Guanine Nucleotide Exchange Factor Activity Research Articles

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.

Phosphorylation of SOS1 on tyrosine 1196 promotes its RAC GEF activity and contributes to BCR-ABL leukemogenesis

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.

The G\u03b1i-GIV binding interface is a druggable protein-protein interaction

Heterotrimeric G proteins are usually activated by the guanine-nucleotide exchange factor (GEF) activity of GPCRs. However, some non-receptor proteins are also GEFs. GIV (a.k.a Girdin) was the first non-receptor protein for which the GEF activity was ascribed to a well-defined protein sequence that directly binds Gαi. GIV expression promotes metastasis and disruption of its binding to Gαi blunts the pro-metastatic behavior of cancer cells. Although this suggests that inhibition of the Gαi-GIV interaction is a promising therapeutic strategy, protein-protein interactions (PPIs) are considered poorly “druggable” targets requiring case-by-case validation. Here, we set out to investigate whether Gαi-GIV is a druggable PPI. We tested a collection of >1,000 compounds on the Gαi-GIV PPI by in silico ligand screening and separately by a chemical high-throughput screening (HTS) assay. Two hits, ATA and NF023, obtained in both screens were confirmed in secondary HTS and low-throughput assays. The binding site of NF023, identified by NMR spectroscopy and biochemical assays, overlaps with the Gαi-GIV interface. Importantly, NF023 did not disrupt Gαi-Gβγ binding, indicating its specificity toward Gαi-GIV. This work establishes the Gαi-GIV PPI as a druggable target and sets the conceptual and technical framework for the discovery of novel inhibitors of this PPI.

Structure-based analysis of the guanine nucleotide exchange factor SmgGDS reveals armadillo-repeat motifs and key regions for activity and GTPase binding

Small GTPases are molecular switches that have critical biological roles and are controlled by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs). The smg GDP dissociation stimulator (SmgGDS) protein functions as a GEF for the RhoA and RhoC small GTPases. SmgGDS has various regulatory roles, including small GTPase trafficking and localization and as a molecular chaperone, and interacts with many small GTPases possessing polybasic regions. Two SmgGDS splice variants, SmgGDS-558 and SmgGDS-607, differ in GEF activity and binding affinity for RhoA depending on the lipidation state, but the reasons for these differences are unclear. Here we determined the crystal structure of SmgGDS-558, revealing a fold containing tandem copies of armadillo repeats not present in other GEFs. We also observed that SmgGDS harbors distinct positively and negatively charged regions, both of which play critical roles in binding to RhoA and GEF activity. This is the first report demonstrating a relationship between the molecular function and atomic structure of SmgGDS. Our findings indicate that the two SmgGDS isoforms differ in GTPase binding and GEF activity, depending on the lipidation state, thus providing useful information about the cellular functions of SmgGDS in cells.

Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF)

Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ADP ribosylation factor family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of ∼20 kDa and at least one key residue difference in the consensus GTP-binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine Arl13b protein. We report results from assays for solution-based nucleotide binding, intrinsic and GTPase-activating protein-stimulated GTPase, and ARL3 guanine nucleotide exchange factor activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine Arl13b is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as a phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.

Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

Regulation of DENND3, the exchange factor for the small GTPase Rab12 through an intramolecular interaction

The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking. Proteins bearing a differentially expressed in normal and neoplastic cells (DENN) domain have emerged as the largest family of Rab-activating guanine nucleotide exchange factors (GEFs). Rab12 functions in the initiation of starvation-induced autophagy, and our previous work revealed that its activator, DENN domain-containing protein 3 (DENND3), is phosphorylated and activated upon starvation. However, how the GEF activity of DENND3 toward Rab12 is regulated at the molecular level is still not understood. Here, we combine size-exclusion chromatography, Förster resonance energy transfer, pulldown, and in vitro GEF assays to demonstrate that regulation of GEF activity is achieved through an intramolecular interaction that is controlled by a key residue in DENND3, tyrosine 940. Our study sheds light on the regulation of Rab12 activation and lays the groundwork for characterizing the regulation of other DENN domain-containing proteins.

Biallelic loss-of-function variants in DOCK3 cause muscle hypotonia, ataxia, and intellectual disability.

DOCK3 encodes the dedicator of cytokinesis 3 protein, a member of the DOCK180 family of proteins that are characterized by guanine-nucleotide exchange factor activity. DOCK3 is expressed exclusively in the central nervous system and plays an important role in axonal outgrowth and cytoskeleton reorganization. Dock3 knockout mice exhibit motor deficiencies with abnormal ataxic gait and impaired learning. We report 2 siblings with biallelic loss-of-function variants in DOCK3. Diagnostic whole-exome sequencing (WES) and chromosomal microarray were performed on a proband with severe developmental disability, hypotonia, and ataxic gait. Testing was also performed on the proband's similarly affected brother. A paternally inherited 458 kb deletion in chromosomal region 3p21.2 disrupting the DOCK3 gene was identified in both affected siblings. WES identified a nonsense variant c.382C>G (p.Gln128*) in the DOCK3 gene (NM_004947) on the maternal allele in both siblings. Common features in both affected individuals include severe developmental disability, ataxic gait, and severe hypotonia, which recapitulates the Dock3 knockout mouse phenotype. We show that complete DOCK3 deficiency in humans leads to developmental disability with significant hypotonia and gait ataxia, probably due to abnormal axonal development.

Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering.

Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes.

Identification of a Novel, Small Molecule Partial Agonist for the Cyclic AMP Sensor, EPAC1

Screening of a carefully selected library of 5,195 small molecules identified 34 hit compounds that interact with the regulatory cyclic nucleotide-binding domain (CNB) of the cAMP sensor, EPAC1. Two of these hits (I942 and I178) were selected for their robust and reproducible inhibitory effects within the primary screening assay. Follow-up characterisation by ligand observed nuclear magnetic resonance (NMR) revealed direct interaction of I942 and I178 with EPAC1 and EPAC2-CNBs in vitro. Moreover, in vitro guanine nucleotide exchange factor (GEF) assays revealed that I942 and, to a lesser extent, I178 had partial agonist properties towards EPAC1, leading to activation of EPAC1, in the absence of cAMP, and inhibition of GEF activity in the presence of cAMP. In contrast, there was very little agonist action of I942 towards EPAC2 or protein kinase A (PKA). To our knowledge, this is the first observation of non-cyclic-nucleotide small molecules with agonist properties towards EPAC1. Furthermore, the isoform selective agonist nature of these compounds highlights the potential for the development of small molecule tools that selectively up-regulate EPAC1 activity.

The RopGEF2-ROP7/ROP2 Pathway Activated by phyB Suppresses Red Light-Induced Stomatal Opening

Circadian rhythm of stomatal aperture is mainly regulated by light/darkness. Blue and red light induce stomatal opening through different mechanisms that are mediated by special receptors. ROP2, a member of Rho GTPase family in Arabidopsis (Arabidopsisthaliana), has been found to negatively regulate light-induced stomatal opening. However, the upstream guanine nucleotide exchange factor (GEF) RopGEFs have not been revealed, and it is unclear which photoreceptor is required for the action of RopGEFs-ROPs. Here, we showed that RopGEF2 acted as a negative regulator in phytochrome B (phyB)-mediated red light-induced stomatal opening. Meanwhile, ROP7, another member of ROP family, acting redundantly with ROP2, was regulated by RopGEF2 in this process. RopGEF2 interacted with ROP7 and ROP2 and enhanced their intrinsic nucleotide exchange rates. Furthermore, the direct interactions between phyB and RopGEF2 were detected in vitro and in plants, and phyB enhanced the GEF activity of RopGEF2 toward both ROP7 and ROP2 under light. In addition, RopGEF4 functioned redundantly with RopGEF2 in red light-induced stomatal opening by activating both ROP7 and ROP2, and RopGEF2/RopGEF4 acted genetically downstream of phyB; however, the GEF activity of RopGEF4 was not directly enhanced by phyB. These results revealed that red light-activated phyB enhances the GEF activities of RopGEF2 and RopGEF4 directly or indirectly, and then activate both ROP7 and ROP2 in guard cells. The negative mechanism triggered by phyB prevents the excessive stomatal opening under red light.

DOCK8 Protein Regulates Macrophage Migration through Cdc42 Protein Activation and LRAP35a Protein Interaction

DOCK8 is an atypical guanine nucleotide exchange factor for Cdc42, and its mutations cause combined immunodeficiency in humans. Accumulating evidence indicates that DOCK8 regulates the migration and activation of various subsets of leukocytes, but its regulatory mechanism is poorly understood. We here report that DOCK8-deficient macrophages exhibit a migration defect in a 2D setting. Although DOCK8 deficiency in macrophages did not affect the global Cdc42 activation induced by chemokine stimulation, rescue experiments revealed that the guanine nucleotide exchange factor activity of DOCK8 was required for macrophage migration. We found that DOCK8 associated with LRAP35a, an adaptor molecule that binds to the Cdc42 effector myotonic dystrophy kinase-related Cdc42-binding kinase, and facilitated its activity to phosphorylate myosin II regulatory light chain. When this interaction was disrupted in WT macrophages, they showed a migration defect, as seen in DOCK8-deficient macrophages. These results suggest that, during macrophage migration, DOCK8 links Cdc42 activation to actomyosin dynamics through the association with LRAP35a.

Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53-Driven Lung Adenocarcinoma

The guanine nucleotide exchange factor (GEF) epithelial cell transforming sequence 2 (Ect2) has been implicated in cancer. However, it is not clear how Ect2 causes transformation and whether Ect2 is necessary for tumorigenesis invivo. Here, we demonstrate that nuclear Ect2 GEF activity is required for Kras-Trp53 lung tumorigenesis invivo and that Ect2-mediated transformation requires Ect2-dependent rDNA transcription. Ect2 activates rRNA synthesis by binding the nucleolar transcription factor upstream binding factor 1 (UBF1) on rDNA promoters and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA. Protein kinase Cι (PKCι)-mediated Ect2 phosphorylation stimulates Ect2-dependent rDNA transcription. Thus, Ect2 regulates rRNA synthesis through a PKCι-Ect2-Rac1-NPM signaling axis that is required for lung tumorigenesis.

Phosphatidic acid binds to and regulates guanine nucleotide exchange factor 8 (GEF8) activity in Arabidopsis.

Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.

LRCH1 interferes with DOCK8-Cdc42–induced T cell migration and ameliorates experimental autoimmune encephalomyelitis

Directional autoreactive CD4+ T cell migration into the central nervous system plays a critical role in multiple sclerosis. Recently, DOCK8 was identified as a guanine-nucleotide exchange factor (GEF) for Cdc42 activation and has been associated with human mental retardation. Little is known about whether DOCK8 is related to multiple sclerosis (MS) and how to restrict its GEF activity. Using two screening systems, we found that LRCH1 competes with Cdc42 for interaction with DOCK8 and restrains T cell migration. In response to chemokine stimulation, PKCα phosphorylates DOCK8 at its three serine sites, promoting DOCK8 separation from LRCH1 and translocation to the leading edge to guide T cell migration. Point mutations at the DOCK8 serine sites block chemokine- and PKCα-induced T cell migration. Importantly, Dock8 mutant mice or Lrch1 transgenic mice were protected from MOG (35-55) peptide-induced experimental autoimmune encephalomyelitis (EAE), whereas Lrch1-deficient mice displayed a more severe phenotype. Notably, DOCK8 expression was markedly increased in PBMCs from the acute phase of MS patients. Together, our study demonstrates LRCH1 as a novel effector to restrain PKCα-DOCK8-Cdc42 module-induced T cell migration and ameliorate EAE.

Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα.

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.

Cdk5 Regulation of the GRAB-Mediated Rab8-Rab11 Cascade in Axon Outgrowth.

While axon outgrowth requires membrane supply for surface expansion, the molecular mechanisms regulating the membrane transport in growing axons remain unclear. Here, we demonstrate that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. GRAB promotes the axonal membrane transport by mediating the interaction between Rab11 and Rab8 in neurons. The activity of GRAB is regulated by phosphorylation with Cdk5. We describe an in vivo role for GRAB and its Cdk5 phosphorylation during neuronal migration and process formation in embryonic brains. Thus, the membrane supply for axonal outgrowth is regulated by Cdk5 through the Rab11-GRAB-Rab8 cascade.

A Critical Role for Toxoplasma gondii Vacuolar Protein Sorting VPS9 in Secretory Organelle Biogenesis and Host Infection.

Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii.

Epileptic Encephalopathy Caused by Mutations in the Guanine Nucleotide Exchange Factor DENND5A

Epileptic encephalopathies are a catastrophic group of epilepsies characterized by refractory seizures and cognitive arrest, often resulting from abnormal brain development. Here, we have identified an epileptic encephalopathy additionally featuring cerebral calcifications and coarse facial features caused by recessive loss-of-function mutations in DENND5A. DENND5A contains a DENN domain, an evolutionarily ancient enzymatic module conferring guanine nucleotide exchange factor (GEF) activity to multiple proteins serving as GEFs for Rabs, which are key regulators of membrane trafficking. DENND5A is detected predominantly in neuronal tissues, and its highest levels occur during development. Knockdown of DENND5A leads to striking alterations in neuronal development. Mechanistically, these changes appear to result from upregulation of neurotrophin receptors, leading to enhanced downstream signaling. Thus, we have identified a link between a DENN domain protein and neuronal development, dysfunction of which is responsible for a form of epileptic encephalopathy.

PLD Protein-Protein Interactions With Signaling Molecules and Modulation by PA.

We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein-lipid interactions or as a substrate. The involvement of PLD as an effector of protein-protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2-Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2-PLD2 protein-protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.