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Continuous synthesis of two protein-kinase-C-related proteins after down-regulation by phorbol esters
C Borner1, U Eppenberger, R Wyss
1Department of Research, University Clinic Medical School, Basel, Switzerland.
Abstract:
The phorbol 12-myristate 13-acetate (PMA)-dependent down-regulation of immunoprecipitable protein kinase C was studied in human breast cancer cell lines that display different growth inhibitions toward the tumor promoter. PMA induces translocation of [35S]methionine-prelabeled cytosolic protein kinase C to membranes, followed by complete degradation of the enzyme (t1/2, 2 hr). PMA does not affect the protein kinase C synthesis; 20-80% of total protein kinase C of control cells was still immunoprecipitable as membrane-bound 74- and 80-kDa protein kinase C-related polypeptides if cells were allowed to incorporate [35S]methionine during PMA exposure for greater than 6 hr. These two proteins lack protein kinase activity and phorbol ester binding but reveal V8 peptide patterns identical to the active forms of protein kinase C (77/80 kDa) of PMA-untreated cells. The amounts of the immunoprecipitable membrane-bound 80-kDa protein kinase C-related polypeptide synthesized during the prolonged PMA treatment appear to inversely correlate with the extent of PMA-mediated growth inhibition of the respective human breast cancer cell line. These data suggest that after homologous down-regulation, functional protein kinase C (77/80 kDa) is replaced by a population of membrane-associated but enzymatically inactive protein kinase C-related polypeptides (74/80 kDa).
Insights
Phorbol 12-myristate 13-acetate (PMA) causes protein kinase C degradation in breast cancer cells. Functional enzyme is replaced by inactive forms, correlating with growth inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Protein kinase C (PKC) is a key signaling enzyme involved in cell growth and differentiation.
- Phorbol 12-myristate 13-acetate (PMA) is a tumor promoter that activates PKC.
- Down-regulation of PKC is a known response to prolonged PMA exposure.
Purpose of the Study:
- To investigate the mechanism of PMA-induced PKC down-regulation in human breast cancer cell lines.
- To determine if PKC degradation is linked to growth inhibition by PMA.
- To characterize the nature of remaining PKC-related proteins after PMA treatment.
Main Methods:
- Human breast cancer cell lines were treated with PMA.
- Metabolic labeling with [35S]methionine was used to track PKC synthesis and degradation.
- Immunoprecipitation was employed to quantify PKC and related polypeptides.
- Enzymatic activity assays and V8 peptide mapping were performed.
Main Results:
- PMA induced rapid translocation and degradation of functional PKC (t1/2 = 2 hr).
- Prolonged PMA exposure resulted in membrane-associated, enzymatically inactive PKC-related polypeptides (74/80 kDa).
- These inactive forms shared V8 peptide patterns with active PKC but lacked kinase activity and phorbol ester binding.
- The amount of the 80-kDa inactive PKC polypeptide inversely correlated with PMA-mediated growth inhibition.
Conclusions:
- PMA induces homologous down-regulation of functional PKC in breast cancer cells.
- Inactive PKC-related polypeptides replace the active enzyme after down-regulation.
- This replacement mechanism may contribute to the differential growth inhibition observed in breast cancer cell lines exposed to PMA.