Differential effects of dimerization on B-Raf and C-Raf function in normal and disease signaling

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NCI	BIOCHEM/CHEM-8

* FARE Award Winner

Authors

• A Freeman
• D Ritt
• D Morrison

Abstract

Proper regulation of Raf kinase signaling is critical for normal cellular function as mutation of the Raf kinases can result in cancer and certain developmental disorders. To study the Raf activation process, we have investigated the importance of dimerization. Growth factor stimulation induces Raf hetero- and homo-dimerization and increases the kinase activities of B-Raf and C-Raf. To further explore these interactions, we utilized mutations in the dimer interface that either enhance (E586K-B-Raf and E478K-C-Raf) or prevent (R509H-B-Raf and R401H-C-Raf) dimerization. Interestingly, these mutations had only a modest effect on B-Raf activity; however, the E-K mutation greatly enhanced C-Raf activity and the R-H mutation eliminated it. Expression of E478K-C-Raf or knockdown of C-Raf had little effect on B-Raf; however, expression of E586K-B-Raf significantly increased endogenous C-Raf activity and knockdown of B-Raf dramatically inhibited C-Raf activation, highlighting the dependence of C-Raf activation on B-Raf. Strikingly, although the dimer interface mutations affected the ability of all disease-associated B-Raf and C-Raf mutant proteins to heterodimerize, they only altered the focus forming ability of B-Raf and C-Raf proteins with moderate to low, but not high, kinase activity. These data indicate that dimerization is important for Raf activation and blocking dimerization may be of therapeutic use.

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