Abstract
Raf1 kinase inhibitor protein (RKIP) negatively regulates the Raf1/MEK/ERK pathway which is vital for cell growth and differentiation. It is also a biomarker in clinical cancer diagnosis. RKIP binds to the N-terminus of Raf1 kinase but little is known about the structural basis of RKIP binding with Raf1. Here, we demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop “127–149”, and the C-terminal helix by NMR experiments. D70, D72, E83, Y120, and Y181 were further verified as the key residues participating in the interaction of hRKIP and hRaf11-147aa. G143-R146 fragment was also critical for hRKIP binding with hRaf11-147aa, for its deletion decreased the binding affinity around 300 times, from 154 to 0.46 mM−1. Our results provide important structural clues for designing the lead compound that disrupts RKIP–Raf1 interaction.
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Acknowledgments
This work was supported by grants from the Natural Science Foundation of China (Nos. 30900233, 31170717, 91129713), Natural Science Foundation of Fujian Province (No. 2011J01246) and the Program of Shanghai Subject Chief Scientist (No. 09XD1405100).
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Wu, Z., Fu, C., Shi, L. et al. Structural basis for RKIP binding with its substrate Raf1 kinase. Biotechnol Lett 36, 1869–1874 (2014). https://doi.org/10.1007/s10529-014-1558-6
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DOI: https://doi.org/10.1007/s10529-014-1558-6