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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

Chemistry & Biology
|July 28, 1999
PubMed
Summary
This summary is machine-generated.

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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.

Related Experiment Videos

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.