Structural basis for RKIP binding with its substrate Raf1 kinase

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.

Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein

“…Except for L14 which is located at the N-terminal random coil, other significantly broadened peaks were associated with residues located in both the conserved ligand-binding pocket of hRKIP and the loop covering residues 127-149, indicating that suramin interacted with hRKIP primarily through binding to its conserved ligand-binding pocket at an intermediate affinity (Figure 2b,c). Notably, most of the residues associated with broadened peaks were consistent with the binding sites of hRaf1NTD on hRKIP, including D70, S75, Y81, V107, S109, K113, R146, K179, Y181, and E182 (Figure 2c,d) [36]. These results suggest that suramin and hRaf1NTD share basically similar binding sites on hRKIP.…”

“…These results suggest that suramin and hRaf1NTD share basically similar binding sites on hRKIP. Residues involved in the hRKIP-hRaf1 NTD interaction are mapped to the same 3D structure 36 . Cyan balls: common residues involved in the interactions of hRKIP with both suramin and hRaf1 NTD .…”

Lack of dcf1 leads to neuronal migration delay, axonal swollen and autism-related deficits

“…Overexpression of DCF1 led to downregulation of ERK activity, which could not be reversed even by PMA stimulation. PEBP1, a RAF kinase inhibitor that inhibits ERK activation by directly binding RAF1, thereby preventing its phosphorylation (Vandamme et al, 2014;Wu et al, 2014). PEBP1 was involved in the regulation of the ERK signaling pathway in this research.…”

Understanding perspectives of signalling mechanisms regulating PEBP1 function

“…12,14 Metastatic tumours have deregulated PEBP1/RKIP as a result of the hypermethylation events in its CpG sites. Hence, conserved sequences spanning phosphorylation sites forming the ligand binding pocket and loop 127-149 along with C-terminal helical region in PEBP1 are required for its binding with other ligands, particularly the N terminus of Raf kinase.…”

“…Hence, conserved sequences spanning phosphorylation sites forming the ligand binding pocket and loop 127-149 along with C-terminal helical region in PEBP1 are required for its binding with other ligands, particularly the N terminus of Raf kinase. 12,14 Metastatic tumours have deregulated PEBP1/RKIP as a result of the hypermethylation events in its CpG sites. CpG methylation seems to be frequent in tumour cells in gastric cardia adenocarcinoma.…”