Elsevier

Methods in Enzymology

Volume 345, 2002, Pages 349-359
Methods in Enzymology

[26] - Assay of Cdc42, Rac, and Rho GTPase Activation by Affinity Methods

https://doi.org/10.1016/S0076-6879(02)45028-8Get rights and content

Introduction

The Rho GTPases are members of the Ras superfamily and include several isoforms of Cdc42, Rac, and Rho. The Rho subfamily regulates a variety of signal transduction pathways in eukaryotic cells, including cell adhesion and migration, by modulating cytoskeletal dynamics. Cdc42 induces formation of fllopodia, whereas Rac regulates actin polymerization at the plasma membrane where ruffles are formed. Both induce the formation of cytoskeletal/signaling aggregates known as focal complexes. Rho controls the assembly of focal adhesions and the reorganization of actin into stress fibers. More recently, it has been recognized that Rho GTPases also initiate signaling pathways that impact on gene expression and cell growth regulation, and that each of these GTPases is essential for Ras to induce malignant transformation. A specific role for Rac has been identified in phagocytic cells, such as neutrophils or monocytes, where it activates the NADPH oxidase enzymatic complex.

GTPases cycle from inactive (GDP-bound) to active (GTP-bound) forms that interact with and regulate components of intracellular signaling pathways. This cycle is under the control of several classes of regulatory protein. Under basal conditions, the Rho GTPases are complexed with their cytosolic GDP-dissociation inhibitor (GDI), which stabilizes the inactive GDP-bound form. On cell stimulation, the GTPase dissociates from the GDI and translocates to the plasma membrane. Subsequently the GTPase releases GDP and binds GTP, a reaction promoted by guanine nucleotide exchange μlctors (GEFs), which leads to the interaction with specific molecular targets. Inactivation of the GTPase results from hydrolysis of GTP into GDP, and the intrinsic rate of hydrolysis by the GTPase is enhanced by the action of GTPase-activating proteins (GAPs). The active form of the GTPase appears to be transient and labile, which makes its measurement difficult. Indirect methods based on monitoring association with or activation of regulatory proteins or on relocalization within the cell have been used to assess Rho GTPase activation during the dynamic cell response. These methods have their problems and limitations, and to characterize Rho GTPase activation a direct measurement of the formation of the active form within the cell is necessary. A classic method to analyze direct GTPase activation consists in measuring the GTP : GDP ratio after 32P labeling of cells and immunoprecipitation of the GTPase. In addition to the need for high levels of radioactivity, this approach is limited for Rho GTPases by the lack of efficient immunoprecipitating antibodies and the rapid turnover of the GTP-bound state. More recently a new assay measuring Rho GTPase activation has been developed on the basis of the μlct that only the active form of the GTPase interacts with downstream effectors. Identification of the GTPase-binding region on target proteins, and the expression of these binding domains in active soluble forms, allows them to be used as specific probes to detect the active form of the GTPase. This article describes an assay developed for Rac and Cdc42 activation using the GTPase/p21-binding region of p21-activated kinase 1 (PAK1). Similar strategies developed as assays for Rho GTP are discussed.

Section snippets

Principle of Assay

The Rac and Cdc42 (p21) -activated kinase PAK1 contains in the N-terminal regulatory region a specific site for interaction with the active form of these two GTPases. This region, referred to as the CRIB domain (Cdc42/Rac interactive binding domain) or p21-binding domain (PBD), was first described by Burbelo et al.1 and the minimal consensus sequence identified for specific GTPase binding corresponds to amino acids 74 to 89 in PAK1. A homologous, but Cdc42-selective, CRIB domain is also found

Construction of GST-PAK1 PBD Fusion Protein

The cDNA of the coding sequence for PAK1 PBD corresponding to amino acids 67-150 is amplified by polymerase chain reaction (PCR), cloned into a pGEX2T vector at the BanHI-EcoRI sites, and used to transform the DH-10B strain of Escherichia coli. The bacteria then produces a fusion protein with glutathione S-transferase (GST) upstream of the PBD. Transformed bacteria are stored at −80° in 20% (v/v) glycerol L-broth medium.

Expression and Purification of Fusion Protein

L-broth medium (30 ml) containing ampicillin (100 μg/ml) is inoculated with

Preparation of Cell Extract to Measure GTP-Bound GTPase

Preparation of cell extract represents a critical point in the assay and determination of the most appropriate lysis buffer must be established before any affinity precipitation. Cells are pretreated or stimulated to generate the active form of the GTPase. For cells in suspension the stimulation is stopped by addition of an equal volume of cold 2× lysis buffer. For adherent cells, the stimulation medium is removed, cold 1× lysis buffer is added, and the cells are scraped from the plates while

Affinity Precipitation Assay for Rac and Cdc42

Cell extract or recombinant GTPases are incubated with GST-PBD in a maximal final volume of 500 μl for 1 hr at 4°. If necessary the solution containing the GTPase is diluted in binding buffer [25 mM Tris-HCl (pH 7.5), 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 0.5% (v/v) Nonidet P-40, leupeptin (1 μg/ml), aprotinin (1 μg/ml), and 1 mM PMSF] to have an equal volume in each sample. The bead pellet is then centrifuged for 2 min at 2000 rpm, 4° and washed three times with washing buffer [25 mMTris-HCl (pH

Comparison with Affinity Precipitation Assay for Rho

The N-terminal region of Rhotekin (amino acids 7 to 89) produced as a fusion protein with GST interacts with GTP-Rho and has been used as the basis for an affinity isolation assay specific for Rho.10 The principle of the assay using the Rho-binding domain (RBD) of Rhotekin is similar to the assay using the PAK1 PBD. However, major differences are noted in the optimum buffer compositions. The Rhotekin/GST-RBD assay was described with Swiss 3T3 and COS-7 cells lysed in a classic

Conclusion

We have described a nonradioactive affinity-based method for analysis of Rac, Cdc42, or Rho activation in cell lysates. We have tried to point out areas in which problems might occur, and simple procedures to check whether the PBD/RBD probe is functioning correctly. The assay is, of course, limited by the levels of GTPases present in the samples analyzed, as well as by the quality and sensitivity of the antibodies used for detection. The use of these affinity-based assays provides a simple and

First page preview

First page preview
Click to open first page preview

References (10)

  • P.D. Burbelo et al.

    J. Biol. Chem.

    (1995)
  • T. Reid et al.

    J. Biol. Chem.

    (1996)
  • K. Fujisawa et al.

    J. Biol. Chem.

    (1996)
  • A.J. Self et al.

    Methods Enzymol.

    (1995)
  • V. Benard et al.

    J. Biol. Chem.

    (1999)
There are more references available in the full text version of this article.

Cited by (152)

  • Fundamental aspects of protein isolation and purification

    2021, Three Phase Partitioning: Applications in Separation and Purification of Biological Molecules and Natural Products
  • Endothelium and Nitric Oxide: Interactions in Cancer Evolution

    2018, Endothelium and Cardiovascular Diseases: Vascular Biology and Clinical Syndromes
View all citing articles on Scopus
View full text