Abstract
A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.
Highlights
A mixed micellar assay for the binding of phorbol- until Blumberg and co-worker8 (15) demonstrated the existesters to protein kinase C was developed to investigate ence of a high affinity specific receptor for biologically active the specificity and stoichiometry of phospholipd cofac- phorbol esters using [3H]phorbol dibutyrate
The ['Hlphorbol dibutyrate .protein & kinase C Triton X-1OO.PSmixedmicellarcomplex f eluted on a SephacrylS-200 molecular sieve at anM, of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate
Understanding the molecular mechanism of phorbol esteraction advanced significantly when protein kinase C, the calcium- and phospholipid-dependent protein kinase, was demonstrated to be activated by phorbol esters (19,20) andto copurify with the phorbol ester receptor (21-23)
Summary
Pasteur pipettes) in the presence of 100 ~ L MCaC12 (A). Nonspecific binding was determined with excess (2 PM) unlabeled PDBu (A).B , specific binding of [3H]PDBu a t variable concentrations of PDBu. In the presence of Ca2+,protein kinase C (MI= 80,000) associated with p-octylglucoside/PS mixed micelles and eluted with an apparent M, of 120,000 (Fig.3C),well included in thecolumn This position is equivalent to that expected from the sum of one mixedmicelle and one protein kinase C molecule. The stoichiometry of protein kinase C mixed micelleinteraction is important to analyze and quantiatethe lipid cofactor requirement for phorbol ester activation of, and binding to, protein kinase C This is because the mole per cent of lipid cofactor:Triton X-100 (or /I-octylglucoside) can be approximately converted to the number of molecules per micelle interacting with protein kinase C by the equation’: number of molecules/micelle = mole per cent/100 X aggregation number X (1 - CMC/detergent concentration). For Triton X-100 this is a negligible effect
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