title: The Pleckstrin homology domains of phospholipases C-beta and -delta confer activation through a common site.
abstract: Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA.
authors.forename: Yuanjian
authors.initials: Y
authors.lastname: Guo
authors.forename: Finly
authors.initials: F
authors.lastname: Philip
authors.forename: Suzanne
authors.initials: S
authors.lastname: Scarlata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D054799:Phospholipase C beta; D054803:Phospholipase C delta; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; C518036:PLCB2 protein, human; C518024:PLCD1 protein, human; D054799:Phospholipase C beta; D054803:Phospholipase C delta