Ras signalling on the endoplasmic reticulum and the Golgi

Abstract

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.

Early endosome localization and activity of RasGEF1b, a toll-like receptor-inducible Ras guanine-nucleotide exchange factor

“…27 It is well known that GEFs should be found in some cellular compartments where their cognate GTPases are located, so it is reasonable to speculate that RasGEF1b can be primarily located at early endosome to activate Ras during vesicular traffic to membrane, as endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and ER, respectively. 28 In response to extracellular stimulation, such as a growth factor, GDP is released from Ras being activated by the subsequent binding of GTP, that is the guaninenucleotide exchange reaction. Such reaction is catalysed by the direct physical association between Ras and its respective GEF and seems to be essential for Ras-MEK-ERK pathway activation.…”

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…In agreement with Raf/ERK activation profiles, Ras effectively supported proliferation and cellular transformation of murine fibroblasts from the ER, disordered membrane and lipid rafts, but not from GC. [51] A discrepancy exists with respect to Ras transforming potential at the GC, [45] probably due to differences in the GC tethers utilized, although in both studies transformation correlates with ERK activation. Ras tethered to the GC by the E1 ABV protein, was also unable to transform.…”

“…[44] A consequence of the site-specific activity of GEFs, GAPs, and other regulatory proteins is that such compartmentalization results in a remarkable variability in Ras signal outputs. Thus, Ras activation at the PM is fast and transient, whereas at endomembranes [45,46] and at endosomes [47] is slow and sustained. Galectin-1, by stabilizing H-Ras nanoclusters, leads to enhanced recruitment of effectors and greater signal output.…”

“…ERK activation was also observed when H-RasV12 was tethered to the GC. [45] A later study from our laboratory utilized the same strategy to study effector usage at PM microdomains as well: H-RasV12 at lipid rafts and at the ER efficiently activated ERK, PI3K, and Ral-GDS; at bulk membrane it behaved similarly, although with lower PI3K activation. [51] H-Ras restricted to the GC, profusely activated Ral-GDS but not ERK.…”

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

“…During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments [4][5][6][7] . FRET-based genetically encoded biosensors for second messengers, protein phosphorylation and activity of small GTPases have provided insights into the spatial and temporal regulation of signaling processes [8][9][10][11][12] .…”