Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase

Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kina se in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the seri ne residue at position 911 was converted into alanine (S911A). The kinase c ould be enriched from detergent-extracted brain membranes and bind and phos phorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel k inase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preli minary analysis of the phosphorylation sites on PLD 1 suggested that the ki nase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta -D-ribofuranosyl-benzimidazole (DRB). Membrane frac tions from Chinese hamster ovary (CHO) cell lines that inducibly express PL D 1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came f rom observations that immunoprecipitates using PLD1 antibodies contained im munoreactive CK2 alpha, and immunoprecipitates using CK2 alpha antibodies c ontained immunoreactive PLD1. Go-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha, or beta, suggests that the associatio n of PLD1 with the kinase is through the beta subunit. Supporting this, pho sphorylation of PLD1 by purified recombinant CK2 alpha was enhanced by puri fied recombinant CK2 beta. Assays measuring PLD1 catalytic activity followi ng phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro.