KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
|
| ||
| Antibodies | ||
| M-Ras | Abcam | Ab176570 |
| R-Ras | Abcam | Ab191399 |
| Ras | Abcam | Ab108602 |
| Rheb | CST | 1379S |
| RalA | Abcam | Ab126627 |
| Rap1A | Abcam | Ab96223 |
| N-cadherin | CST | D4R1H |
| Vimentin | CST | 5741S |
| Cofilin (pS3) | Abcam | Ab12866 |
| Cofilin | Abcam | Ab42824 |
| PAK1 (pThr423)/Pak2 pThr402 | CST | 2601S |
| PAK1 | CST | 2692S |
| Rac1 | Proteintech | 24072–1-AP |
| GFP | CST | 2955S |
| P38 (pThr180/pTyr182) | CST | 4511S |
| P38 | CST | 9212S |
| MAP3K1 (pThr1400) | Proteintech | 28844–1-AP |
| MAP3K1 | Proteintech | 19970–1-AP |
| GAPDH | Proteintech | 60004–1-lg |
| beta-Actin | CST | 4967S |
| IRDye 800CW Goat anti-Human | LI-COR | 926–32211 |
| IRDye 680RD Goat anti-Human | LI-COR | 926–68070 |
|
Bacterial and virus strains | ||
| Escherichia coli BL21(DE3) | NEB | Cat# C2527H |
|
Chemicals, peptides, and recombinant proteins | ||
| GDP | Sigma-Aldrich | Cat# G7127–100MG |
| Guanosine 5’-[β,γ-imido]triphosphate (GNP, GppNHp) | Axorra | Cat# JBS-NU-401–50 |
| GTPγS | Abcam | Cat# ab146662 |
| RAPGEF5 | Cytoskeleton | Cat# CS-GE09 |
| His-Rac1(WT) | Cytoskeleton | Cat# RC01 |
| His-RabL5(WT) | Abcam | Cat# ab156964 |
| His-Rab1A(WT) | Abcam | Cat# ab90975 |
| Biotinylated Avi-Rap1A(WT) | This study | N/A |
| Biotinylated Avi-RhoA(WT) | This study | N/A |
| Biotinylated Avi-RalA(WT) | Amid Biosciences | Cat# RALA-B-301 |
| Phospho p44/42 MAPK ERK1/2 (Thr202 Tyr204) (D13.14.4E) | Cell Signaling Technology | Cat# 4370S |
| R-PE-Fab Goat Anti-Rabbit IgG, Fc Secondary Antibody | Jackson ImmunoResearch | Cat# 111–117-008 |
| EDTA 0.5 M in water, pH 8.0 | Sigma-Aldrich | Cat# 03690 |
| Sypro Orange | Thermo Fisher Scientific | S6650 |
| TEV Protease | Sigma-Aldrich | Cat# T4455 |
| cOmplete Protease Inhibitor Cocktail | Sigma-Aldrich | Cat# 5056489001 |
| Carbenicillin | Goldbio | Cat# C-103–100 |
| Kanamycin | Goldbio | Cat# K-120–25 |
| IPTG | Goldbio | Cat# I2481C100 |
| DTT | Goldbio | Cat# DTT10 |
| 1M HEPES pH 7.4 | Teknova | Cat# H1030 |
| 1M Magnesium Chloride | Thermo Fisher Scientific | Cat# AM9530G |
| 5M Sodium Chloride | Sigma-Aldrich | Cat# S6546–4L |
| 10% Tween-20 | Bio-Rad | Cat# 1610781 |
| Bond-Breaker TCEP Solution, Neutral pH | Thermo Fisher Scientific | Cat# 77720 |
| NeutrAvidin Protein | Thermo Fisher Scientific | Cat# 31000 |
| ProteOn Sodium Hydroxide Solution, 50mM | Bio-Rad | Cat# 1762230 |
| 10X RIPA buffer | EMD Millipore | 20–188 |
| 4X LDS sample loading buffer | G Biosciences | 786–323 |
| cOmplete protease inhibitor cocktail | Roche | 45–11836170001 |
| InstantBlue Coomassie Protein Stain | Abcam | Ab119211 |
| GFP-Trap agarose beads | ChromoTek | gta |
| Sotorasib (AMG510) | Cayman | Cat# 29465 |
| Adagrasib (MRTX849) | Cayman | Cat# 31440 |
| MRTX1257 | Cayman | Cat# 33527 |
| ARS853 | Cayman | Cat# 19137 |
| ARS1620 | Cayman | Cat# 27915 |
| GDC6036 | MedChemExpress | Cat# HY-145928 |
| JDQ443 | MedChemExpress | Cat# HY-139612 |
| Garsorasib | MedChemExpress | Cat# HY-145571 |
| ASP2453 | MedChemExpress | Cat# HY-132966 |
| RMC-6291 | MedChemExpress | Cat# HY-153346 |
| Compound 11 | Medicilon Inc | Custom Order |
| Compound 12 | Medicilon Inc | Custom Order |
| Compound 13 | Medicilon Inc | Custom Order |
| Compound 14 | Medicilon Inc | Custom Order |
| Compound 15 | Medicilon Inc | Custom Order |
| Compound 16 | Medicilon Inc | Custom Order |
| Compound 17 | Medicilon Inc | Custom Order |
| Compound 18 | Medicilon Inc | Custom Order |
| Compound 19 | Medicilon Inc | Custom Order |
| Compound 20 | Medicilon Inc | Custom Order |
| Compound 21 | Medicilon Inc | Custom Order |
| Compound 22 | Medicilon Inc | Custom Order |
| Deuterium Oxide 99.9% | Sigma Aldrich | 151882–10X1ML |
|
Critical commercial assays | ||
| RalA G-LISA Activation Assay Kit 96 Assays | Cytoskeleton | Cat# BK129 |
| Rac1 G-LISA Activation Assay Kit 96 Assays | Cytoskeleton | Cat# BK128 |
| Active Rap1 Detection Kit | Cell Signaling Technology | Cat# 8818 |
| Active Rac1 Detection Kit | Cell Signaling Technology | Cat#8815S |
| Quantitative colorimetric peptide assay | Pierce | 23275 |
| Quantitative protein BCA assay | Pierce | 23225 |
| PreOmics iST kit | PreOmics | P.O.00027 |
| CellTiter-Glo | Promega | G7570 |
|
Deposited data | ||
| AMG510-bound H-Ras(G12C) | This study | PDB 8TLR |
| Mass spectrometry proteomics data | PRIDE | PXD041440 and PXD054414 |
|
Experimental models: Cell lines | ||
| HeLa | ATCC | Cat# CCL-2 |
| MOLM-13 | DSMZ | Cat# ACC 554 |
| MOLM-13 Inducible NRAS G12C | This study | N/A |
| MOLM-13 Inducible KRAS G12C | This study | N/A |
| MOLM-13 Inducible KRAS G12D | This study | N/A |
| COS-7 | Gifted by the Jura lab | N/A |
|
Recombinant DNA | ||
| pProEx H-Ras(G12C) CL | This study | Addgene #224261 |
| pProEx N-Ras(G12C) CL | This study | Addgene #224262 |
| pProEx RalA(G23C) CL | This study | Addgene #224263 |
| pProEx Rap1A(G12C) CL | This study | N/A |
| pProEx Rap1A(G12C, L96F) CL | This study | Addgene #224264 |
| pProEx Ypt1(S17C) CL | This study | Addgene #224265 |
| pProEx Rheb(R15C) CL | This study | Addgene #224266 |
| pProEx Rit1(G47C) CL | This study | Addgene #224267 |
| pProEx M-Ras(G22C) CL | This study | Addgene #224268 |
| pcDNA3.1(+) EGFP-RalA(WT) | This study | Addgene #224274 |
| pcDNA3.1(+) EGFP-RalA(G23C) | This study | Addgene #224275 |
| pcDNA3.1(+) EGFP-Rap1A(WT) | This study | Addgene #224276 |
| pcDNA3.1(+) EGFP-Rap1A(G12C, L96F) | This study | Addgene #224277 |
| pProEx Rac1(G12C) CL | This study | Addgene #224269 |
| pProEx Rac1(G12C, W97Y) CL | This study | N/A |
| pProEx Rac1(G12C, E100Q) CL | This study | N/A |
| pProEx Rac1(G12C, K96H) CL | This study | Addgene #224270 |
| pProEx Rac1(G12C, P29S) CL | This study | N/A |
| pProEx RhoA(G14C) CL | This study | Addgene #224271 |
| pProEx Rab1A(S20C) CL | This study | Addgene #224272 |
| pProEx Rab1A(S20C, E108Q) CL | This study | N/A |
| pProEx Rab5C (S30C) CL | This study | Addgene #224273 |
| pcDNA3.1(+) EGFP-Rac1 (WT) | This study | Addgene #224278 |
| pcDNA3.1(+) EGFP-Rac1 (G12C) | This study | Addgene #224279 |
| pcDNA3.1(+) EGFP-Rac1 (G12C, K96H) | This study | Addgene #224281 |
| pcDNA3.1(+) EGFP-Rac1 (P29S) | This study | Addgene #224280 |
| pcDNA3.1(+) EGFP-Rac1 (G12C, P29S, K96H) | This study | Addgene #224282 |
| pcDNA3.1(+) K-Ras(WT) | This study | Addgene #224283 |
| pcDNA3.1(+) K-Ras(G12C) | This study | Addgene #224284 |
| pcDNA3.1(+) R-Ras1(WT) | This study | Addgene #224285 |
| pcDNA3.1(+) R-Ras1(G38C) | This study | Addgene #224286 |
| pcDNA3.1(+) M-Ras(WT) | This study | Addgene #224287 |
| pcDNA3.1(+) M-Ras(G22C) | This study | Addgene #224288 |
| pcDNA3.1(+) Rheb(WT) | This study | Addgene #224289 |
| pcDNA3.1(+) Rheb(R15C) | This study | Addgene #224290 |
| NRAS(G12C) in pCW57.1 vector | This study | N/A |
| KRAS(G12C) in pCW57.1 vector | This study | N/A |
| KRAS(G12D) in pCW57.1 vector | This study | N/A |
| Avi-RhoA(WT) in pDest-566 vector | This study | N/A |
| Avi-Rap1A(WT) in pDest-566 vector | This study | N/A |
|
Software and algorithms | ||
| GraphPad Prism | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ |
| Phenix | Adams et al.37 | https://www.phenix-online.org/ |
| Coot | Emsley et al.38 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| Excel | Microsoft | https://www.microsoft.com/en-us/ |
| ChemDraw | Revvity Signals | https://www.revvitysignals.com |
| SnapGene | Dotmatics | https://www.snapgene.com/ |
| Zotero 5.0 | Corporation for Digital Scholarship | https://www.zotero.org/ |
| Word | Microsoft | https://www.microsoft.com/en-us/ |
| Illustrator 2022 | Adobe | https://www.adobe.com/products/illustrator.html |
| AMBER20 | Case et al.39 | https://ambermd.org/ |
| Gaussian16 | Gaussian | https://gaussian.com/gaussian16/ |
| VMD | Humphrey et al.40 | https://www.ks.uiuc.edu/Research/vmd/ |
| Pymol | The PyMOL Molecular Graphics System; Version 1.8; Schrodinger, LLC | https://pymol.org/2/ |
| HDExaminer | Sierra Analytics | http://massspec.com/hdexaminer |
| Bruker Compass DataAnalysis 4.2 | Bruker | https://www.bruker.com |
| FragPipe (v19.1) | Nesvizhskii Lab - University of Michigan | https://fragpipe.nesvilab.org/ |
| ImageStudioLite | Li-Cor | Version 5.2.5 |
| PEAKS online | Bioinformatics Solutions Inc. | Xpro 1.6 |
| Biacore S200 Control Software | Cytiva | Version 1.1.1 |
| Biacore S200 Evaluation Software | Cytiva | Version 1.1.1 |
|
Other | ||
| TALON Metal Affinity Resin | Clontech Laboratories | Cat# 635503 |
| Superdex 75 Increase 10/300 GL | Cytiva | Cat# 29148721 |
| Zeba Spin Desalting Columns, 7K MWCO | Thermo Fisher Scientific | Cat# 89882 |
| Slide-A-Lyzer Dialysis Cassettes, 10K MWCO | Thermo Fisher Scientific | Cat# 66380 |
| Pierce Protein Concentrators, 10 K MWCO, 15 mL | Thermo Fisher Scientific | Cat# 88528 |
| Pierce Protein Concentrators, 10 K MWCO, 5 mL | Thermo Fisher Scientific | Cat# 88517 |
| Lipofectamine 3000 Transfection Reagent | Thermo Fisher Scientific | Cat# L3000015 |
| NuPAGE 4 to 12%, Bis-Tris, Protein Gels | Thermo Fisher Scientific | Cat# NP0322BOX |
| Spark 20 M plate reader | TECAN | Cat# 20173336– 03 |
| C18 trap column | Waters | 186003975 |
| Pepsin column | Waters | 186007233 |
| Nepenthesin Column | Affipro | AP-PC-004 |
| C18 UPLC column | Waters | 186005593 |
| Mini Bio-Spin column | Bio-Rad | 7326207 |
| 10X RIPA buffer | EMD Millipore | 20–188 |
| 4X LDS sample loading buffer | G Biosciences | 786–323 |
| InstantBlue Coomassie Protein Stain | Abcam | Ab119211 |
| GFP-Trap agarose beads | ChromoTek | gta |
| Amine Coupling Kit | Cytiva | Cat# BR100050 |
| Series S CM5 Chips | Cytiva | Cat# 29149603 |
| iBlot 3 Transfer Stacks, midi nitrocellulose | Thermo Scientific | IB33001X3 |
| iBlot 3 Western Blot Transfer Device | Thermo Scientific | IB31001 |