Sites on phospholipase D2 phosphorylated by PKCα

The phosphorylation sites in phospholipase D2 (PLD2) induced by activation of protein kinase Cα (PKCα) in COS 7 cells were analyzed by mass spectrometry. Ser134, 146, and 243, and Thr72, 99/100, and 252 were identified. These sites were mutated to Ala and the double mutation of Ser243 and Thr252 eliminated the phosphorylation. However, the PLD2 activity, and the binding between PKCα and PLD2 were unaffected by the mutations. We conclude that phosphorylation of these residues is not required for PLD2 activation by PKCα, and that protein-protein interaction between PLD2 and PKCα is sufficient to activate PLD2.