Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein.
We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.
Yeung
K
K
Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
Janosch
P
P
McFerran
B
B
Rose
D W
DW
Mischak
H
H
Sedivy
J M
JM
Kolch
W
W
D000483:Alleles; D000727:Androgen-Binding Protein; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D008954:Models, Biological; D049294:Phospholipid Transfer Proteins; D010957:Plasmids; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D049294:Phospholipid Transfer Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf