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

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Abstract
We have previously reported that a heat-stable activator for ganglioside metabolism, GM2 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 GM2 activator and various other regulators including ARF. PLD2 was potently stimulated in vitro by GM2 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 GM2 activator, suggesting a possible role for GM2 activator as a coupling factor. GM2 activator was also demonstrated to physically associate with PLD2 in a stoichiometric manner. Further, PMA stimulation of COS-7 cells overexpressing both GM2 activator and PLD2 resulted in a marked increase in the association of the two molecules. Interestingly, ARF association with PLD2 was greatly increased by GM2 activator. Moreover, GM2 activator enhanced PMA-induced PLD activity in a synergistic manner with ARF in streptolysin-O-permeabilized, cytosol-depleted HL-60 cells, suggesting that GM2 activator may regulate PLD in a concerted manner with other factors, including ARF, inside the cells.