Diverging gain-of-function mechanisms of two novel KRAS mutations associated with Noonan and cardio-facio-cutaneous syndromes

Abstract

Activating somatic and germline mutations of closely related RAS genes (H, K, N) have been found in various types of cancer and in patients with developmental disorders, respectively. The involvement of the RAS signalling pathways in developmental disorders has recently emerged as one of the most important drivers in RAS research. In the present study, we investigated the biochemical and cell biological properties of two novel missense KRAS mutations (Y71H and K147E). Both mutations affect residues that are highly conserved within the RAS family. KRAS(Y71H) showed no clear differences to KRAS(wt), except for an increased binding affinity for its major effector, the RAF1 kinase. Consistent with this finding, even though we detected similar levels of active KRAS(Y71H) when compared with wild-type protein, we observed an increased activation of MEK1/2, irrespective of the stimulation conditions. In contrast, KRAS(K147E) exhibited a tremendous increase in nucleotide dissociation generating a self-activating RAS protein that can act independently of upstream signals. As a consequence, levels of active KRAS(K147E) were strongly increased regardless of serum stimulation and similar to the oncogenic KRAS(G12V). In spite of this, KRAS(K147E) downstream signalling did not reach the level triggered by oncogenic KRAS(G12V), especially because KRAS(K147E) was downregulated by RASGAP and moreover exhibited a 2-fold lower affinity for RAF kinase. Here, our findings clearly emphasize that individual RAS mutations, despite being associated with comparable phenotypes of developmental disorders in patients, can cause remarkably diverse biochemical effects with a common outcome, namely a rather moderate gain-of-function.

Rectal cancer profiling identifies distinct subtypes in India based on age at onset, genetic, epigenetic and clinicopathological characteristics

“…Functional validation exhibited decreased GTPase activity through reducing GAP sensitivity in codon 15 mutants, thereby supporting its analogous role in carcinogenesis apart from codon12 and 13 mutations []. The oncogenic role of KRAS was also found to be dependent on its specific mutation due to differential regulation of KRAS downstream pathways []. The consequence might be discrete changes in crucial cellular events, tumor morphology, and aggressiveness as well.…”

Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

“…GTP-bound proteins and total recombinant proteins were analysed by immunoblotting with anti-FLAG antibody. Antibodies against MEK1/2, ERK1/2, AKT, phospho-MEK1/2 (Ser217/221), phospho-ERK1/2 (Thr202/Tyr204) and phospho-AKT (Thr308) were purchased from Cell Signaling Technology (68). …”

Nucleolar stress with and without p53

“…To investigate the dose- and time-dependent effects of RGB-286638 treatment, Cirstea et al 260 used both p53 -wild type (MM.1S, MM.1R, and H929) and p53 -null MM cells (U266, OPM1, RPMI). Cell viability was measured 48 h after a 50 nM RGB-286638 treatment; p53 -wild type cells (MM1.S, which had p53 stabilization and activation) were slightly more sensitive than p53 -null cells.…”