Characterization of Two Alternately Spliced Forms of Phospholipase D1

Abstract

We previously reported the cloning of a cDNA encod- ing human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Ham- mond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640–29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585– 624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and pu- rified to homogeneity by detergent extraction and im- munoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2- dependent but insensitive to changes in free Ca2 concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-S-liganded ADP-ribosylation fac- tor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-bind- ing proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggest- ing that these three classes of regulators interact with different sites on the enzyme.