title: Identification of a novel phosphatidic acid binding domain in protein phosphatase-1.
abstract: 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 (gamma isoform) of protein phosphatase-1 (PP1c gamma) 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 PP1c gamma. 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 PP1c gamma 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 Ki 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 PP1c gamma residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1c gamma fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1c gamma 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 PP1c gamma 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 PP1c gamma that contains a unique loop-strand structural fold responsible for the interaction with PA.
authors.affiliation: Molecular and Cellular Biology and Pathobiology Program, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Jones
authors.forename: Robert
authors.initials: R
authors.lastname: Rawles
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010712:Phosphatidic Acids / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D008055:Lipids; D010712:Phosphatidic Acids; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1