Quantitation of the interaction of protein kinase C with diacylglycerol and phosphoinositides by time-resolved detection of resonance energy transfer.
Quantitative studies of the binding of protein kinase C (PKC) to lipid cofactors were performed by monitoring resonance energy transfer with time-resolved fluorescence techniques. For that purpose, diacylglycerol (DG), phosphatidylinositol 4,5-biphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were labeled with a pyrenyl decanoyl moiety at the sn-2 position of the lipid glycerol. These labeled lipids proved excellent energy acceptors of light-excited tryptophan residues in PKC. The quenching efficiency of the tryptophan fluorescence was determined as function of lipid probe concentration in mixed micelles consisting of poly(oxyethylene)-9-lauryl ether, PS, and various mole fractions of probe lipid. The experimental conditions and method of data analysis allowed the estimation of binding constants of single or multiple pyrene lipids to PKC. The affinity of PKC for inositide lipids increases in the order PI < PIP < PIP2. The affinity of PKC for PIP and PIP2 is higher than that for DG. Determination of PKC activity in the presence of labeled lipids and PS showed that only PIP2 and DG activate PKC. Double-labeling experiments suggest that PIP2 and DG are not able to bind simultaneously to PKC, indicating a reciprocal binding relationship of both cofactors. The results support the notion that, besides DG, PIP2 can be a primary activator of PKC.
Pap
E H
EH
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Bastiaens
P I
PI
Borst
J W
JW
van den Berg
P A
PA
van Hoek
A
A
Snoek
G T
GT
Wirtz
K W
KW
Visser
A J
AJ
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism*; D004735:Energy Transfer; D066298:In Vitro Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010716:Phosphatidylinositols / Q000378:metabolism*; D010718:Phosphatidylserines / Q000737:chemistry; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D004075:Diglycerides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D014364:Tryptophan; D011493:Protein Kinase C; D002118:Calcium