title: Propofol activates and allosterically modulates recombinant protein kinase C epsilon.
abstract: BACKGROUND
  Myocardial protection by anesthetics is known to involve activation of protein kinase C epsilon (PKC epsilon). A key step in the activation process is autophosphorylation of the enzyme at serine 729. This study's objectives were to identify the extent to which propofol interacts with PKC epsilon and to identify the molecular mechanism(s) of interaction.
  
  
  METHODS
  Immunoblot analysis of recombinant PKC epsilon was used to assess autophosphorylation of PKC epsilon at serine 729 before and after exposure to propofol. An enzyme-linked immunosorbant assay kit was used for measuring PKC epsilon activity. Spectral shifts in fluorescence emission maxima of the C1B subdomain of PKC epsilon in combination with the fluorescent phorbol ester, sapintoxin D, was used to identify molecular interactions between propofol and the phorbol ester/diacylglycerol binding site on the enzyme.
  
  
  RESULTS
  Propofol (1 microM) caused a sixfold increase in immunodetectable serine 729 phosphorylated PKC epsilon and increased catalytic activity of the enzyme in a dose-dependent manner. Dioctanoylglycerol-induced or phorbol myristic acetate-induced activation of recombinant PKC epsilon activity was enhanced by preincubation with propofol. Both propofol and phorbol myristic acetate quenched the intrinsic fluorescence spectra of the PKC epsilon C1B subdomain in a dose-dependent manner, and propofol caused a further leftward-shift in the fluorescence emission maxima of sapintoxin D after addition of the C1B subdomain.
  
  
  CONCLUSIONS
  These results demonstrate that propofol interacts with recombinant PKC epsilon causing autophosphorylation and activation of the enzyme. Moreover, propofol enhances phorbol ester-induced catalytic activity, suggesting that propofol binds to a region near the phorbol ester binding site allowing for allosteric modulation of PKC epsilon catalytic activity.
authors.affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio 44242, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Wickley
authors.forename: Ryo
authors.initials: R
authors.lastname: Yuge
authors.forename: Brad A
authors.initials: BA
authors.lastname: Martin
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Meyer
authors.forename: Derek S
authors.initials: DS
authors.lastname: Damron
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D007313:Insecta; D008969:Molecular Sequence Data; D015742:Propofol / Q000494:pharmacology*; D051744:Protein Kinase C-epsilon / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D051744:Protein Kinase C-epsilon; D015742:Propofol