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Functional expression in mammalian cells of a full-length cDNA coding for the pp42/MAP kinase (p42mapk) protein
G L'Allemain1, J H Her, R L Del Vecchio
1Department of Microbiology, University of Virginia, Charlottesville 22908.
Abstract:
We recently cloned from a mouse 3T3 cell cDNA library a cDNA with sequence similarity to the p42mapk protein and other members of the MAP kinase family. To determine with certainty which member of the family this clone encodes, we have expressed the cDNA in COS cells and characterized the protein product. When the pSV2MAP plasmid carrying the full-length clone was transfected into COS cells, a protein of 42,000 Da was expressed. This 42 kDa protein displayed chromatographic properties indistinguishable from the endogenous p42mapk, and could be separated from the closely related pp44. In addition, upon serum stimulation, the 42 kDa protein became tyrosine-phosphorylated and enzymatically active towards the substrate myelin basic protein. We conclude that this clone codes for a functional p42mapk protein kinase.
Insights
Researchers cloned a cDNA encoding the p42 mitogen-activated protein kinase (MAPK). This functional protein kinase was expressed and characterized in COS cells, confirming its identity and enzymatic activity.
Area of Science:
- Molecular Biology
- Cell Signaling
- Enzymology
Background:
- Mitogen-activated protein kinases (MAPKs) are crucial signaling molecules.
- Identifying specific MAPK family members is essential for understanding cellular processes.
Purpose of the Study:
- To definitively identify a cloned cDNA with sequence similarity to p42mapk.
- To characterize the functional properties of the expressed protein product.
Main Methods:
- Cloning a cDNA from a mouse 3T3 cell library.
- Expression of the full-length cDNA in COS cells via pSV2MAP plasmid transfection.
- Chromatographic analysis and enzymatic activity assays using myelin basic protein.
Main Results:
- A 42 kDa protein was successfully expressed in COS cells.
- The expressed protein exhibited chromatographic properties identical to endogenous p42mapk.
- The 42 kDa protein underwent tyrosine phosphorylation and became enzymatically active upon serum stimulation.
Conclusions:
- The cloned cDNA encodes a functional p42mapk protein kinase.
- This finding contributes to the understanding of MAPK signaling pathways.