Paradoxical activation of Raf by a novel Raf inhibitor.
BACKGROUND
Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade.


RESULTS
The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580.


CONCLUSIONS
Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.
MRC Protein Phosphorylation Unit, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee, DD1 5EH, UK. cahalljackson@bad.dundee.ac.uk
C A
CA
Hall-Jackson
P A
PA
Eyers
P
P
Cohen
M
M
Goedert
F T
FT
Boyle
N
N
Hewitt
H
H
Plant
P
P
Hedge
D016475:3T3 Cells; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006133:Growth Substances / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007536:Isomerism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D020929:Mitogen-Activated Protein Kinase Kinases; D010641:Phenotype; D010703:Phorbol Esters / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000096:biosynthesis; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011519:Proto-Oncogenes / Q000235:genetics*; D011725:Pyridines / Q000494:pharmacology
D001549:Benzamides; D004791:Enzyme Inhibitors; D006133:Growth Substances; D006150:Guanine Nucleotides; D007093:Imidazoles; C121825:N-(5-(3-dimethylaminobenzamido)-2-methylphenyl)-4-hydroxybenzamide; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; C093642:SB 203580