Cell genetic evidence of correlation of intracellular translocation of protein kinase C (PKC) and PKC-mediated phosphorylation of 80-kDa protein with mitogenic action of tumor promoters

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Abstract
Recently, we isolated a series of 3T3-L1 cell variants that are unable to respond to mitogenic stimulation by the tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA). Since protein kinase C (PKC) is the major receptor for TPA and appears to play a key role in cellular proliferation, we have examined the distribution of PKC in the parental 3T3-L1 cells and the variant VT-1 cells. PKC was located predominantly in the cytosol of growth-arrested confluent 3T3-L1 cells, and upon TPA treatment it was rapidly translocated into the plasma membrane. In contrast, PKC was located predominantly in the plasma membrane of confluent VT-1 variant cells and was no longer activated by TPA. Two-dimensional gel analysis showed that a Mr 80,000 acidic protein (80-kDa protein) was rapidly phosphorylated in 3T3-L1 cells upon TPA treatment, whereas phosphorylation of this protein was barely detected in VT-1 cells. In growing cultures, the majority of PKC was found in the plasma membrane of both cell lines, and no change occurred upon TPA treatment. Hydroxyapatite column chromatography revealed the presence of α-type PKC as the major component in both cell lines. These results suggest that the intracellular translocation of α-type PKC and the PKC-mediated phosphorylation of the 80-kDa protein may be involved in the mechanism of mitogenic signal transfer.