title: Substrate-induced translocation of PKC-alpha to the membrane.
abstract: The protein kinase C-alpha (PKC-alpha)-catalyzed phosphorylation of the peptide [Arg]4-Tyr-Gly-Ser-[Arg]5-Tyr is independent of Ca2+ and phospholipid. The binding of this peptide to PKC-alpha induces a conformational change in the enzyme that results in the exposure of hydrophobic groups that subsequently insert into a membrane. Induction of a conformational change in the enzyme by this peptide is demonstrated by susceptibility to trypsin cleavage. Additionally, exposure of hydrophobic sites on the enzyme is shown by the binding of the fluorescent probes PRODAN and bis-ANS and by the partitioning of the enzyme into a Triton X-114-enriched phase. In the presence of a phospholipid bilayer containing phosphatidylserine, this peptide promotes the translocation of PKC-alpha to the membrane in the absence of Ca2+ as observed by increased resonance energy transfer between Trp on the enzyme and dansyl-groups attached to the lipid, as well as by changes in the intrinsic tryptophan fluorescence of the enzyme. Also, once bound to the membrane the peptide.PKC-alpha complex undergoes further conformational change which is evident by an increased sensitivity to trypsin cleavage at the hinge region. These results demonstrate that substrate binding can also induce translocation of PKC to the membrane and suggest that the removal of the pseudosubstrate domain is coupled to a conformational change in the enzyme that results in the exposure of hydrophobic groups.
authors.affiliation: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
authors.forename: R H
authors.initials: RH
authors.lastname: Bruins
authors.forename: R M
authors.initials: RM
authors.lastname: Epand
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D014357:Trypsin
chemical_list: D005456:Fluorescent Dyes; D007527:Isoenzymes; D008081:Liposomes; D009842:Oligopeptides; D010455:Peptides; C097606:acetyl-phenylalanyl-arginyl-arginyl-seryl-phenylalanyl-arginyl-leucinamide; C097605:acetyl-phenylalanyl-lysyl-lysyl-seryl-phenylalanyl-lysyl-leucinamide; C097604:arginyl-arginyl-arginyl-arginyl-tyrosyl-glycyl-seryl-arginyl-arginyl-arginyl-arginyl-arginyl-tyrosine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D014357:Trypsin