Uncovering the Complexity of RAS Signaling Networks
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The Raf/MEK/MAP Kinase signal transduction cascade is a key Ras effector pathway that mediates diverse cellular responses to environmental cues, and makes a major contribution to Ras-dependent oncogenic transformation. Here we describe two important aspects to understanding the signaling complexity of any multiphenotypic arrangement, namely, the identification and examination of new effectors and the direct authentication of protein-protein interactions. We have identified a new Ras effector, IMP (Impedes Mitogenic signal Propagation) that negatively regulates MAP kinase activation by limiting formation of Raf/MEK complexes. The mechanism of inhibition appears to be through inactivation of the KSR1 adaptor/scaffold protein, demonstrating that in addition to promoting signal transmission, scaffold proteins can function to restrict signal propagation. Ras can inactivate IMP through induction of IMP auto-ubiquitination, facilitating KSR-dependent engagement of MEK by activated Raf. These observations reveal dual Ras effector inputs to the MAP kinase cascade: induction of Raf kinase activity concomitant with derepression of KSR-dependent Raf/MEK complex formation. This relationship provides a mechanism to limit engagement of the MAP kinase cascade in the absence of Ras activation. MAP kinase activation contributes to multiple diverse cellular responses to environment. The capacity to control the amplitude of this response via molecules like IMP likely contributes to flexible and adaptive cellular behavior in the context of complex regulatory signals. Analysis of the primary amino acid sequence of IMP indicated that it may regulated on multiple levels in different ways. We show that IMP can be phosphorylated by ERK and that this correlates with IMP translocation to the nucleus. We also demonstrate that SUMO-1 exerts negative effects on IMP inhibitory activity. Additionally, a predicted UBP-ZnF was examined for regulation of autoubiquitination. Together, these data point to the existence of multiple regulatory inputs that control IMP activity. Finally, we discuss the importance of establishing true binding relationships between associated proteins for the characterization of signaling pathways. Using Ras and its putative effector RalGDS as an example, we describe a simple method to this end. By identifying loss-of-function mutations in both proteins that reciprocally compensate to restore a biological outcome, we confirm that RalGDS is a bona fide effector of Ras, thereby proving the validity of this technique for authenticating other protein-protein interactions.