NEW FLUORESCENT-PROBES FOR PROTEIN-KINASE-C - SYNTHESIS, CHARACTERIZATION, AND APPLICATION

Fluorescent derivatives of the bisindolylmaleimide inhibitors of prote in kinase C (PKC) were synthesized and tested with respect to their in hibitory potency, specificity, and usefulness as fluorescent cytologic al stains for PKC. Several of the fluorescent bisindolylmaleimide deri vatives (fim-1, fim-2, and rim-1) acted as ATP-competitive catalytic s ite inhibitors and retained much of the potency and specificity of the parental compound. The R6-C1 and the PKCbeta1-overexpressing R6-PKC3 cell lines were used for testing fim-1 and rim-1 as cytological stains for PKC. Comparisons showed that the R6-PKC3 cells stained much more brightly than R6-C1 cells. When R6-PKC3 cells were treated with the ph orbol ester phorbol 12-myristate 13-acetate (PMA) for 30 min, staining with fim-1 or anti-PKCbeta1 revealed a dramatic translocation of PKC to the cell periphery. When R6-PKC3 cells were exposed to PMA for 24 h to down-regulate PKC, cytoplasmic staining was drastically reduced. S taining patterns obtained with an antibody specific for PKCbeta1 and w ith fim-1 were remarkably similar except for mitochondrial staining, w hich was only seen with fim-1. A closer examination of the mitochondri al staining showed that mitochondria convert from filamentous to punct ate shapes and cluster around the nucleus when cells are treated with PMA. This punctate morphology, perinuclear clustering, and staining wi th fim-1 persists when PKC is down-regulated. Overall, these results i ndicate that fim-1 and rim-1 can serve as useful fluorescent probes fo r PKC. The mitochondrial staining may be due to a PKC isoform resistan t to down-regulation.