Related Experiment Video
Updated: Aug 17, 2026
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Published on: December 31, 2014
Transformation of NIH 3T3 fibroblasts by an activated form of p59hck
S F Ziegler1, S D Levin, R M Perlmutter
1Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Abstract:
Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.
Insights
Altering a specific tyrosine in src-family kinases (p59hck) to phenylalanine (p59hckF501) dramatically increased its transforming activity. This mutation enhanced protein phosphotyrosine levels and colony formation in cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Src-family protein tyrosine kinases play a crucial role in regulating cellular activities.
- Phosphorylation of a specific tyrosine residue near the carboxy terminus is a key regulatory mechanism for these kinases.
Purpose of the Study:
- To investigate the regulatory role of tyrosine phosphorylation in src-family protein tyrosine kinases.
- To determine the transforming activity of a mutated src-family kinase lacking this regulatory phosphorylation site.
Main Methods:
- Oligonucleotide-directed mutagenesis was employed to convert tyrosine 501 (Tyr-501) in p59hck to a phenylalanine residue, creating p59hckF501.
- Quantitative analysis using a soft-agar cloning assay was performed to assess colony formation.
- Protein phosphotyrosine levels in cells were analyzed.
- A second mutation targeting the ATP-binding domain was introduced to assess its impact on transforming ability.
Main Results:
- The mutated p59hckF501 exhibited potent transforming activity, significantly exceeding that of related transforming elements like p56lckF505.
- Cells expressing p59hckF501 showed elevated levels of protein phosphotyrosine.
- The transforming ability of p59hckF501 was abrogated by a mutation affecting the ATP-binding domain, indicating its essential role.
Conclusions:
- The phosphorylation status of Tyr-501 in p59hck is a critical determinant of its biological activity and transforming potential.
- Disrupting this regulatory phosphorylation site leads to hyperactivation and potent oncogenic transformation.
- The ATP-binding domain is essential for the observed transforming activity of the mutated kinase.
