The interaction between protein kinase C and lipid cofactors studied by simultaneous observation of lipid and protein fluorescence.
The interaction of protein kinase C (PKC) with lipids was probed by a dual approach. Pyrene-labeled lipid analogues of diacylglycerol, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylcholine (PC) were used both as acceptors of tryptophan excitation energy of PKC and as membrane probes for intra- and intermolecular lipid chain collisions by measuring the ratio of excimer-to-monomer fluorescence intensity (EM). Both in micelles of polyoxyethylene 9-lauryl ether and in dioleoyl-PC vesicles, interaction of PKC with monopyrenyl PS (pyr-PS) in the absence of calcium resulted in a relatively slow decrease of the EM value. This effect on the lipid dynamics was accompanied by quenching of the tryptophan fluorescence of PKC. Addition of calcium resulted in a rapid further decrease of the EM ratio of pyr-PS and in additional quenching of the tryptophan fluorescence. When 4 mol % of pyr-PS was replaced by 0.5 mol % of dipyrenyl-labeled diacylglycerol a decrease of the intramolecular excimer formation rate and tryptophan fluorescence could only be detected in the presence of calcium and PS. Strong binding was also observed with dipyrenyl-labeled PIP (dipyr-PIP), but not with the other dipyrenyl-labeled lipids: PI, PS, or PC. In addition, the EM ratios of dipyr-PIP were not affected by phorbol 12-myristate 13-acetate, indicating that phorbol 12-myristate 13-acetate and dipyr-PIP can bind simultaneously to PKC.
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
E H
EH
Pap
P A
PA
van den Berg
J W
JW
Borst
A J
AJ
Visser
D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D004075:Diglycerides; D010743:Phospholipids; D011506:Proteins; D012965:Sodium Chloride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium