Regulation of mammalian phospholipase D2: interaction with and stimulation by GM2 activator

We have previously reported that a heat-stable activator for ganglioside metabolism, G_M2 activator, potently stimulates ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) activity (presumably PLD1) in an in vitro system [Nakamura, Akisue, Jinnai, Hitomi, Sarkar, Miwa, Okada, Yoshida, Kuroda, Kikkawa and Nishizuka (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12249–12253]. However, little is known about the regulation of PLD2. In the present studies we have investigated the regulation of PLD2 by G_M2 activator and various other regulators including ARF. PLD2 was potently stimulated in vitro by G_M2 activator in a time- and dose-dependent manner. Neither ARF nor protein kinase C caused any significant changes in PLD2 activity. Importantly, PLD2 responsiveness to ARF was greatly enhanced by G_M2 activator, suggesting a possible role for G_M2 activator as a coupling factor. G_M2 activator was also demonstrated to physically associate with PLD2 in a stoichiometric manner. Further, PMA stimulation of COS-7 cells overexpressing both G_M2 activator and PLD2 resulted in a marked increase in the association of the two molecules. Interestingly, ARF association with PLD2 was greatly increased by G_M2 activator. Moreover, G_M2 activator enhanced PMA-induced PLD activity in a synergistic manner with ARF in streptolysin-O-permeabilized, cytosol-depleted HL-60 cells, suggesting that G_M2 activator may regulate PLD in a concerted manner with other factors, including ARF, inside the cells.