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(Received for publication, December 18, 1996, and in revised form, February 12, 1997)
From the Department of Biochemistry, Gunma University School of
Medicine, Maebashi 371, Japan
Phospholipase D (PLD) is implicated in important
cellular processes such as signal transduction, membrane
trafficking, and mitosis regulation. Recently, cDNA for human
PLD1 (hPLD1) was cloned from HeLa cells (Hammond, S. M.,
Altshuller, Y. M., Sung, T-C., Rudge, S. A., Rose, K., Engebrecht, J.,
Morris, A. J., and Frohman, M. A. (1995) J. Biol.
Chem. 270, 29640-29643). hPLD1 is stimulated by
phosphatidylinositol 4,5-bisphosphate and the small GTP-binding protein
known as ADP-ribosylation factor 1. Here we report the cloning and
characterization of cDNA for a different type of PLD (rat PLD2
(rPLD2)) from rat brain. We synthesized highly degenerate amplimers
corresponding to the conserved regions of eukaryote PLDs and performed
polymerase chain reaction on a rat brain cDNA library. Using the
amplified sequence as the probe, we cloned a rat brain cDNA clone
that contained an open reading frame of 933 amino acids with an
Mr of 105,992. The deduced amino acid sequence
showed significant similarity to hPLD1 with a large deletion in the
middle of the sequence. When the sequence was expressed in the fission
yeast Schizosaccharomyces pombe, PLD activity was greatly
increased. The activity was markedly stimulated by phosphatidylinositol
4,5-bisphosphate, but not by ADP-ribosylation factor 1 and RhoA. Rat
brain cytosol known to stimulate small GTP-binding
protein-dependent PLD did not stimulate rPLD2 expressed in
S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and
testis, but at low levels in thymus, liver, and muscle. Only a
negligible level was found in spleen and pancreas. Thus rPLD2 is a
novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and
RhoA.
Volume 272, Number 17,
Issue of April 25, 1997
pp. 11408-11413
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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