Ras/mitogen-activated protein kinase mediates norepinephrine-induced phospholipase D activation in rabbit aortic smooth muscle cells by a phosphorylation-dependent mechanism.
Phospholipase D (PLD) activity is regulated by phosphatidylinositol 4,5-biphosphate, protein kinase C (PKC), ADP-ribosylation factor, and Rho. The present study was designed to investigate the mechanism of norepinephrine (NE)-mediated PLD activation in rabbit aortic vascular smooth muscle cells (VSMC). NE (10 microM) caused activation of PLD, as measured by the production of phosphatidylethanol in [(3)H]oleic acid-labeled cells. NE also increased PKC activity in VSMC. However, treatment of cells with bisindolylmaleimide, a PKC inhibitor, or long-term treatment with phorbol-12-myristate-13-acetate that depletes PKC did not decrease NE-induced activation of PLD. NE-stimulated PLD activity was attenuated by farnesyl transferase inhibitors (FPT III and SCH-56582), which reduce activation of both Ras and mitogen-activated protein (MAP) kinase. Moreover, transfection of VSMC with a dominant negative Ras resulted in inhibition of NE-stimulated MAP kinase and PLD activities. Treatment of cells with PD-98059, a MAP kinase kinase inhibitor, also reduced NE-stimulated PLD activity. These data suggest that NE-stimulated PLD activity is mediated via activation of Ras and MAP kinase in rabbit VSMC. To study the mechanism of activation of PLD by Ras/MAP kinase, NE-induced phosphorylation of PLD was examined. In VSMC, PLD of molecular mass 120 kDa was identified with polyclonal PLD antibody. Phosphorylation of PLD by NE, measured as (32)P incorporation into PLD, was inhibited by PD-98059. Moreover, PLD immunoprecipitated from VSMC lysates was phosphorylated in vitro by MAP kinase. Collectively, these results show a novel pathway for activation of PLD that appears to be mediated through Ras/MAP kinase pathway by a mechanism involving phosphorylation.
Muthalif
M M
MM
Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis, Tennessee, USA.
Parmentier
J H
JH
Benter
I F
IF
Karzoun
N
N
Ahmed
A
A
Khandekar
Z
Z
Adl
M Z
MZ
Bourgoin
S
S
Malik
K U
KU
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D011905:Genes, ras / Q000235:genetics*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009638:Norepinephrine / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D001616:beta-Galactosidase / Q000378:metabolism
D000316:Adrenergic alpha-Agonists; D020928:Mitogen-Activated Protein Kinases; D010739:Phospholipase D; D001616:beta-Galactosidase; D002118:Calcium; D009638:Norepinephrine