Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids

J Biol Chem. 1995 Jun 16;270(24):14679-84. doi: 10.1074/jbc.270.24.14679.

Abstract

Binding of phorbol esters to protein kinase C (PKC) has been regarded as dependent on phospholipids, with phosphatidylserine being the most effective for reconstituting binding. By using a purified single cysteine-rich region from PKC delta expressed in Escherichia coli we were able to demonstrate that specific binding of [3H]phorbol 12,13-dibutyrate to the receptor still takes place in the absence of the phospholipid cofactor. However, [3H]phorbol 12,13-dibutyrate bound to the cysteine-rich region with 80-fold lower affinity in the absence than in the presence of 100 micrograms/ml phosphatidylserine. Similar results were observed with the intact recombinant PKC delta isolated from insect cells. When different phorbol derivatives were examined, distinct structure-activity relations for the cysteine-rich region were found in the presence and absence of phospholipid. Our results have potential implications for PKC translocation, for inhibitor design, and for PKC structural determination.

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • Cysteine / chemistry
  • Cysteine / metabolism*
  • Escherichia coli / genetics
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides
  • Phorbol 12,13-Dibutyrate / metabolism*
  • Phospholipids / metabolism*
  • Protein Binding
  • Protein Kinase C / chemistry
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Kinase C-delta
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spodoptera

Substances

  • Isoenzymes
  • Oligodeoxyribonucleotides
  • Phospholipids
  • Recombinant Proteins
  • Phorbol 12,13-Dibutyrate
  • Protein Kinase C
  • Protein Kinase C-delta
  • Cysteine