MOLECULAR-CLONING OF 2 ALTERNATIVELY SPLICED FORMS OF HUMAN PHOSPHATIDIC-ACID PHOSPHATASE CDNA THAT ARE DIFFERENTIALLY EXPRESSED IN NORMAL AND TUMOR-CELLS

Phosphatidic acid (PA) and diacylglycerol (DG) are lipids involved in signal transduction and in structural membrane-lipid biosynthesis in c ells. Phosphatidic acid phosphatase (PAP) catalyzes the conversion of PA to DG. This enzyme exists in at least two isoforms, one of which (P AP1) is presumed to be cytosolic and membrane associated and the other (PAP2) to be an integral membrane protein. Homology search of the Gen Bank database using a murine sequence probe enabled the cloning of sev eral putative human isoenzymes. Two isoforms, presumed to be alternati ve splice variants from a single gene, designated as PAP2-alpha 1 and PAP2-alpha 2, have been cloned and expressed. The PAP2-alpha 1 and PAP 2-alpha 2 have a 84% and a 72% overall match, respectively, with the p ublished mouse PAP amino acid sequence. The area of alternative exon u sage was confined to the coding region at amino acids 20 to 70. Ectopi c expression of PAP2-alpha 1 and PAP2-alpha 2 cDNAs in ECV304 endothel ial 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 meas ured the conversion of [C-14]PA to [C-14]DG. The increase in PAP activ ity in PAP2-alpha-transfected cells correlated with a >50% decrease in the steady-state PA level. Northern analysis showed that PAP2-alpha m RNA 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 PA P2-alpha than in matching normal colon tissue. Considering these data and previous demonstrations that certain transformed cell lines have l ower PAP activity, we suggest that human PAP cDNAs may be candidates f or gene therapy for certain tumors.