raf1 entry clone lacking a stop codon  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

doi: 10.1074/jbc.RA119.011080

Figure Lengend Snippet: Switch II internal tandem duplication of both NRAS and KRAS proteins increases the affinity for RAF effector. NanoBRET saturation curves of transiently transfected RAF1 NanoLuc donor constructs (constant) and titrated, transiently transfected Halotag-RAS acceptor constructs. BRETmax and BRET50 values represent the maximum number of protein–protein interactions and protein affinity, respectively. A, top, Halotag-NRAS ITD fusion proteins show increased interaction with effector RAF1 compared with the WT protein when overexpressed in Caco-2 cells. *Interactions with NRAS WT to be significantly different to both NRAS Q61R and NRAS ITD (p < 0.0005 from 0.0625–4 µg DNA; p < 0.02 at 8 µg DNA). A, bottom, both KRAS ITD and NRAS ITD mutants display increased RAF1 interaction compared with KRAS WT in HEK293T cells. *NRAS ITD mBRET values are significantly different to KRAS WT (p < 0.0001 from 0.0094–0.3 µg DNA; p < 0.008 at 0.6 µg DNA). **KRAS ITD mBRET values are significantly different from KRAS WT (p < 0.0001 from 0.0094–0.075 µg DNA; p < 0.03 at 0.15 µg DNA). B, nanoBRET saturation curves of NRAS ITD and NRAS WT in HEK293T cells. *RAF1 interactions are significantly different (p < 0.0005) between 0.00195 and 0.00781 µg DNA. C, nanoBRET saturation curves of KRAS ITD and KRAS WT. *RAF1 interactions are significantly different (p < 0.0001) between 0.00195 and 0.25 µg DNA. Nonlinear regressions were performed in GraphPad Prism (see Tables S2–S5 for curve fit parameters). Error bars represent n = 3 technical replicates. Statistical significance of the differences was determined by 2-way ANOVA in GraphPad Prism.

Article Snippet: NanoLuc-RAF1 fusions were made in a similar fashion using an att5-att1 NanoLuc (Promega) construct and a RAF1 Entry clone (Addgene, no. 70497).

Techniques: Transfection, Construct, Protein-Protein interactions

Journal: The Journal of Biological Chemistry

Article Title: RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

doi: 10.1074/jbc.RA119.011080

Figure Lengend Snippet: The ITD mutation in RAS does not affect RAS–RAF1 interaction but blocks RAS-RasGAP binding. A and B, isothermal titration calorimetry experiments to measure the dissociation constant for GMPPNP-bound WT NRAS and NRAS ITD with (A) RasGAP NF1 (GRD) and (B) effector RAF1 (RBD). Differential power (DP) is a measure of energy required to maintain isothermal conditions between the reference cell and the sample cell. C, mBRET saturation values assessing the ability of NF1 GAP co-expression to squelch RAF–RAS interaction for WT and ITD mutant KRAS and NRAS. The error bars represent the standard deviation of three technical replicates.

Article Snippet: NanoLuc-RAF1 fusions were made in a similar fashion using an att5-att1 NanoLuc (Promega) construct and a RAF1 Entry clone (Addgene, no. 70497).

Techniques: Mutagenesis, Binding Assay, Isothermal Titration Calorimetry, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

doi: 10.1074/jbc.RA119.011080

Figure Lengend Snippet: Crystal structure of GDP-bound NRAS ITD and KRAS ITD provide insights into the effect of ITD on RAS interaction with GAP and effector proteins. A, the tertiary structure of GDP-bound NRAS ITD. B, the tertiary structure of GDP-bound KRAS ITD. C and D, structural superposition of GDP-bound NRAS ITD with (C) GDP-bound WT KRAS and (D) GMPPNP-bound WT NRAS. E–G, models of ITD mutants of NRAS and KRAS in complex with (E) NF1-GRD (PDB ID: 6OB2), (F) PI3Kγ (PDB ID: 1HE8), and (G) RAF1-RBD (PDB ID: 4G0N) generated using the structural superposition of NRAS ITD on K/HRAS present in KRAS-NF1, HRAS-PI3Kγ, and HRAS-RAF1 (RBD) complexes. These models suggest that the ITD (shown in red) in RAS would sterically clash with NF1 GAP and PI3Kγ and not with RAF1.

Article Snippet: NanoLuc-RAF1 fusions were made in a similar fashion using an att5-att1 NanoLuc (Promega) construct and a RAF1 Entry clone (Addgene, no. 70497).

Techniques: Generated