The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.
The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.
Rath
Oliver
O
The Beatson institute for Cancer Research, Cancer Research UK Garscube Estate, Bearsden, Glasgow G61 1BD, UK.
Park
Sungdae
S
Tang
Hui-hui
HH
Banfield
Mark J
MJ
Brady
R Leo
RL
Lee
Yie Chia
YC
Dignam
John D
JD
Sedivy
John M
JM
Kolch
Walter
W
Yeung
Kam C
KC
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D006801:Humans; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D011994:Recombinant Proteins; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases