Role of Hippocalcin, a calcium binding protein, in Phospholipase D Activation by Lysophosphatidic acid

Abstract

We isolated a 22kDa protein from rat brain that involved in the activation of phospholipsae D(PLD), and identified the protein as hippocalcin using peptide sequencing. We have suggested that hippocalcin is involved in PKC-independent Ca2+-induced PLD activation. But the function of hippocalcin for PLD activation is still not resolved. Hippocalcin is a specific protein expressed mainly in the pyramidal nerve cells of the hippocampus that has three EF-hand structural Ca2+ binding domains and a myristoylation site in the amino terminal. In attempt to explore the effect of hippocalcin in facilitating Cdc42-activated PLD activation, we investigated PLD activity in the hippocalcin/Cdc42 co-transfected NIH3T3 cells and the translocation of hippocalcin in EGFP-hippocalcin transfected NIH3T3 cells using flourescence microscopy. When intracellular Ca2+ level was increased by lysophosphatidic acid (LPA), hippocalcin was translocated to the cell membrane. LPA treatment increased PLD activity about 2.3 fold in hippocalcin/Cdc42 co-transfected NIH3T3 cells compared to pcDNA3.1-transfected NIH3T3 cells. LPA also induced shape changes of EGFP-hippocalcin transfected NIH3T3 cells showing the pseudopodia formation and cytoskeletal remodeling. U73122, an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C (PI-PLC) and BAPTA-AM, a chelator of intracellular Ca2+ inhibited LPA-induced PLD activation and cellular morphological change, while EGTA, a chelator of extracellular Ca2+ had little effect on the LPA-induced PLD activation and cellular morphological change in the hippocalcin/Cdc42 co-transfected NIH3T3 cells. These results suggest that hippocalcin is involved in the Ca2+-mediated PLD activation pathway via PI-PLC in conjunction with Cdc42 and increased PLD activity is implicated in the cellular morphological change by resulting in the change in lipid bilayer properties, membrane trafficking, and cytoskeletal remodeling.