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ABSTRACT
The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.
Abbreviations
| Actn1 | = | Alpha-actinin 1 |
| Arp2/3 | = | Actin-related protein 2/3 |
| Arp3 | = | Actin-related protein 3 |
| Arpc1b | = | Actin-related protein 2/3 complex, subunit 1B, 41 kDa |
| Arpc2 | = | Actin-related protein 2/3 complex, subunit 2, 34 kDa |
| CHL1 | = | Homo sapiens skin melanoma cell line |
| Dox | = | Doxycycline |
| ECM | = | Extracellular Matrix |
| FLII | = | Protein flightless-1 homolog |
| GEF | = | Guanine nucleotide Exchange Factor |
| GEF* | = | GEF-dead mutant |
| GTPase | = | Guanosine Triphosphate Phosphohydrolase |
| H | = | Heavy SILAC media |
| HPRD | = | Human Protein Reference Database |
| IQGAP1 | = | Ras GTPase-activating-like protein 1 |
| K | = | Lysine |
| KDa | = | Kilodaltons |
| L | = | Light SILAC media |
| LC-MS/MS | = | Liquid Chromatography-tandem Mass Spectrometry |
| M | = | Medium SILAC media |
| MCF7 | = | Michigan Cancer Foundation-7 breast cancer cell line |
| MDCK-f3 | = | Fibroblastoid MDCKII cells |
| MDCKII | = | Madin-Darby Canine Kidney II |
| MMP | = | Matrix Metalloproteinase |
| NIH3T3 | = | National Institutes of Health 3-day Transfer, Inoculum 3 × 105 cells |
| P-Rex1 | = | Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 |
| PIPs | = | Human Protein-Protein Interaction Predictions (PIPs) database |
| PLA | = | Proximity Ligation Assay |
| R | = | Arginine |
| Rac1 | = | Ras-related C3 botulinum toxin substrate 1 |
| Rho A | = | Ras homolog gene family member A |
| RhoGDI1 | = | Rho GDP-dissociation inhibitor 1 |
| ROCK | = | Rho-associated serine/threonie kinase |
| SDS- PAGE | = | Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis |
| SF | = | StrepII-FLAG |
| SILAC | = | Stable Isotope Labeling by Amino acids in Cell culture |
| TAP | = | Tandem Affinity Purification |
| Tiam1 | = | T-cell lymphoma invasion and metastasis-1 |
| TIMP | = | Tissue Inhibitor of Metalloproteinase |
| TMOD3 | = | Tropomodulin-3 |
| VE | = | Vascular Endothelial |
| WT | = | Wild Type |
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors would like to thank Marius Ueffing for kindly providing the SF-tag construct.
Funding
This work was supported by Cancer Research UK for AM (grant number C5759/A12328).
