Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: ( A-B ) Metascape enrichment network visualization showing the intra-cluster and inter-cluster similarities of enriched terms (up to ten terms per cluster), in starvation ( A ) and FCS stimulation state (B) separately, where nodes are represented by pie charts indicating their associations with three different mutants. The colour code for pie sector represents G12D (red), G13D (blue), and Q61H (green). Cluster labels were added manually. ( C - D ) Heatmap showing the log2(FC MUT/WT) of differentially enriched proximal proteins which were reported in previous proximitome studies ( C ) or proposed based on our study ( D ) Asterisk indicates that the hit meets the criteria of log2(mutant/WT FC) > 0.5 or <-0.5 and - log10 (p value) > 0.7. ( E ) Heatmap showing the relative abundance (log2 LFQ) of proximal proteins involved in KRAS related canonical pathways (such as MEK-RAF, mTOR, PI3K, RALGDS, RASSF and TIAM RAC pathway).

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Western blot indicates the biotinylated proteins in the presence or absence of different c reagents including tetracycline, phenol-biotin and H 2 O 2 . K-Ras APEX-2 stable cell line was treated with tetracycline for 24 hours where is indicated. Then, cells were treated with phenol-biotin and/ or H 2 O 2 (where is indicated) and cells were lysed, and proteins were separated in an SDS Gel. Streptavidin, DyLight 488 Conjugated antibody were used to visualise the biotinylating proteins. * Represents biotinylated background proteins. (B) Western blot analysis showing a-FLAG in cell extract upon 24 hours tetracycline treatment. (C) Imaging of live cells using Opera Phenix™ and processed in Columbus Image Analysis System. Panels 1183 Hoechst 33342 for staining DNA (blue); the green fluorescence signal from GFP (Green), Differential Interference Contrast (DIC). (D) Western blot analysis showing K-Ras GFP, GAPDH and GFP expression upon digitonin cytoplasmic-membrane fractionation of Stable K-Ras GFP or GFP only FRT T-Rex HEK293 cell line. Cells were incubated with tetracycline for 24 hours. (E) Western blot analysis showing a-FLAG, endogenous KRAS, TOM20 and Calreticulin expression upon digitonin cytoplasmic-membrane fractionation of stable KRAS APEX-2 WT, G12D, G13D and Q61H FRT T-Rex HEK293 cell lines. Cells were incubated with or without tetracycline for 24 hours. (F) Western blot analysis showing a-FLAG, GAPDH, TOM20 and endogenous KRAS in sucrose gradient fractions upon 15 hours of starvation or 15 hours of starvation and a subsequent 10-minute treatment with 20% FCS.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Western Blot, Stable Transfection, SDS-Gel, Imaging, Staining, Fluorescence, Expressing, Membrane, Fractionation, Incubation

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A-F) Volcano plotting of fold changes and p values derived from t-test statistic for proximal proteins identified in G12D, G13D, and Q61H KRAS mutant in either starvation or FCS treated condition, in which WT-KRAS was used as a control. Known KRAS effectors (red), repressors (green) and receptors (blue) are coloured and labelled.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Heatmap showing relative abundance of known KRAS interactors in WT KRAS, G12D, G13D and Q61H KRAS mutant-APEX2 samples in either starvation or FCS stimulated condition. (B) APEX-2 labelling was performed in all different cell lines (WT, G12D, G13D and Q61H) under starvation and FCS induction. Western blot analysis showing ERK, pERK, ARAF and LZTR1 in cell extract as well as elution upon streptavidin-immunoprecipitation. (C) Cells were seeded in 96 well plates and treated with tetracycline for 24 hours. Next day cells were transfected with LZTR1 siRNA or Control siRNA. Cells were imaged after 72 hours using the Opera Phenix microscope. The images were analysed in Columbus Image Data Storage and Analysis System. The media fluorescent per well per mean per cell was determined. Finally, a fold difference between the control and the siRNA LZTR1 was measured. (D) Stable HEK293 FRT T-REX cell lines of WT, G12D, G13D and Q61H were treated with tetracycline for 24 hours. The next day, cells were treated with 50 μg/mL cycloheximide (CHX). At the indicated time points after CHX-treatment cells were harvested. Western blot analysis showing GFP, Pan-Ras and ACTB in cell extract was performed. (E) Band densities from were analyzed using ImageStudio, and expression was normalized to ACTB protein levels to determine KRAS half-life. (F) The influence of LZTR1 overexpression on WT K-Ras as well as G12D, G13D and Q61H oncogenic mutants. HEK293 cells were seeded in 6 well plates and transfected with 1µg Flag-K-Ras plasmids as well 0, 0.1 or 0,25 µg Myc-LZTR1 plasmid concentration. Cells were cultured for a total of 48 hours before protein levels were evaluated using Western blot. (G) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2 µg Myc-LZTR1 plasmid concentration. Cells were incubated for 48 hours, then cells were treated with 50 μg/mL CHX. At the indicated time points after CHX-treatment cells were harvested. Western blot analysis showing FLAG (RAS), MYC (LZTR1) and ACTB in cell extract was performed. (H) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2µg Myc-LZTR1 plasmid concentration and let to grow for 48 hours. Cells were then treated for 24 hours with MLN4924 in different concentrations (0, 0.3 1 µM). Cell extract was then analysed on western blot and FLAG (RAS), MYC (LZTR1), CUL3 and ACTB was monitored. (I) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2 µg Myc-LZTR1 plasmid concentration and let to grow for 48 hours. Cells were then treated for indicated time with MLN4924 (0.3 µM). Cell extract was then analysed on western blot and FLAG (RAS), MYC (LZTR1), CUL3 and ACTB was monitored.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Mutagenesis, Western Blot, Immunoprecipitation, Transfection, Microscopy, Expressing, Over Expression, Plasmid Preparation, Concentration Assay, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Microscopy based analysis of GFP-KRAS in the presence and absence of LZTR1. ( B ) KRAS and Pan- Ras protein stability in the presence and absence of LZTR1 assessed by immunoblotting. ( C ) Cullin 3 (CUL3) knockdown stabilises LZTR1. ( D ) LZTR1 (MYC) and KRAS (FLAG) levels are correlated at the protein level. ( E ) KRAS wildtype and G12D (both FLAG tagged) differentially affect LZTR1 (MYC-tag) protein levels. ( F ) KRAS (FLAG) and LZTR1 (MYC) protein levels are dependent on the presence of Cullin 3 (CUL3).

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Microscopy, Western Blot