Ras to inhibit PA activation of ERK was also investigated. This is a novel mechanism for PLD involvement in growth factor pathways. This work was supported from a grant from the NIH, Award number 1 R15 HL074924-01A1.