Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids.
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.
Kazanietz
M G
MG
Molecular Mechanisms of Tumor Promotion Section, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Barchi
J J
JJ
Omichinski
J G
JG
Blumberg
P M
PM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010743:Phospholipids; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D003545:Cysteine