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Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells
K Inoue1, B Wongsasant, T Akiyama
1Department of Oncogene Research, Osaka University, Japan.
Molecular and Cellular Biology
|December 1, 1991
Summary
The mutant c-FGR protein transforms NIH 3T3 cells and has higher tyrosine kinase activity. Normal c-FGR induces monocytic marker alpha-NBE, suggesting tyrosine phosphorylation
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The c-FGR protein is a member of the Src family of protein-tyrosine kinases.
- Mutations in c-FGR can lead to altered protein activity and cellular effects.
- The alpha-naphthyl butyrate esterase (alpha-NBE) enzyme is a marker for monocytic cells.
Purpose of the Study:
- To investigate the transforming activity of a mutant c-FGR protein (p58c-fgr/F523) compared to the normal form (p58c-fgr/wt).
- To determine the effect of normal c-FGR gene transfection on NIH 3T3 cells, specifically the induction of monocytic markers.
- To explore the role of tyrosine phosphorylation in c-FGR-mediated cell transformation and enzyme induction.
Main Methods:
- Transfection of NIH 3T3 cells with normal and mutant c-FGR genes.
- In vitro tyrosine kinase activity assays.
- Isoelectric focusing to characterize induced alpha-NBE.
- Immunoblotting with antiphosphotyrosine antibodies to identify phosphorylated proteins.
Main Results:
- Mutant p58c-fgr/F523 exhibited transforming activity and threefold higher tyrosine kinase activity than normal p58c-fgr/wt.
- Transfection with normal c-FGR gene induced sodium fluoride (NaF)-sensitive alpha-NBE in NIH 3T3 cells, a marker of monocytic origin.
- Isoelectric focusing confirmed the induced alpha-NBE matched that of monocytic cells.
- Tyrosine phosphorylation of specific cellular proteins (58, 62, 75, 120, 200, 230 kDa) occurred in cells transfected with both normal and mutant c-FGR.
- A 95-kDa protein was significantly phosphorylated in cells transfected with the mutated c-FGR.
Conclusions:
- Tyrosine phosphorylation of specific cellular substrates is crucial for the induction of NaF-sensitive alpha-NBE by p58c-fgr.
- Tyrosine phosphorylation of specific cellular substrates is important for cell transformation mediated by p58c-fgr.
- The normal c-FGR protein can induce monocytic differentiation markers in NIH 3T3 cells, suggesting a role beyond simple transformation.