PKN regulates phospholipase D1 through direct interaction

The association of phospholipase (PLD)-1 with protein kinase C-related protein kinases, PKN alpha and PKN beta, was analyzed. PLD1 interacted with PKN alpha and PKN beta in COS-7 cells transiently transfected with PLD1 and PKN alpha or PKN beta expression constructs. The interactions between endogenous PLD1 and PKN alpha or PKN beta were confirmed by co-immunoprecipitation from mammalian cells. In vitro binding studies using the deletion mutants of PLD1 indicated that PKN alpha directly bound to residues 228-598 of PLD1 and that PKN beta interacted with residues 1-228 and 228-598 of PLD1. PKN alpha stimulated the activity of PLD1 in the presence of phosphatidylinositol 4,5-bisphosphate in vitro, whereas PKN beta had a modest effect on the stimulation of PLD1 activity. The stimulation of PLD1 activity by PKN alpha was slightly enhanced by the addition of arachidonic acid. These results suggest that the PKN family functions as a novel intracellular player of PLD1 signaling pathway.