Measure Activation of Small GTPases via their Specific Downstream Effectors

Pull-down kits enrich active GTPases from cell or tissue lysates

by Suzanne M. Smith, M.S.; Kay K. Opperman, Ph.D.; Barbara J. Kaboord, Ph.D.; Rizwan Farooqui, Ph.D. - 04/05/11

The Ras superfamily of small GTPases serve as molecular switches to control diverse eukaryotic cellular behaviors, including cell growth, differentiation and motility. Consequently, small GTPases are involved in several disease states such as cancer and metabolic disorders.^1,2 GTPases are active when bound to guanosine triphosphate (GTP) and inactive when the triphosphate is hydrolyzed to guanosine diphosphate (GDP). The Thermo Scientific Pierce Active GTPase Pull-down and Detection Kits enable GTPase activation studies by preferentially enriching their active form. These kits contain a GST-protein binding-domain (PBD or RBD) fusion that is selective for active Rho, Ras, Rac1, Cdc42, Rap1, Arf1 or Arf6 (Table 1).

This pull-down method is based on the affinity of known downstream effector proteins for the active forms of specific GTPases. The respective protein-binding domain (PBD) of these downstream effectors is expressed as a GST-fusion protein (Table 1). When immobilized on glutathione agarose resin, the PBD will bind active, GTP-bound GTPase from a cell lysate (Figure 1). The pulled-down active GTPase is detected via Western blotting. As a control, cell lysates can be treated with GTPgS, which is a non-hydrolyzable analog of GTP. This method traps all GTPases in the active form and results in high GTPase enrichment. As a negative control, cell lysates are treated with an excess of GDP to shift the majority of GTPase to the inactive state.

Each active GTPase kit includes a GST fusion of the protein-binding domain

Figure 1. Thermo Scientific Active GTPase Pull-down and Detection Kit protocol summary.

To determine the specificity and function of the GTPase pull-down and detection kits, NIH 3T3 cell lysate was incubated with either GTPγS or GDP to activate or inactivate endogenous GTPases, respectively. The specific GST-PBD or -RBD was used to pull down active Rho, Ras, Rac1, Cdc42, Rap1, Arf1 or Arf6. A strong signal is detected in the GTPγS-treated lysate; however, minimal or no signal is detected in the GDP-treated lysate (Figure 2). These results illustrate the specificity of the PBD for active GTPases.

Figure 2. Specific detection of active Rho, Ras, Rac1, Cdc42, Rap1, Arf1 and Arf6 by western blotting. NIH 3T3 cell lysate treated with GTPγS or GDP was incubated with the appropriate GST binding domain and immobilized glutathione resin. Eluted samples and a portion of the lysate were analyzed by Western blot using GTPase-specific antibodies.

The pull-down of endogenous active small GTPases after growth factor or serum stimulations was highly effective in a variety of cell types derived from different species (Figure 3). Changes in the GTPase activities can be detected in time-course studies and differ with cell type and treatment. Because total GTPase levels in each lysate are constant, the amount of GTPase pulled down in each assay reflects activation rather than changes in GTPase expression levels. The activity profiles detected are similar to those reported in the literature.^8-11

Figure 3. Specific, induced changes in the level of endogenously activated GTPases from a variety of cell types are easily monitored by the pull-down assay. In each panel, the top western blot shows the level of active GTPase isolated by pull-down assay; the lower western blot shows the total amount of expressed GTPase in the lysate. Densitometry was performed on the western blots and plotted graphically for each system. Panel A: Rho activity in HeLa (human) cells stimulated with EGF. Panel B: Ras activity in NIH 3T3 (murine) cells stimulated with PDGF. Panel C: Rac1 activity in NS1 (rodent) cells stimulated with NGF. Panel D: Arf1 activity in MDCK (canine) cells stimulated with HGF. Panel E: Arf6 activity in C2C12 (murine) cells stimulated with serum.

These results demonstrate the effectiveness of the GTPase pull-down and detection kits for monitoring sensitive changes in activity using time-dependent activity assays.

Cell culture and treatments

HeLa cells were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) to ~70% confluency and then starved in 1% FBS medium for 24 hours before stimulation with 100ng/mL of epidermal growth factor (EGF) for the indicated times. NIH 3T3 cells were grown in DMEM supplemented with 10% FBS to ~70% confluency and starved in 0.1% FBS medium for 24 hours. Platelet-derived growth factor (PDGF) was added at 50ng/mL for the indicated times. NS1 cells were grown in RPMI supplemented with 10% FBS to ~70% confluency and nerve growth factor (NGF, 50ng/mL) was added for the indicated times. MDCK cells were grown in EMEM supplemented with 10% FBS to ~70% confluency and starved in serum-free medium for 48 hours before stimulation with 50ng/mL of hepatocyte growth factor (HGF) at indicated times. C2C12 cells were grown in DMEM supplemented with 10% FBS to ~70% confluency and were starved in serum-free medium for 48 hours before adding 10% serum at the indicated times.

Active GTPase pull-down and detection

NIH 3T3 cells were lysed on the culture plate with 1mL lysis/binding/wash buffer. The clarified cell lysate (500μg) was treated with either GTPγS (positive control) or GDP (negative control). The treated lysates (or 1mg of the endogenous time-course lysates) were incubated with 400μg GST-Rhotekin-RBD (for active Rho), 80μg GST-Raf1-RBD (for active Ras), 20μg GST-Pak1-PBD (for active Rac1 or Cdc42), 20μg GST-RalGDS-RBD (for active Rap1) or 100μg GST-GGA3-PBD (for active Arf1 or Arf6). Half of each elution was analyzed by SDS-PAGE and detected by Western blot using the specific GTPase primary antibody.

1. Charest, P. and Firtel, R. (2007). Big roles for small GTPases in the control of directed cell movement. Biochem J 401:377-90.
2. Williams, D., et al. (2008). Rho GTPases and regulation of hematopoietic stem cell localization. Methods Enzymol 439:365-93.
3. Van Aelst, L. and D’Souza-Schorey, C. (1997). Rho GTPases and signaling networks. Genes Dev 11:2295-322.
4. Ehrhardt, A., et al. (2002). Ras and relatives – job sharing and networking keep an old family together. Exp Hematol 30:1089-106.
5. Posern, G., et al. (1998). Activity of Rap1 is regulated by bombesin, cell adhesion and cell density in NIH3T3 fibroblasts. J Bio Chem 273:24297-300.
6. Yoon H.Y., et al. (2005). In vitro assays of Arf1 interaction with GGA proteins.
   Methods Enzymol 404:316-32.
7. D’Souza-Schorey, C. and Chavrier, P. (2006). ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7:347-58.
8. Jones, S. and Kazlauskas, A. (2001). Growth factor-dependent signaling and cell cycle progression. FEBS Letters 490:110-6.
9. Palacios, F. and D’Souza-Schorey, C. (2003). Modulation of Rac1 and ARF6 activation during epithelial cell scattering. J Bio Chem 278:17395-400.
10. Bach, A., et al. (2010). ADP-ribosylation factor 6 regulates mammalian myoblast fusion through phospholipase D1 and phosphatidylinositol 4,5-bisphosphate signaling pathways. Mol Biol Cell 21:2412-24.
11. Govek, E., et al. (2005). The role of Rho GTPases in neuronal development. Genes Dev 19:1-49.