HUMAN TYPE-2 PHOSPHATIDIC-ACID PHOSPHOHYDROLASES - SUBSTRATE-SPECIFICITY OF THE TYPE 2A, 2B, AND 2C ENZYMES AND CELL-SURFACE ACTIVITY OF THE 2A ISOFORM

Phosphatidic acid (PA), lysophosphatidic acid, ceramide l-phosphate (C IP), and sphingosine l-phosphate (S1P) are lipid mediators generated b y phospholipases, sphingomyelinases, and lipid kinases, The major path way for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types 1 and 2) of phosphohydrolase activities (PAPs), cDNAs encoding two type 2 PAPs, PAP-2a and -2b, have been expr essed by transient transfection and shown to catalyze hydrolysis of PA , CIP, and SIP (Kai, M., Wada, I., Imai, S., Sakane, F. and Kanoh, H. (1997) J. Biol. Chem. 272, 24572-24578), We report the cloning and exp ression of a third type 2 PAP enzyme (288 amino acids, predicted molec ular mass of 32.6 kDa), PAP-2c, which exhibits 54 and 43% sequence hom ology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low en dogenous PAP activity. Recombinant expression of the three PAP enzymes using baculovirus vectors produces dramatic increases in membrane-ass ociated Mg2+-independent, N-ethylmaleimide-insensitive PAP activity, E xpression of PAP-2a but not PAP-2b or -2c resulted in high levels of c ell surface PAP activity in intact insect cells. Kinetic analysis of P AP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C1P, a nd S1P presented in mixed micelles of Triton X-100 revealed difference s in substrate specificity and susceptibility to inhibition by sphingo sine, Zn2+, and propranol.