Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Expressed genes with significant overall mutation burden in sun-exposed melanoma

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Mutagenesis

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Statistical analysis of RAC1 P29S in the Yale tumor cohort

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Mutagenesis

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Crystal structure of RAC1 P29S . ( a ) Overall schematic of RAC1 P29S showing the P-loop (pink), switch I (light green), switch II (purple), Mg 2+ (cyan) and the slowly hydrolyzing GTP analog, GMP-PNP (stick format). The red sphere indicates the location of p.Pro29Ser. The dashed box indicates the regions shown in b – e . ( b ) Close-up view showing close contacts of ribose hydroxyls with the switch I region in RAC1 P29S . ( c ) RAC1 WT bound to GMP-PNP. ( d ) HRAS in complex with GMP-PNP (PDB 5P21) . Direct hydrogen bonding between the switch I backbone and ribose are commonly observed in activated HRAS but are less frequently observed in Rho family GTPases, including RAC1. A RAS-like hydrogen bonding pattern is observed in activated RAC1 P29S (dashed red lines). ( e ) Superposition of the switch I region and GMP-PNP. RAC1 P29S is shown in light green, RAC1 WT is shown in gray, and HRAS is shown in light blue. The red sphere indicates the location of p.Pro29Ser. The residues discussed in the text are labeled. The figure was made using CCP4mg .

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Labeling

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: In vitro RAC1 P29S binding to downstream effectors. ( a ) PAK1 pulldown assay showing that recombinant histidine-tagged RAC1 P29S (His-RAC1 P29S ) has higher binding to its effector PAK1 compared to His-RAC1 WT for samples that include GTP and GTPγS. NC, PAK1-PBD beads alone as a negative control. The His-RAC1 WT and His-RAC P29S lanes show unbound wild-type and mutant protein, as indicated. The multiple bands in all lanes are RAC1; all bands are observed when RAC1 crystals are run on SDS-PAGE. ( b ) RAC1 pulldown assay showing that recombinant His-GST-RAC1 P29S has higher binding to endogenous MLK3 expressed in melanoma cells compared to His-GST-RAC1 WT in all reaction samples. The immobilized recombinant RAC1 proteins were incubated with cell lysates from two independent melanomas (YULAC that is RAC1 WT and BRAF V600K and YUHEF that is RAC1 P29S and BRAF WT ). The figure shows western blot of bound proteins with antibodies to MLK3. As negative controls, we used lysates incubated with GST-bound beads or recombinant RAC1 proteins that were not incubated with cell extracts. The western blot analysis also shows the expression of the relevant proteins in whole-cell lysate (WCL). The reaction mixtures were likely to contain nucleotides contributed by the cell lysates in addition to those added in vitro , as indicated. ( c ) PAK1 pulldown assay comparing the binding activity of RAC1 WT , RAC1 P29S and RAC1 F28L proteins. The numbers under each lane indicate the band density as determined by scanning the membranes with ImageJ.

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: In Vitro, Binding Assay, Recombinant, Negative Control, Mutagenesis, SDS Page, Incubation, Western Blot, Expressing, Activity Assay

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Cellular function of RAC1 P29S compared to RAC1 WT . ( a ) Western blot analysis of RAC1 in mouse melanocytes infected with wild-type (WT) or mutant RAC P29S retroviral expression vectors compared to nontransfected cells (parental). Actin indicates protein loading in each lane. ( b ) RAC1 P29S enhances melanocyte proliferation. The proliferation of melanocytes transiently expressing RAC1 WT (green) or RAC1 P29S (red) compared to noninfected parental cells (blue). Error bars indicate standard errors. ( c ) RAC1 P29S enhances cell migration. The graphs show the rate of migration of parental, RAC1 WT - and RAC1 P29S -expressing melanocytes at daily intervals. ( d ) RAC1 P29S enhances ERK activation. Western blots of lysates from mouse melanocytes expressing RAC1 WT or RAC1 P29S probed with RAC1, pERK, ERK and actin antibodies, as indicated on the side of the gel. ( e ) Localization of GFP-tagged RAC1 WT (GFP-RAC1 WT ) and GFP-RAC1 P29S in COS-7 cells. COS-7 cells transiently expressing GFP-RAC1 WT (i, ii) or GFP-RAC1 P29S (iii, iv) were fixed with paraformaldehyde and imaged with a spinning-disk confocal-based inverted Olympus microscope. Insets (yellow boxes) illustrate the localization of RAC1 P29S , but not RAC1 WT , in membrane ruffles (compare i and ii to the yellow arrows in iii and iv, respectively). pERK, phosphorylated ERK. Scale bars, 10 μm.

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Cell Function Assay, Western Blot, Infection, Mutagenesis, Expressing, Migration, Activation Assay, Microscopy