IMP Modulates KSR1-dependent Multivalent Complex Formation to Specify ERK1/2 Pathway Activation and Response Thresholds

Abstract

The Raf-MEK 2 -ERK kinase cascade is one of the most extensively studied signal transduction modules on account of its core participation in both normal and pathological cell regulatory networks. A great deal of information has been gathered about the acute participation of the Raf-MEK-ERK signal transduction circuitry in the modulation and/or propagation of a broad range of dynamic cell regulatory events ranging from cellular proliferation and tumorigenesis to differentiation and cell specialization (1-3). Attention is now necessarily becoming focused on understanding the molecular architecture that allows the core enzymatic components of this cascade to be shared among multiple regulatory pathways yet produce distinct biological outcomes in response to discrete inductive signals. There is accumulating evidence that control of the amplitude, duration, and subcellular compartmentalization of ERK activation are all critical determinants of the biological response (4 -7). The discovery and characterization of non-catalytic accessory components, or scaffolds, that generate higher order molecular organization to modulate the assembly, activation, and compartmentalization of MAPK cascades are beginning to reveal mechanisms that generate specificity in the coupling of MAPK activation to appropriate biological responses (8 -11).We have recently described IMP (impedes mitogenic signal propagation) as a specifier of ERK activation amplitude through inhibition of functional coupling between Raf and MEK kinases (12). The mode of action of IMP is dependent upon the presence of the Raf-MEK-ERK scaffolding protein KSR1 (kinase suppressor of Ras 1) and is a consequence of impairing the capacity of this scaffold to promote signal propagation through Raf to MEK (13). Here, we have examined how IMP association with KSR1 can limit Raf⅐MEK⅐ERK complex formation and function. We find that KSR1 homo-oligomerization is required to couple distinct KSR⅐MEK and KSR⅐B-Raf complexes to allow MEK activation. Furthermore, KSR1 promotes formation of B-Raf/c-Raf hetero-oligomers that contribute to c-Raf kinase activation. IMP association blocks both KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization to impair both MEK activation and the contribution of Raf oligomers to c-Raf kinase activation. These observations reveal the participation of KSR1 both upstream and downstream of Raf kinase activation in human cells and highlight Raf family oligomerization as a basic control point for modulation of signal amplitude by IMP. EXPERIMENTAL PROCEDURESCell Culture and Transfection-HEK293 cells were cultured in Dulbecco's modified Eagle's medium without sodium pyruvate (Invitrogen) with 10% fetal bovine serum and transfected with Lipofectamine and Plus Reagent (Invitrogen). HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and RNA interference was performed with DharmaFECT 1 (Dharmacon).siRNAs, Plasmids, and Antibodies-The following siRNA sequences were used: IMP-FW (GGACACAGCAGAG-GAAAU...

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

“…4D-F). This is consistent with the constitutive binding of KSR1 to B-RAF and the Ras-dependent binding of KSR1 to C-RAF [33][34][35][36].…”

Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

“…However, we found that Rta, although conjugated by SUMO-1 (Chang et al, 2004), was not conjugated by ubiquitin (unpublished results), suggesting that BRAP2 does not influence the function of Rta via ubiquitination. Another important function of BRAP2 is its ability to bind to KSR1 to inhibit ERK signal transduction (Chen et al, 2008;Matheny et al, 2004). KSR1 is a scaffolding protein that facilitates the phosphorylation of MEK1/2 and ERK1/2 in the ERK signal transduction cascade (Muller et al, 2001;Nguyen et al, 2002;Roy et al, 2002).…”

“…Müller et al (2001) demonstrated that KSR1 functions as a scaffold, providing a platform for the phosphorylation of MEK1/2 and ERK1/2 (Muller et al, 2001;Nguyen et al, 2002;Roy et al, 2002). However, BRAP2 appears to prevent KSR1 from interacting with the cytoplasmic membrane and homooligomerization, thus inhibiting the ERK signal transduction pathway (Chen et al, 2008;Matheny & White, 2006;Matheny et al, 2004). This study demonstrated that Rta prevents the binding of BRAP2 to KSR1, activating the ERK signal transduction pathway and the transcription of BZLF1 to influence the viral lytic cycle.…”

Signal control through Raf: in sickness and in health

“…The E3 ligase and Ras effector protein, impedes mitogenic signal propagation (IMP), attenuates Raf-MEK association via inactivation of KSR homo-and heterodimerization with RAF proteins [34]. The association between IMP and KSR1 also blocks KSR1 interaction with MEK1, preventing activation of ERK1/2 [35].…”