Direct Visualization by Fluorescence Videomicroscopy of the Subcellular Localization of a New Derivative of Phorbol-12,13-Dibutanoate

Abstract

A new fluorescent phorbol ester derivative, dansylaza-PDB, was synthesized by the esterification of a phorbol moeity by N-dansyl-methylglycine and butyric acid. The binding of dansylaza-PDB to intact C3H/10T1/2 fibroblasts was similar to that of [³H]-PDB to the same cells. At 47 nM, dansylaza-PDB induced half-maximum inhibition of specific [³H]-PDB. In the same cell line, it activated protein kinase C both in vivo and in vitro. The apparent K_a value of dansylaza-PDB for protein kinase C partially purified from these cells was estimated at 20 n<i>M</i>. In in vitro assays, this derivative also completed with [³H]-PDB for the binding of partially purified protein kinase C, exhibiting a K_i value of 2.7 n<i>M</i>. Dansylaza-PDB is therefore suitable for fluorescence studies of living cells. The time-course of dansylaza-PDB binding to living interphase C3H/10T1/2 fibroblasts was studied by fluorescence videomicroscopy with two concentrations of the derivative, 22.5 and 116 n<i>M</i>. Direct kinetic analysis of this binding allowed characterization of specific binding sites in the same range of concentrations as those reported for [³H]-PDB binding to the same cell line. The specific binding sites were dissociated by an excess of unlabelled PDB, whereas nonspecific binding sites were not. ∼80% of the derivative was removed by washing the cells with medium. Direct subcellular localization of dansylaza-PDB by fluorescence videomicroscopy showed, for the first time, that the phorbol ester derivative is present in different membranes of living cells, including plasma membrane, endoplasmic reticulum and mitochondria. This distribution of dansylaza-PDB reflects the multiple locations of phorbol ester receptor sites. These results substantiate the possibility that the biological effects of phorbol esters might not only be triggered by their stimulation of protein kinase C activity but also by direct binding of phorbol esters to lipid domains of cell membranes.