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Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins
1Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520.
Abstract:
We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.
Insights
The 14-3-3 protein binds unactivated Raf-1 kinase, acting as a latent co-activator. It mediates mitogen-triggered, Ras-independent activation of Raf-1, impacting cellular signaling pathways.
Area of Science:
- Cellular signaling
- Protein kinase regulation
- Molecular interactions
Background:
- Raf-1 protein kinase is a key regulator in cellular signaling pathways.
- 14-3-3 proteins are known to interact with various signaling molecules.
- Understanding Raf-1 activation mechanisms is crucial for deciphering cell growth and differentiation.
Purpose of the Study:
- To identify novel regulators of Raf-1 protein kinase activity.
- To investigate the role of the 14-3-3 protein family in Raf-1 signaling.
- To elucidate the mechanism by which 14-3-3 proteins modulate Raf-1 function.
Main Methods:
- Yeast two-hybrid screening to identify Raf-1 binding proteins.
- In vitro binding assays with purified proteins.
- Co-immunoprecipitation from cell extracts.
- Transfection assays to assess reporter gene expression and cell differentiation.
Main Results:
- The beta isoform of 14-3-3 protein was identified as a Raf-1 binding protein.
- 14-3-3 binds to unactivated Raf-1, with binding sensitive to Raf-1 activation status.
- 14-3-3 acts as a Ras-independent activator of Raf-1 in cell-free extracts and in vivo.
- 14-3-3 enhances reporter gene expression and accelerates differentiation triggered by Raf-1.
Conclusions:
- 14-3-3 protein serves as a latent co-activator for Raf-1 kinase.
- 14-3-3 binds to quiescent, unactivated Raf-1, mediating regulatory effects on the kinase domain.
- This interaction represents a novel mechanism for regulating Raf-1 activity in response to mitogenic stimuli.