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Regulation of Raf through phosphorylation and N terminus-C terminus interaction
1Department of Biological Chemistry and the Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Abstract:
Raf kinase is a key component in regulating the MAPK pathway. B-Raf has been reported as an oncogene and is mutated in 60% of human melanomas. The main focus of Raf regulation studies has been on phosphorylation, dephosphorylation, and scaffolding proteins; however, Raf also has its own auto-regulatory domain. Removal of the N-terminal regulatory domain, initially discovered in the viral Raf oncogene (v-Raf), results in a kinase domain with high basal activity independent of Ras activation. In this report, we show that activating phosphorylations are still required for activity of the truncated C-terminal kinase domain (called 22W). The interaction between the N-terminal regulatory domain and the C-terminal kinase domain is disrupted by activated Ras. Mutations in the Ras binding domain, cysteine-rich domain, or S259A do not affect the inhibition of 22W by the N-terminal domain. When phosphomimetic residues are substituted at the activating sites (DDED) in 22W, this results in a higher basal activity that is no longer inhibited by expression of the N-terminal domain, although binding to the N-terminal domain still occurs. Although the interaction between 22W/DDED and the N-terminal domain may be in a different conformation, the interaction is still disrupted by activated Ras. These data demonstrate that N-terminal domain binding to the kinase domain inhibits the activity of the kinase domain. However, this inhibition is relieved when the C-terminal kinase domain is activated by phosphorylation.
Insights
Raf kinase auto-regulation involves its N-terminal domain inhibiting the C-terminal kinase domain. This inhibition is overcome by activating phosphorylations, even when Ras is activated.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Raf kinase is crucial for the MAPK pathway, with B-Raf mutations common in melanoma.
- Raf auto-regulation via its N-terminal domain is less studied than phosphorylation or scaffolding.
- Removing the N-terminal domain of viral Raf (v-Raf) yields a highly active kinase domain.
Purpose of the Study:
- To investigate the auto-regulatory mechanism of Raf kinase.
- To determine the role of the N-terminal regulatory domain in Raf kinase activity.
- To understand how Ras activation and phosphorylation affect Raf auto-regulation.
Main Methods:
- Studied a truncated Raf kinase domain (22W) lacking the N-terminal regulatory domain.
- Investigated the requirement of activating phosphorylations for 22W activity.
- Analyzed the effect of activated Ras on the interaction between N-terminal and C-terminal domains.
- Introduced phosphomimetic mutations (DDED) in 22W to assess basal activity and N-terminal domain inhibition.
Main Results:
- Activating phosphorylations are essential for the truncated 22W kinase domain's activity.
- Activated Ras disrupts the interaction between the N-terminal regulatory domain and the C-terminal kinase domain.
- Phosphomimetic mutations (DDED) in 22W result in high basal activity, resistant to N-terminal inhibition despite continued binding.
- N-terminal domain binding inhibits kinase activity, but this inhibition is relieved by C-terminal domain phosphorylation.
Conclusions:
- The N-terminal domain of Raf kinase auto-regulates by inhibiting the C-terminal kinase domain.
- Activating phosphorylations on the C-terminal domain are necessary for kinase activity and can overcome N-terminal inhibition.
- Ras activation provides a mechanism to relieve N-terminal auto-inhibition, promoting kinase activity.