Identification of a novel phosphatidic acid binding domain in protein phosphatase-1

Phosphatidic acid (PA) has been recognized as a lipid second messenger, yet few cellular targets for PA have been identified. Previous work demonstrated PA as a potent and noncompetitive tight-binding inhibitor of the catalytic subunit (γ isoform) of protein phosphatase-1 (PP1cγ) in vitro. The high potency of inhibition, coupled with high specificity for PA over other phospholipids, suggested the presence of a high-affinity PA binding domain on PP1cγ. In the current study, quantification of the binding interaction and identification of the binding domain were pursued. Surface plasmon resonance was employed to quantitate the interaction between PP1cγ and immobilized mixed lipid vesicles of PA/phosphatidylcholine (PC) or PC alone. The data disclosed a high-affinity interaction with a KD measured in the low (1-40) nanomolar range, consistent with the range of K_i previously obtained from in vitro enzymatic assays. Next, identification of the segment of PP1 necessary for PA binding was determined using a deletion mutagenesis strategy. Binding assays revealed that PP1cγ residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1cγ fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1cγ residues 286-296 were sufficient to confer PA binding to GFP, a protein that does not interact with PA. The minimal PA binding domain of PP1cγ lacked similarity to the previously described PA binding segments of Raf-1 kinase and cyclic-AMP phosphodiesterase 4A1. When these results were taken together with the known crystallographic structure of PP1, they identified a novel PA binding region on PP1cγ that contains a unique loop-strand structural fold responsible for the interaction with PA.