Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts
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Published on: March 19, 2014
S Rose-John1, A Dietrich, F Marks
1Department of Biochemistry, German Cancer Research Center, Heidelberg.
Abstract:
A set of complementary DNAs (cDNAs) that encode the complete 672-amino acid sequence for mouse protein kinase C (PKC) type alpha have been isolated from a mouse Swiss 3T3 cDNA library. Extensive rescreening of this cDNA library only resulted in the isolation of clones of the same PKC species, indicating that Swiss 3T3 fibroblasts express exclusively PKC-alpha. This enzyme is encoded by two different mRNAs that exhibit a substantial heterogeneity in their 3'-noncoding regions. This heterogeneity could have been derived from alternative splicing of the pre-mRNAs or be due to differential usage of the polyadenylation motif. The expression of PKC mRNA in fibroblasts is very low and it is not influenced by treatment with the tumor promoter 12-O-tetradecanoyl-13-phorbol-acetate.
Insights
Researchers isolated mouse protein kinase C (PKC) alpha cDNA from Swiss 3T3 fibroblasts, finding this specific enzyme is exclusively expressed. PKC mRNA levels were low and unaffected by tumor promoters.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein Kinase C (PKC) is a family of enzymes involved in cell signaling.
- PKC alpha is a specific isoform implicated in various cellular processes.
- Understanding the expression and regulation of PKC isoforms is crucial for cell biology research.
Purpose of the Study:
- To isolate and characterize complementary DNAs (cDNAs) encoding mouse protein kinase C (PKC) type alpha.
- To determine the specific PKC species expressed in Swiss 3T3 fibroblasts.
- To investigate the characteristics of PKC mRNA and its regulation.
Main Methods:
- Isolation of cDNAs encoding mouse PKC alpha from a Swiss 3T3 cDNA library.
- Rescreening of the cDNA library to identify expressed PKC species.
- Analysis of mRNA heterogeneity in the 3'-noncoding regions.
- Assessment of PKC mRNA expression levels in fibroblasts before and after treatment with 12-O-tetradecanoyl-13-phorbol-acetate.
Main Results:
- Complete 672-amino acid sequence of mouse PKC alpha was obtained.
- Swiss 3T3 fibroblasts exclusively express PKC alpha.
- Two distinct PKC alpha mRNAs with heterogeneous 3'-noncoding regions were identified.
- PKC mRNA expression in fibroblasts is low and not modulated by tumor promoter treatment.
Conclusions:
- Swiss 3T3 fibroblasts predominantly express PKC alpha, suggesting isoform specificity in this cell type.
- mRNA heterogeneity in the 3'-noncoding regions may arise from alternative splicing or polyadenylation.
- PKC alpha expression in these fibroblasts is constitutive and not responsive to phorbol ester treatment.
