Regulation of
phospholipase D (
PLD) activity participating in signal transduction involves complex interactions with
small G-proteins (ARF, Rho) and
protein kinase C isoforms (PKCalpha). In SK-N-MC human
neuroblastoma cells,
phorbol ester (TPA) activation of
PLD was enhanced by overexpressing
myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with
PLD, we investigated
PLD isoform expression and activation by TPA and
GTPgammaS in intact and
digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated
PLD activity was higher in both intact and
digitonin-permeabilized M22 cells than in vector controls. In contrast,
GTPgammaS-stimulated
PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with
GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to
GTPgammaS, stimulation of
PLD mediated by
GTPgammaS was independent of PKC, and
PLD activation by PKC in permeabilized cells was
kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a
detergent-insoluble fraction (DIF);
GTPgammaS increased recovery of PLD2 in DIF. Disruption of
cholesterol-rich DIFs with
digitonin,
cyclodextrin or
filipin potentiated activation of
PLD by TPA. Our studies suggest that activation of
PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As
PLD activation by
GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with
G-protein-mediated mechanisms for regulation of
PLD.