The Subcellular Distribution of Protein Kinase Cα, -ϵ, and -ζ Isoforms during Cardiac Cell Differentiation

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the “classical,” “novel,” and “atypical” protein kinase Cʹs in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387–2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Cα, -ϵ, and -ζ were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Cα, -ϵ, and -ζ to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1,2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1,2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Cα with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cϵ or -ζ. The results indicate that protein kinase C activity and protein kinase Cα, -ϵ and -ζ isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Cα may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.