Role of hippocalcin in Ca2+ -induced activation of phospholipase D

The role of hippocalcin as a novel mediator in the PKC-independent Ca2+ -induced phospholipase D (PLD) activation pathway was investigated. Hippocalcin was expressed in the Sf9 insect cell expression system because the myristoylation of this protein is essential for its function. PLD and Cdc42 proteins were prepared from a rat brain cell membrane and cytosol, respectively. The recombinant hippocalcin was expressed in the Sf9 cell using expression vector pVL1393. The hippocalcin expressed was purified as a single band on PAGE following the hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. The molecular size of the rat brain hippocalcin expressed in this system was estimated to be 22 kDa. Myristoylated hippocalcin migrated faster than the non-myristoylated form on SDS-PAGE. Less than 10% of the total hippocalcin expressed was myristoylated in this baculovirus expression system. PLD was extracted from rat brain membranes and chromatographically enriched 70-fold. From the rat brain cytosol, Cdc42 was purified to near homogeneity. While hippocalcin alone did not activate PLD, it increased PLD activity activated with Cdc42 1.8-fold in the presence of calcium (300 nM free calcium). In the absence of calcium in the reaction mixture, the effect of hippocalcin to facilitate Cdc42-activated PLD activity was abolished. This result suggests that hippocalcin might be one of the regulatory proteins in the PKC-independent Ca2+ -mediated PLD activation pathway in conjunction with the Cdc42 protein.