Regulation of human PLD1 and PLD2 by calcium and protein kinase C

Numerous studies show that PLD is activated in cells by calcium and by prot ein kinase C (PKC). We found that human PLD1 and PLD2 expressed in Sf9 cell s can be activated by calcium-mobilizing agonists and by co-expression with PKC alpha. The calcium-mobilizing agonists A23187 and CryIC toxin triggere d large increases in phosphatidylethanol (PtdEth) production in Sf9 cells o ver-expressing PLD1 and PLD2, but not in vector controls. PLD activation by these agonists was largely dependent on extracellular calcium. Membrane as says demonstrated significant PLD1 and PLD2 activity in the absence of diva lent cations, which could be enhanced by low levels of calcium either in th e presence or absence of magnesium, PLD1 but not PLD2 activity was slightly enhanced by magnesium. Treatment of Sf9 cells expressing PLD1 and PLD2 wit h PMA resulted in little PtdEth production. However, a significant and comp arable formation of PtdEth occurred when PLD1 or PLD2 were co-expressed wit h PKC alpha, but not PKC delta, and was further augmented by PMA. In contra st to PLD1, co-expressing PLD2 with PKC alpha or PKC delta further enhanced A23187-induced PtdEth production. Immunoprecipitation experiments demonstr ated that PLD1 and PLD2 associated with the PKC isoforms in Sf9 cells. Furt hermore, in membrane reconstitution assays, both PLD1 and PLD2 could be sti mulated by calmodulin and PKC alpha-enriched cytosol. The results indicate that PLD2 as well as PLD1 is subject to agonist-induced activation in intac t cells and can be regulated by calcium and PKC. (C) 2000 Elsevier Science B.V. All rights reserved.