Analysis of the Modulation of RAF Signaling by 14-3-3 Proteins

The regulation of cellular biochemical signaling reactions includes the modulation of protein activity through a variety of processes. For example, signaling by the RAF kinases, which are key transmitters of extracellular growth signals downstream from the RAS GTPases, is modulated by dimerization, protein conformational changes, post-translational modifications, and protein-protein interactions. 14-3-3 proteins are known to play an important role in RAF signal regulation, and have the ability to stabilize both inactive (monomeric) and active (dimeric) states of RAF. It is poorly understood how these antagonistic roles ultimately modulate RAF signaling. To investigate, we develop a mathematical model of RAF activation with both roles of 14-3-3, perform algebraic and numeric analyses, and compare with available experimental data. We derive the conditions necessary to explain experimental observations that 14-3-3 overexpression activates RAF, and we show that even arbitrarily strong binding of 14-3-3 to RAF dimers alone could not necessarily explain this observation. Our integrated analysis also suggests that RAF-14-3-3 binding is relatively weak (significant amounts of RAF would remain unbound if only the first affinity were a factor), and instead that changing avidity more directly controls the bound fraction. Lastly we consider the limit at which RAF-14-3-3 interactions are driven solely by avidity, which allows for significant simplifications to the interaction model. Overall, our work presents a mathematical model that can serve as a foundational piece for future, extended, studies of signaling reactions involving regulated RAF kinase activity.