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A mutant protein kinase C that can transform fibroblasts
1Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.
Insights
Mutations in protein kinase C (PKC) alpha can cause cell transformation and oncogenicity. This study identified specific PKC mutations leading to fibrosarcoma-like cell behavior and tumor formation in mice.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Protein kinase C (PKC) isoenzymes influence fibroblast growth regulation.
- Normal PKC expression in fibroblasts alters growth but does not induce complete transformation.
Purpose of the Study:
- To investigate the role of specific mutations in protein kinase C (PKC) alpha in cell transformation and oncogenicity.
- To identify the molecular basis for the unusual subcellular distribution of PKC in a fibrosarcoma cell line.
Main Methods:
- Cloning and sequencing of alpha-PKC complementary DNA (cDNA) from fibrosarcoma cells and normal mouse brain.
- Expression of normal and mutant alpha-PKC cDNA in Balb/c 3T3 fibroblasts.
- Assessment of cell transformation markers (dense foci, anchorage-independent growth) and tumor formation in nude mice.
Main Results:
- A fibrosarcoma cell line exhibited an unusual PKC distribution with 87% activity at the membrane.
- Four point mutations were identified in the fibrosarcoma alpha-PKC, three in the regulatory domain and one in the catalytic domain.
- Expression of mutant alpha-PKC induced fibrosarcoma-like membrane localization and cell transformation, leading to tumor formation in vivo.
Conclusions:
- Specific point mutations in the alpha-PKC gene can lead to its aberrant membrane localization.
- Mutations in alpha-PKC are sufficient to induce cell transformation and oncogenicity, resulting in fibrosarcoma-like characteristics.
- Altered PKC function due to primary structure mutations plays a critical role in cancer development.