Analysis of Activated GAPs and GEFs in Cell Lysates

doi:10.1016/S0076-6879(06)06031-9

Methods in Enzymology

Volume 406, 2006, Pages 425-437

https://doi.org/10.1016/S0076-6879(06)06031-9 Get rights and content

Abstract

An assay was developed that allows the precipitation of the active pools of Rho‐GEFs, Rho‐GAPs, or effectors from cell or tissue lysates. This assay can be used to identify GEFs, GAPs, and effectors involved in specific cellular pathways to determine their GTPase specificity and to monitor the temporal activation of GEFs and GAPs in response to upstream signals.

Introduction

Rho GTPases control many aspects of cellular behavior, including the organization of the cytoskeleton, cell migration, cell adhesion, cell cycle, and gene expression (Burridge 2004, Hall 1998, Van Aelst 1997). Like all GTPases, Rho proteins act as molecular switches by cycling between an active (GTP bound) and an inactive (GDP bound) state. Exchange of GTP for GDP allows the GTPase to interact with downstream effectors to modulate their activity and localization. Cycling between the GDP‐bound and GTP‐bound state is regulated primarily by two distinct families of proteins: guanine‐nucleotide exchange factors (GEFs) activate Rho proteins by catalyzing the exchange of GDP for GTP, the GTPase activating proteins or GAPs negatively regulate GTPase function by stimulating GTP hydrolysis.

Since the development of the Rho pull‐down assay in 1999 (Ren et al., 1999), a lot of progress has been made in determining the identity of the Rho‐GTPases that are activated in response to certain stimuli or signals. However, much less has been done in terms of identifying the molecules involved in the activation and inactivation of each particular Rho‐GTPase. The issue becomes more complicated considering that the human genome contains more than 60 RhoGEFs and approximately 80 RhoGAPs (Moon 2003, Peck 2002, Rossman 2005, Schmidt 2002).

We have developed an affinity precipitation assay that allows us to specifically pull down RhoGEFs, RhoGAPs, or effectors that are being activated in the cell at any given time or condition. The assay takes advantage of constitutively active and dominant negative Rho‐family mutants that bind either to Rho‐GAPs and effectors or to RhoGEFs, respectively. Constitutively active Rho mutants were originally designed based on their analogous Ras mutants (G12V and Q61L) (Garrett 1989, Ridley 1992, Ridley 1992). These mutants have lost both their intrinsic capacity and their GAP‐mediated ability to hydrolyze GTP, and they bind with high affinity to GAPs and effectors (Barbacid 1987, Trahey 1987). Traditional dominant negative mutants like S17N‐Ras and the analogous Rho mutants have been shown to bind GDP with similar affinity to the corresponding wild‐type form. However, their affinity for GTP is extremely low, so virtually all of the protein is found in the GDP‐bound form (Ridley 1992, Feig 1999). Another dominant negative Ras mutant (RasG15A) has been previously shown to bind very poorly to both GDP and GTP, existing virtually in a nucleotide free state (Chen et al., 1994). A nucleotide‐free GTPase is one of the intermediates of the nucleotide exchange reaction and is able to form a high affinity binary complex with the GEF (Cherfils and Chardin, 1999). This intermediate is rapidly dissociated by GTP and does not accumulate in cells. We took advantage of the properties of these mutants and used them to pull down Rho effectors and GAPs from tissue and cell lysates (Arthur 2002, Noren 2003). We generated mutant versions of various representative GTPases of the Rho subfamily that harbor mutations equivalent to the Q61L and G15A mutations in Ras. We then used GST fusion proteins of these mutants to specifically pull down Rho family GEFs, GAPs, or effectors from cell or tissue lysates. This assay can be used to determine Rho protein specificity on GEFs, GAPs, or effectors (Arthur 2002, Ellerbroek 2004, Noren 2003, Wennerberg 2003). It can also be used as a simple way to find interactors such as GEFs, GAPs, and effectors to GTPases where little is known about upstream and downstream regulation. In addition, many GEFs and GAPs seem to be activated by making the binding site to their target GTPase available. This can be achieved either by unmasking an intramolecular inhibitory domain (Vav1, Dbl, Asef), by association with or dissociation from other proteins (p115‐RhoGEF, Sos, Dock180), or by release of the protein from a sequestering cellular compartment (GEF‐H1/Lfc, Net1, Ect2) (Schmidt 2002, Rossman 2005). Given this type of regulation, it is likely that the activated and nucleotide‐free mutants of Rho proteins will have highest affinity toward “activated” GAPs and GEFs, respectively, and that our assay can, indeed, be used to detect activation of GEFs and GAPs by specific stimuli.

Activation and inactivation of GEFs and GAPs can be studied either by analyzing the precipitation of known candidate components by immunoblot or by determining the identity of the bound proteins by mass spectrometry. In addition, GEF and GAP pull‐downs can be an extremely useful tool to follow the pattern of GAP or GEF activation over time or in response to different upstream signals.

Section snippets

DNA Constructs

Human cDNA for RhoA, Rac1, and Cdc42 were subcloned into pGEX 4T‐1 (Amersham) between the EcoRI and XhoI sites. Empty‐nucleotide mutants, G17A (RhoA), G15A (Rac1 and Cdc42), and constitutively active mutants, Q63L (RhoA), and Q61L (Rac1 and Cdc42) were generated by site‐directed mutagenesis using the Quick Change Site–directed mutagenesis kit (Stratagene) following the manufacturer's instructions.

Antibodies

Antibodies against p190RhoGAP, ROCK, Sos1, and GFP were from BD Biosciences; Lsc, LARG, and RhoGDI

Expression and Purification of GST‐Rho Constructs

1
Transform and grow up Escherichia coli with the appropriate pGEX construct. For RhoA, Rac1, and Cdc42 proteins, DH5α or any regular strain will work. For some less‐soluble Rho proteins (RhoB, RhoC, RhoG), it is good to use a codon optimized strain such as codon plus BL21 (Stratagene).
2
Grow up a 50‐ml culture overnight to full density in LB with 50 μg/ml ampicillin (LB‐Amp)(O.D. > 1.0).
3
Dilute the culture into 450 ml of LB‐Amp and let grow for 30 min at 37°.
4
Induce the protein by adding IPTG to a

GAP and GEF Pull‐Downs

Wild‐type RhoA, nucleotide‐free RhoA (G17A‐RhoA), and constitutively active Q63L‐RhoA were expressed as GST fusion proteins, and affinity precipitations were performed on lysates of CHO cells to assess the ability of these GST fusion proteins to bind to known regulators of Rho proteins. We immunoblotted the precipitated proteins with antibodies against a RhoA‐GAP (p190RhoGAP), a RhoA effector ROCK (ROK or Rho kinase), a RhoA‐GEF (Lsc/p115 RhoGEF), and RhoGDI. Our results revealed that

Stability of GST‐Rho Proteins

Some of the GST‐Rho mutants, in particular the empty nucleotide constructs, are not as stable as their wild‐type counterparts when expressed in bacteria. Protein stability decreases even more when proteins are eluted from beads. One way to deal with this problem is to prepare the GST proteins fresh when needed and to keep them at −20° with glycerol (see “Methods”) for no longer than 1 week.

Bacterial Contaminants

The method described here to purify the GST fusion proteins allows fast purification. However, depending

Acknowledgments

We thank Dr. Janiel Shields and Dr. Channing Der for the Ras‐transformed cell lines. We would also like to thank Adi Dubash for the critical reading of this manuscript. This work was supported by NIH grants GM29860 and HL45100. RGM was supported by a postdoctoral fellowship from the Susan G. Komen Breast Cancer Foundation.

References (27)

ArthurW.T. et al.
XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC
J. Biol. Chem.
(2002)
BurridgeK. et al.
Rho and Rac take center stage
Cell
(2004)
ChenJ.C. et al.
Oncogenic Ras leads to Rho activation by activating the mitogen‐activated protein kinase pathway and decreasing Rho‐GTPase‐activating protein activity
J. Biol. Chem.
(2003)
CherfilsJ. et al.
GEFs: Structural basis for their activation of small GTP‐binding proteins
Trends Biochem. Sci.
(1999)
GarrettM.D. et al.
Identification of distinct cytoplasmic targets for ras/R‐ras and rho regulatory proteins
J. Biol. Chem.
(1989)
MoonS.Y. et al.
Rho GTPase‐activating proteins in cell regulation
Trends Cell Biol.
(2003)
NorenN.K. et al.
Cadherin engagement inhibits RhoA via p190RhoGAP
J. Biol. Chem.
(2003)
PeckJ. et al.
Human RhoGAP domain‐containing proteins: Structure, function and evolutionary relationships
FEBS Lett.
(2002)
RidleyA.J. et al.
The small GTP‐binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors
Cell
(1992)
RidleyA.J. et al.
The small GTP‐binding protein rac regulates growth factor‐induced membrane ruffling
Cell
(1992)

WennerbergK. et al.

Rnd proteins function as RhoA antagonists by activating p190 RhoGAP

Curr. Biol.

(2003)

BarbacidM.

Ras genes

Annu. Rev. Biochem.

(1987)

ChenS.Y. et al.

Ras‐15A protein shares highly similar dominant‐negative biological properties with Ras‐17N and forms a stable, guanine‐nucleotide resistant complex with CDC25 exchange factor

Oncogene

(1994)

Cited by (163)

Src stimulates Abl-dependent phosphorylation of the guanine exchange factor Net1A to promote its cytosolic localization and cell motility
2023, Journal of Biological Chemistry
The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor–stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.
A molecular clock controls periodically driven cell migration in confined spaces
2022, Cell Systems
Navigation through a dense, physically confining extracellular matrix is common in invasive cell spread and tissue reorganization but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTPase RhoA, which is dependent on the oscillatory changes in the activity and abundance of the RhoA guanine nucleotide exchange factor, GEF-H1, and triggered by a persistent increase in the intracellular Ca²⁺ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with a frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.
Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis
2021, Cellular Signalling
The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.
Gα<inf>s</inf>directly drives PDZ-RhoGEF signaling to Cdc42
2020, Journal of Biological Chemistry
Citation Excerpt :
Activation of RhoA and Cdc42, using cells grown in 10-cm dishes, was assessed by pulldown using recombinant Rhotekin and PAK effector domains (13, 46). Active RhoGEFs were detected by pulldown with nucleotide-free GTPases (RhoA G17A and Cdc42 G15A) fused to GST (20, 45). Before lysis, the cells were washed with PBS containing 10 mm MgCl2 and lysed with 1 ml of ice-cold lysis buffer (50 mm Tris, 150 mm NaCl, pH 7.5, containing 1% Triton X-100, 5 mm EDTA, and protease and phosphatase inhibitors (13)).
Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα₁₃ binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα₁₃. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gα_s (Gα_sQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by G_s-coupled receptors, but not by those coupled to G_i or G_q, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gα_s-binding region, PRG-linker. Active Gα_s interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with Gα_sQ227L's ability to guide PRG's interaction with Cdc42. Endogenous G_s-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gα_s can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.
Endothelial cell sprouting driven by RhoJ directly activated by a membrane-anchored Intersectin 1 (ITSN1) RhoGEF module
2020, Biochemical and Biophysical Research Communications
Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.
Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells
2019, Journal of Biological Chemistry
The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na⁺ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-α time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK¹ tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 down-regulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-α treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

View all citing articles on Scopus

View full text

Analysis of Activated GAPs and GEFs in Cell Lysates

Abstract

Introduction

Section snippets

DNA Constructs

Antibodies

Expression and Purification of GST‐Rho Constructs

GAP and GEF Pull‐Downs

Stability of GST‐Rho Proteins

Bacterial Contaminants

Acknowledgments

J. Biol. Chem.

Cell

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

Trends Cell Biol.

J. Biol. Chem.

FEBS Lett.

Cell

Cell

Curr. Biol.

Ras genes

Annu. Rev. Biochem.

Ras‐15A protein shares highly similar dominant‐negative biological properties with Ras‐17N and forms a stable, guanine‐nucleotide resistant complex with CDC25 exchange factor

Oncogene