Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation
Pan-Yu Chen, Benjamin J. Huang, Max Harris, Christopher Boone, Weijie Wang, Heidi Carias, Brian Mesiona, Daniela Mavrici, Amanda C. Kohler, Gideon Bollag, Chao Zhang, Ying Zhang, Kevin Shannon
Pan-Yu Chen, Benjamin J. Huang, Max Harris, Christopher Boone, Weijie Wang, Heidi Carias, Brian Mesiona, Daniela Mavrici, Amanda C. Kohler, Gideon Bollag, Chao Zhang, Ying Zhang, Kevin Shannon
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Research Article Oncology

Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation

Abstract

A T50I substitution in the K-Ras interswitch domain causes Noonan syndrome and emerged as a third-site mutation that restored the in vivo transforming activity and constitutive MAPK pathway activation by an attenuated KrasG12D,E37G oncogene in a mouse leukemia model. Biochemical and crystallographic data suggested that K-RasT50I increases MAPK signal output through a non-GTPase mechanism, potentially by promoting asymmetric Ras:Ras interactions between T50 and E162. We generated a “switchable” system in which K-Ras mutant proteins expressed at physiologic levels supplant the fms like tyrosine kinase 3 (FLT3) dependency of MOLM-13 leukemia cells lacking endogenous KRAS and used this system to interrogate single or compound G12D, T50I, D154Q, and E162L mutations. These studies support a key role for the asymmetric lateral assembly of K-Ras in a plasma membrane–distal orientation that promotes the formation of active Ras:Raf complexes in a membrane-proximal conformation. Disease-causing mutations such as T50I are a valuable starting point for illuminating normal Ras function, elucidating mechanisms of disease, and identifying potential therapeutic opportunities for Rasopathy disorders and cancer.

Authors

Pan-Yu Chen, Benjamin J. Huang, Max Harris, Christopher Boone, Weijie Wang, Heidi Carias, Brian Mesiona, Daniela Mavrici, Amanda C. Kohler, Gideon Bollag, Chao Zhang, Ying Zhang, Kevin Shannon

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Figure 4

Coexpression of WT EGFP–K-Ras or mCherry–K-Ras in MOLM-13 KRASKO clone 24 cells results in variable growth inhibition that correlates with the strength of individual mutant K-Ras proteins.

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Coexpression of WT EGFP–K-Ras or mCherry–K-Ras in MOLM-13 KRASKO clone 2...
(A) Expression of K-RasWT antagonizes the pro-survival activity of K-RasG12D with mCherry–K-Ras expression showing a more potent effect than EGFP–K-Ras. Clone 24 cells were treated with DMSO or 10 nM AC220 in the absence or presence of dox for 48 hours before flow cytometry–based analysis of CC3. Results are shown from 3 independent experiments each performed in technical triplicate. (B) Absolute increases in the percentage of CC3+ cells coexpressing EGFP–K-Ras proteins and either mCherry–K-RasWT or mCherry–K-RasG12D. (C and D) IC50 values were measured using CTG in cells coexpressing the indicated combinations of EGFP– and mCherry–K-Ras proteins. Normalized IC50 values for 3 independent CTG experiments that were each performed in technical triplicate. Values shown are mean ± SEM. Multiple t tests were performed using the Holm-Šidák method to correct for multiple comparisons. Adjusted P values: ****P < 0.0001, ***P ≥ 0.0001 and < 0.001, **P ≥ 0.001 and < 0.01, *P ≥ 0.01 and < 0.05.