Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present.

ABSTRACT

We have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non-contact-inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies alpha and delta increases as the cells increase in density while that of PKC-zeta remains the same. Unusually, protein expression of PKC-epsilon is completely down-regulated as cells grow beyond about 50% confluency and no PKC-epsilon protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC-epsilon is expressed at all stages of fibroblast growth as revealed by RT-PCR. When high-density 3T6 fibroblasts are passaged to low density in fresh medium, re-expression of PKC-epsilon protein is observed within 15 min and becomes down-regulated again as cells become more dense. This very rapid synthesis of PKC-epsilon is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC-epsilon has some characteristics of a novel 'early response' protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level.