The phosphorylation sites in phospholipase D2 (PLD2) induced by activation of protein kinase C alpha (PKC alpha) 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 phospborylation. However, the PLD2 activity, and the binding between PKC alpha and PLD2 were unaffected by the mutations. We conclude that phosphorylation of these residues is not required for PLD2 activation by PKC alpha, and that protein-protein interaction between PLD2 and PKCa is sufficient to activate PLD2. (c) 2005 Elsevier Inc. All rights reserved.