Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: real-time analysis using fluorescent ligands and proteins.
The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKCdelta or RasGRP3. However, PKCalpha and, initially, PKCdelta, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKCdelta translocation, and that the specificity for PKCalpha translocation is dominated by factors other than the localization of the ligand.
Braun
Derek C
DC
Department of Biology, Gallaudet University, Washington, District of Columbia, USA.
Cao
Yeyu
Y
Wang
Shaomeng
S
Garfield
Susan H
SH
Hur
Gang Min
GM
Blumberg
Peter M
PM
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D015240:Phorbol 12,13-Dibutyrate / Q000493:pharmacokinetics / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D020663:ras Guanine Nucleotide Exchange Factors
D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; C413212:RASGRP3 protein, human; D011993:Recombinant Fusion Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate