Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.
Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.
Balázs
M
M
Department of Laboratory Medicine, University of California, San Francisco 94143.
Szöllösi
J
J
Lee
W C
WC
Haugland
R P
RP
Guzikowski
A P
AP
Fulwyler
M J
MJ
Damjanovich
S
S
Feuerstein
B G
BG
Pershadsingh
H A
HA
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D001665:Binding Sites; D001923:Brain Chemistry; D002453:Cell Cycle; D005260:Female; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000276:immunology; D007091:Image Processing, Computer-Assisted; D010069:Oxadiazoles / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010703:Phorbol Esters / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000956:Antigens, Viral; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010703:Phorbol Esters; C072539:phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-nitrobenz-2-oxa-1,3-diazole-aminododecanoate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate