Regulation of mammalian phospholipase D2: interaction with and stimulation by G(M2) 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.
Sarkar
S
S
Division of Biochemistry, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Miwa
N
N
Kominami
H
H
Igarashi
N
N
Hayashi
S
S
Okada
T
T
Jahangeer
S
S
Nakamura
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D049289:G(M2) Activator Protein; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D049289:G(M2) Activator Protein; D007527:Isoenzymes; D011506:Proteins; D011994:Recombinant Proteins; C105606:phospholipase D2; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate