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Published on: June 24, 2022
Generation of point-mutant FAK knockin mice
B Tavora1, S Batista, A N Alexopoulou
1Adhesion and Angiogenesis Laboratory, Centre for Tumour Biology, Barts Cancer Institute, -A CR-UK Centre of Excellence, Queen Mary University of London, London, United Kingdom.
Abstract:
Focal adhesion kinase is a non-receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non-kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead, non-phosphorylatable mutants Y397F and Y861F, or mutant Y397E-containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism.
Insights
This study introduces novel FAK-mutant mice to investigate focal adhesion kinase (FAK) in vivo. These models will elucidate FAK
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Focal adhesion kinase (FAK) is a crucial protein tyrosine kinase involved in cell adhesion, migration, and proliferation.
- FAK signaling is activated by integrins and growth factor receptors, with key phosphorylation sites at Y397 and Y861.
- While in vitro studies highlight FAK's kinase and scaffolding roles, its in vivo functions remain less understood.
Purpose of the Study:
- To generate and characterize FAK-mutant knockin mice for in vivo functional studies.
- To enable spatiotemporal investigation of FAK's roles in the whole organism.
- To provide tools for dissecting the in vivo significance of FAK kinase activity and specific phosphorylation sites.
Main Methods:
- Generation of Cre-inducible FAK-mutant knockin mice using the ROSA26 promoter.
- Creation of mutants including kinase-dead FAK, non-phosphorylatable Y397F and Y861F, and phosphomimetic Y397E.
- Strategy involves intercrossing with FAKflox/flox and Cre-expressing mice for conditional FAK knockout and mutant expression.
Main Results:
- Successfully generated a series of FAK-mutant knockin mouse lines.
- Established a Cre-inducible system for controlled expression of mutant FAK variants.
- Developed a platform for future in vivo studies of FAK function.
Conclusions:
- The generated FAK-mutant mice are valuable tools for in vivo research.
- These models will facilitate a deeper understanding of FAK's physiological and pathological roles.
- Future studies will leverage these mice to explore FAK signaling in various biological contexts.
