Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2.
Phospholipase D (PLD) has been implicated in a variety of cellular processes including vesicular transport, the respiratory burst, and mitogenesis. PLD1, first cloned from human, is activated by small GTPases such as ADP-ribosylation factor (ARF) and RhoA. Rodent PLD2, which is approximately 50% identical to PLD1 has recently been cloned from mouse embryo (Colley, W., Sung, T., Roll, R., Jenco, J., Hammond, S., Altshuller, Y., Bar-Sagi, D., Morris, A., and Frohman, M. (1997) Curr. Biol. 7, 191-201) and rat brain (Kodaki, T., and Yamashita, S. (1997) J. Biol. Chem. 272, 11408-11413). We describe herein the cloning from a B cell library and expression of human PLD2 (hPLD2). The open reading frame is predicted to encode a 933-amino acid protein (Mr of 105,995); this corresponds to the size of the protein expressed in insect cells using recombinant baculovirus. The deduced amino acid sequence shows 53 and 90% identity to hPLD1 and rodent PLD2, respectively. The mRNA for PLD2 was widely distributed in various tissues including peripheral blood leukocytes, and the distribution was distinctly different from that of hPLD1. hPLD1 and hPLD2 both showed a requirement for phosphatidylinositol 4,5-bisphosphate. Both isoforms showed optimal activity at 10-20 mol % phosphatidylcholine in a mixed lipid vesicle system and showed comparable basal activities in the presence of phosphatidylinositol 4,5-bisphosphate. Unexpectedly, ARF-1 stimulated the activity of hPLD2 expressed in insect cells about 2-fold, compared with a 20-fold stimulation of hPLD1 activity. Thus, not only PLD1 but also hPLD2 activity can be positively regulated by both phosphatidylinositol 4,5-bisphosphate and ARF.
Lopez
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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Arnold
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RS
Lambeth
J D
JD
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
D018076:DNA, Complementary; D010713:Phosphatidylcholines; D012333:RNA, Messenger; C105606:phospholipase D2; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors