Regulation of Raf through phosphorylation and N terminus-C terminus interaction.
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.
Chong
Huira
H
Department of Biological Chemistry and the Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Guan
Kun-Liang
KL
D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases