title: Identification of interaction sites of protein kinase Calpha on phospholipase D1.
abstract: Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCalpha to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCalpha strongly interacted with the amino acid sequence 1-318 at the N-terminus of PLD1 and weakly with the sequence 841-1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1-318 and 1-215 PLD1 fragments revealed that there were binding sites in the 1-49 N-terminal sequence and also in the 216-318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1-49 sequence and a further site in the 1-318 sequence. Both deletion mutants showed impaired activation by PKCalpha in vivo, but unchanged activation by active V(14)RhoA. These findings identify the 1-49 sequence is a major binding/activation site for PKCalpha on PLD1, but also indicate involvement of the PH domain.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Seunghyi
authors.initials: S
authors.lastname: Kook
authors.forename: John H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009154:Mutation; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate