LOW-AFFINITY FINDING 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 cyste ine-rich region from PKC delta expressed in Escherichia coli we were a ble to demonstrate that specific binding of [H-3]phorbol 12,13-dibutyr ate to the receptor still takes place in the absence of the phospholip id cofactor. However, [H-3]phorbol 12,13-dibutyrate bound to the cyste ine rich region with 80-fold lower affinity in the absence than in the presence of 100 mu g/ml phosphatidlylserine. Similar results were obs erved with the intact recombinant PRC delta isolated from insect cells . When different phorbol derivatives were examined, distinct structure -activity relations for the cysteine-rich region were found in the pre sence and absence of phospholipid. Our results have potential implicat ions for PKC translocation, for inhibitor design, and for PKC structur al determination.