Ras isoform abundance and signalling in human cancer cell lines

Abstract

The ubiquitously expressed major Ras isoforms: H-, Kand N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N-and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically KXNbH. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

TCR-Induced Activation of Ras Proceeds at the Plasma Membrane and Requires Palmitoylation of N-Ras

“…1B) (14). This expression pattern is reminiscent of findings in other lymphoid leukemia cell lines (21) and peripheral T cells (20). Importantly, T cell costimulation via CD28, SLAM, or LFA-1 did not lead to significantly increased Ras-GTP formation as triggered by 5 mg/ml anti-CD3 (data not shown).…”

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling

“…Unfortunately, we were unable to efficiently suppress the expression of choline kinase in the hT/LT/Ras cells, which is likely due to poor transfection efficiency, as has been previously described (12.7 ± 3% using fluorescein-labeled nonsense siRNA; data not shown) (Telang et al, 2007). Unlike human bronchial epithelial cells, HeLa cervical carcinoma cells are easily transfected with high efficiency and express unmutated H-Ras, K-Ras and N-Ras, but maintain an activated Ras signaling pathway in the presence of serum (Omerovic et al, 2008). Initially, we examined the HeLa cells for choline kinase-a and choline kinase-b mRNA expression using reverse transcriptase-PCR and found that these cells expressed choline kinase a-1 and choline kinase-b, but not the choline kinase a-2 splice variant (35 cycles, data not shown).…”

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

“…[29] Interestingly, Ras association to endosomes exhibits isoform specificity: whereas N-and H-Ras establish stable interactions with the endosomal compartment, K-Ras does not. [11,30,31] These differences have been attributed to the modification of N-and H-Ras, but not of K-Ras, by ubiquitination. [31] At the PM, Ras proteins partition into microdomains referred to as ''nanoclusters.''…”