PKCα translocation is microtubule‐dependent in passaged smooth muscle cells

Abstract

The translocation of protein kinase C (PKC) isozymes from their inactive cell locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specificity. We have utilized confocal microscopy to compare phorbol 12, 13 dibutyrate (PDB) – stimulated translocation of PKCα in cultured cells derived from rat vascular smooth muscle. In enzymatically dispersed, passaged smooth muscle cells, PKCα was uniformly distributed throughout the unstimulated cell. PDB stimulation resulted in extensive association of the PKCα into filamentous strands with subsequent accumulation of the isoform in the peri-nuclear region of the cell. Dual immunostaining indicated that PKCα was extensively colocalized with microtubules in the interval immediately following PDB stimulation but was largely disassociated from microtubules at 10 min, at which time the translocation of PKCα 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 PKCα translocation to the peri-nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The data suggest that PDB stimulation results in a transient association of PKCα with cell microtubules and that the microtubules play an important role in the translocation of PKCα from the cytosol in passaged cells derived from rat aortic smooth muscle.