Journal: Nature Communications

Article Title: SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers

doi: 10.1038/s41467-019-10367-x

Figure Lengend Snippet: SHOC2 is required for feedback relief ERK activation induced by MEKi’s. a SHOC2 deletion impairs ERK-reactivation after treatment with Selumetinib. Indicated cells were treated with 1 µM Selumetinib and lysates collected at indicated time points. b Quantification of P-BRAF/BRAF over time for cell lines shown in ( a ) relative to NT control. c Quantification of P-ERK/ERK over time for cell lines shown in ( a ) relative to NT control. d – f SHOC2 deletion impairs MEK, but not PanRAF induced ERK-reactivation. A549 and A427 cells were pre-treated for 12 h with either 1 µM Selumetinib ( d ) / 100 nM Trametinib ( e ) / or 2.5 µM LY3009120 ( f ). Cells were either lysed at this point (NT - Non Treated, NW - Non washed) or the inhibitor was washed-out for the indicated time points before lysate collection. Lysates were used to perform RAS-RBD pull downs and the additional cell lysate probed with indicated antibodies. g H520 cells or h H522 cells, which have no known driver mutations in the ERK pathway show a reduced dependency on SHOC2 for MEKi-induced ERK-reactivation. Parental or SHOC2 KO H520/H522 cells were treated as ( e )

Article Snippet: MEK 1⁄2 , Cell Signaling Technology , 4694 , Rabbit , 1:1000.

Techniques: Activation Assay, Control

Journal: Nature Communications

Article Title: SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers

doi: 10.1038/s41467-019-10367-x

Figure Lengend Snippet: SHOC2 is required for RAF dimerization induced by MEKi’s. a SHOC2 depletion abrogates MEKi-induced RAF dimerization and impairs ERK pathway reactivation after MEKi withdrawal. shSCR of shSHOC2 H358 cells were pre-treated with 1 µM Selumetinib for 12 h, before the inhibitor was washed-out at indicated time points and lysates used to perform endogenous RAF IPs. (NT - Non Treated, NW - Non washed). Con = IgG control IP. b As ( a ) using A549 and HCC4006 cells with a single wash-out time point of 30 min. c SHOC2 is required for MEK but not PanRAFi-induced RAF dimerization. Parental and SHOC2 KO H358 cells were pre-treated with 1 µM Selumetinib, 100 nM Trametinib 2.5 μM LY3009120 and subject to endogenous RAF IPs as ( a ). d SHOC2 is required for ERK inhibitor induced RAF dimerization. As ( c ), H358 cells were treated with 1 μM Selumetinib and 2 μM LY3214996. e B & C but not ARAF knockdown partially diminish MEKi induced signalling rebound and ERK reactivation. H358 cells transfected with indicated siRNAs were treated 3 days later with 1 µM Selumetinib for 12 h before the inhibitor was washed-out for 30 min. (NT - Non Treated, NW - Non washed). f Quantification of P-ERK and P-T380 RSK in ( e ). g B & C, but not ARAF knockdown partially sensitise H358 cells to Selumetinib. Viability curves for Selumetinib of H358 cells transfected with siRNAs as in ( e ). h Schematic to illustrate the requirement of the SHOC2 phosphatase complex for feedback relief ERK-activation on MEKi treatment. ERK activity in RAS-mutant cells is maintained at steady state by negative feedbacks at multiple levels including RTK and RAF pathway nodes. MEKi treatment leads to feedback relief ERK-pathway activation that is both dependent upon RAS-GTP and SHOC2 phosphatase-dependent ‘S259’ dephosphorylation for RAF dimerization. Following inhibitor withdrawal, release of this ‘primed’ P-MEK (phosphorylated but unable to activate ERK when inhibitor-bound) generates a wave of ERK phosphorylation that is dampened by negative feedbacks. Even in the presence of mutant RAS in SHOC2 KO cells MEKi induced feedback relief RAF dimerization is prevented, leading to reduced P-MEK rebound and more potent and durable ERK inhibition

Article Snippet: MEK 1⁄2 , Cell Signaling Technology , 4694 , Rabbit , 1:1000.

Techniques: Control, Knockdown, Transfection, Activation Assay, Activity Assay, Mutagenesis, De-Phosphorylation Assay, Phospho-proteomics, Inhibition

Journal: Cell host & microbe

Article Title: Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation

doi: 10.1016/j.chom.2018.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-MEK 1⁄2 , Cell Signaling , Cat # 8727, RRID:AB_10829473.

Techniques: Virus, Isolation, Recombinant, Modification, Ab Array, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, BIA-KA, Extraction, Membrane, Protein Extraction, Luciferase, Endotoxin Assay, LDH Cytotoxicity Assay, shRNA, Control, Plasmid Preparation, Transgenic Assay, Knock-Out, Software