Characterization of human PLD2 and the analysis of PLD isoform splice variants.
Phospholipase D (PLD) cleaves phosphatidylcholine in response to a variety of cell stimuli to release phosphatidic acid, which is associated with a number of cellular responses including regulated secretion, mitogenesis, and cytoskeletal changes. Recent advances in this field include the reports of cDNA sequences for two mammalian PLD isoforms: human PLD1 and rodent PLD2. We report the characterization of cDNA encoding human PLD2. In these experiments, we uncovered alternate splice variants of both human isoforms and evaluated the relative abundance of these messages by reverse transcriptase polymerase chain reaction, thereby indicating the physiologically relevant forms. Further, Northern hybridization experiments defined the tissue distribution of the human PLD messages. Human PLD1 does not appear to be an abundant message in any tissue tested whereas levels of human PLD2 mRNA apparently were higher and more variable. The specific activity and regulation of recombinant human PLD2 are indistinguishable from that of recombinant mouse PLD2. Analysis of the amino acid sequences of both human isoforms revealed important putative Pleckstrin homology domains and identified additional members of the PLD gene family that help to delimit the catalytic domain. The presence of Pleckstrin homology domains in the PLDs resolves several contradictory observations regarding PLD regulation and the domain structure of the proteins.
Novartis Institute for Biomedical Research, Summit, New Jersey 07901, USA. paul.steed@pharma.novartis.com
P M
PM
Steed
K L
KL
Clark
W C
WC
Boyar
D J
DJ
Lasala
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics*; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; C105606:phospholipase D2; D010739:Phospholipase D