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Paradoxical activation of Raf by a novel Raf inhibitor
C A Hall-Jackson1, P A Eyers, P Cohen
1MRC Protein Phosphorylation Unit, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee, DD1 5EH, UK. cahalljackson@bad.dundee.ac.uk
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.
Insights
A novel feedback loop suppresses Raf activation, meaning Raf inhibitors may not be effective anticancer drugs. This study identifies ZM 336372 as a Raf inhibitor that paradoxically activates c-Raf.
Area of Science:
- Cellular signaling pathways
- Oncogene research
- Drug discovery
Background:
- Raf is a proto-oncogene activated by growth factors.
- Raf is believed to activate the MAP kinase kinase-1 (MKK1) and MAP kinase (MAPK) cascade.
Purpose of the Study:
- To investigate the mechanism of Raf activation and inhibition.
- To characterize the compound ZM 336372 as a Raf inhibitor.
Main Methods:
- In vitro kinase assays to assess Raf isoform inhibition.
- Cell-based assays to measure c-Raf, MKK1, and p42 MAPK/ERK2 activation.
- Analysis of Ras GTP-loading and effects of pathway inhibitors.
- Testing ZM 336372 on growth factor/phorbol ester-induced signaling and transformed cell lines.
- Inhibition assays against a panel of 20 protein kinases.
Main Results:
- ZM 336372 is a potent and specific in vitro inhibitor of Raf isoforms.
- ZM 336372 induced >100-fold activation of c-Raf without activating MKK1 or p42 MAPK/ERK2.
- This c-Raf activation was independent of Ras, protein kinase C, or phosphatidylinositide 3-kinase.
- ZM 336372 did not inhibit growth factor/phorbol ester-induced MKK1/MAPK activation or reverse transformed cell phenotypes.
- SAPK2/p38 was the only other kinase inhibited; a mutation rendered it insensitive to ZM 336372 and SB 203580.
Conclusions:
- Raf may possess a novel feedback loop that suppresses its own activation.
- Inhibition of Raf kinase activity might not be a viable anticancer strategy.
- Some reported activators of c-Raf may function as inhibitors.
- ZM 336372 and SB 203580 likely share overlapping binding sites on Raf and SAPK2/p38.