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.
Table 1. GTPases and their corresponding downstream effectors
Each active GTPase kit includes a GST fusion of the protein-binding domain
|GTPase||Downstream effector binding domain||Cellular function|
|Rho||GST-Rhotekin-RBD||Filopodia, lamellipodia formation, and stress fibers|
|Ras||GST-Raf1-RBD||Cell proliferation and differentiation|
|Rac1||GST-Pak1-PBD||Filopodia, lamellipodia formation, and stress fibers|
|Rap1||GST-RalGDS-RBD||Cell proliferation and differentiation|
|Arf1||GST-GGA3-PBD-PBD||Assembly of coat proteins onto budding vesicles on trans-golgi network and endosomes|
|Arf6||Membrane traffic, actin remodeling and structural organization at the cell surface|
|Cellular functions identified in this table are based on cited references 3-7.|
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.
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
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 hepatacyte 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.
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For Research Use Only. Not for use in diagnostic procedures.