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Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Published on: January 27, 2012
Regulation of the cell cycle by focal adhesion kinase
1Cancer Biology Laboratories, Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Abstract:
In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant DeltaC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in DeltaC14. Biochemical analyses indicated that FAK mutant DeltaC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKDeltaC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that DeltaC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.
Insights
Integrin signaling via focal adhesion kinase (FAK) regulates the cell cycle. FAK overexpression accelerates cell cycle progression, while dominant-negative mutants inhibit it at G1 phase, impacting cyclin D1 and p21 expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Integrins are crucial cell surface receptors mediating cell adhesion and signaling.
- Focal Adhesion Kinase (FAK) is a key signaling molecule downstream of integrins.
- Cell cycle progression is tightly regulated by various signaling pathways.
Purpose of the Study:
- To investigate the role and mechanisms of integrin-mediated FAK signaling in cell cycle regulation.
- To determine how FAK activity influences specific phases of the cell cycle.
Main Methods:
- Utilized tetracycline-regulated expression of wild-type and mutant FAK (DeltaC14, Y397F) and FRNK.
- Performed biochemical analyses, including assessing protein localization and interactions (Src, Fyn).
- Assayed cell cycle progression using BrdU incorporation and analyzed protein expression (cyclin D1, p21) and Erk activation.
Main Results:
- Overexpression of wild-type FAK accelerated the G1 to S phase transition.
- Dominant-negative FAK mutant DeltaC14 inhibited cell cycle at G1 phase, dependent on Y397.
- DeltaC14 competed with endogenous FAK, reducing Erk activation and blocking cyclin D1 upregulation while inducing p21.
- FAK Y397F mutant and FRNK also inhibited BrdU incorporation and Erk activation.
Conclusions:
- FAK signaling is a critical mediator of integrin-dependent cell cycle regulation.
- FAK activity directly impacts the expression of key cell cycle regulators like cyclin D1 and p21.
- These findings elucidate a novel mechanism linking cell adhesion to cell cycle control.
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