title: Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D.
abstract: Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.
authors.affiliation: Department of Pharmacology, University of Pittsburgh, E1354 Biomedical Science Tower, Pittsburgh, PA 15261, USA. qjw1@pitt.edu
authors.forename: Qiming J
authors.initials: QJ
authors.lastname: Wang
authors.forename: Tzan-Wei
authors.initials: TW
authors.lastname: Fang
authors.forename: Dazhi
authors.initials: D
authors.lastname: Yang
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Lewin
authors.forename: Johan
authors.initials: J
authors.lastname: Van Lint
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
mesh_terms: D000818:Animals; D054713:Bryostatins; D002460:Cell Line; D006801:Humans; D007783:Lactones / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D018942:Macrolides; D015240:Phorbol 12,13-Dibutyrate / Q000737:chemistry / Q000378:metabolism; D010703:Phorbol Esters / Q000737:chemistry* / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D018411:Spodoptera; D013329:Structure-Activity Relationship
chemical_list: D054713:Bryostatins; D007783:Lactones; D008024:Ligands; D018942:Macrolides; D010703:Phorbol Esters; D010743:Phospholipids; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; C089212:protein kinase D; D011493:Protein Kinase C