Molecular Cloning of Two Alternatively Spliced Forms of Human Phosphatidic Acid Phosphatase cDNAs that Are Differentially Expressed in Normal and Tumor Cells

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Abstract
Phosphatidic acid (PA) and diacylglycerol (DG) are lipids involved in signal transduction and in structural membrane-lipid biosynthesis in cells. Phosphatidic acid phosphatase (PAP) catalyzes the conversion of PA to DG. This enzyme exists in at least two isoforms, one of which (PAP1) is presumed to be cytosolic and membrane associated and the other (PAP2) to be an integral membrane protein. Homology search of the GenBank database using a murine sequence probe enabled the cloning of several putative human isoenzymes. Two isoforms, presumed to be alternative splice variants from a single gene, designated as PAP2-alpha1 and PAP2-alpha2, have been cloned and expressed. The PAP2-alpha1 and PAP2-alpha2 have a 84% and a 72% overall match, respectively, with the published mouse PAP amino acid sequence. The area of alternative exon usage was confined to the coding region at amino acids 20 to 70. Ectopic expression of PAP2-alpha1 and PAP2-alpha2 cDNAs in ECV304 endothelial cells led to a 6- to 8-fold and a 2-fold increase in PAP activity, respectively, in cell-free extracts using an in vitro assay that measured the conversion of [14C]PA to [14C]DG. The increase in PAP activity in PAP2-alpha-transfected cells correlated with a >50% decrease in the steady-state PA level. Northern analysis showed that PAP2-alpha mRNA expression was suppressed in several tumor tissues, notably those derived from the lower alimentary tract. Subsequent analysis of colon tumor tissue derived from four donors confirmed lower expression of PAP2-alpha than in matching normal colon tissue. Considering these data and previous demonstrations that certain transformed cell lines have lower PAP activity, we suggest that human PAP cDNAs may be candidates for gene therapy for certain tumors.