Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.