Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling

Abstract

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

Figure 1

Analysis of Endogenous Ras-dependent Raf Dimerization and Activation (A) Serum-starved HeLa cells were treated or not with EGF for five minutes prior to lysis. Endogenous A-Raf, B-Raf, or C-Raf proteins were immunoprecipitated and probed for the binding of other endogenous Raf members. (B) Serum-starved HeLa cells were treated or not with EGF prior to fixation. Red foci indicate binding interactions in the PLA assay. Cell nuclei were stained with DAPI. (C) HeLa cells stably expressing the pLKO.1 shA-Raf, shB-Raf, shC-Raf or empty vector were serum-starved and then treated or not with EGF prior to lysis. Endogenous Raf proteins were immunopurified and their intrinsic kinase activity determined using kinase-inactive MEK as a substrate. Error bars represent the standard deviation of the mean for three independent experiments.

Figure 2

Raf Dimer Interface Mutations Modulate Raf Dimerization and Activation (A-D) Serum-starved HeLa cells expressing the indicted Pyo- and FLAG-tagged Raf proteins were treated or not with EGF prior to lysis. (A) Immunoprecipitated FLAG-Raf complexes were probed for the binding of endogenous Raf proteins. (B) Immunoprecipitated Pyo-Raf complexes were probed for the binding of FLAG-Raf. (C) FLAG-Raf proteins were immunopurified and their intrinsic kinase activity determined. Error bars represent the standard deviation of the mean for three independent experiments. (D) Immunoprecipitated FLAG-Raf complexes were probed for the binding of the indicated endogenous proteins. (E) Pyo-B-Raf complexes from EGF-treated HeLa cells were subjected to mass spectrometry analysis.

Figure 3

Differential Effects of Dimerization on Disease-associated Raf Mutants (A) FLAG-Raf proteins were immunoprecipitated from cycling HeLa cells and probed for the binding of the endogenous Rafs. (B) NIH3T3 cells were infected with retroviruses expressing the indicated Raf proteins. After two weeks of culture, focus formation was visualized by methylene blue staining. Shown are focus plates from a representative experiment.

Figure 4

DI1 Peptide Inhibits Raf Dimerization and Signaling (A) B-Raf structure is shown with the amino acids of DI1 highlighted in red. (B-D) Cos cells were transfected to express GFP or GFP-DI1 as indicated. (B) Endogenous Raf proteins were immunoprecipitated and probed for the binding of GFP-DI1. (C,D) Endogenous C-Raf proteins were immunoprecipitated from serum-starved or EGF-treated cells and probed for the binding of GFPDI1 or B-Raf. Lysates were probed for pMEK levels in (C). (E) Cos cells stably expressing the indicated B-Raf or C-Raf mutants were transfected to express GFP or GFP-DI1. Lysates were probed for levels of pMEK. (F-H) Cancer cell lines were treated with the indicated concentrations of TAT-DI1 or TAT-Scram. (F) Cell viability was determined by cell counting. Error bars represent the standard deviation of at least three independent experiments. (G) Lysates were probed for pMEK levels. (H) Lysates were examined for PARP cleavage.