PKC alpha translocation is microtubule-dependent in passaged smooth musclecells

The translocation of protein kinase C (PKC) isozymes from their inactive ce ll locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specif icity. We have utilized confocal microscopy to compare phorbol 12, 13 dibut yrate (PDB) - stimulated translocation of PKC alpha in cultured cells deriv ed from rat vascular smooth muscle. In enzymatically dispersed, passaged sm ooth muscle cells, PKC alpha was uniformly distributed throughout the unsti mulated cell. PDB stimulation resulted in extensive association of the PKC alpha into filamentous strands with subsequent accumulation of the isoform in the peri-nuclear region of the cell. Dual immunostaining indicated that PKC alpha was extensively colocalized with microtubules in the interval imm ediately following PDB stimulation but was largely disassociated from micro tubules at 10 min, at which time the translocation of PKC alpha to the peri -nucleus/nucleus was nearly complete. It was further found that the use of colchicine to disrupt the microtubules caused the loss of PKC alpha translo cation to the peri-nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The d ata suggest that PDB stimulation results in a transient association of PKC alpha with cell microtubules and that the microtubules play an important ro le in the translocation of PKC alpha from the cytosol in passaged cells der ived from rat aortic smooth muscle.