Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Clinical profiling of MRD48 and functional characterization of two novel pathogenic RAC1 variants.

RAC1 is a member of the Rac/Rho GTPase subfamily within the RAS superfamily of small GTP-binding proteins, comprising 3 paralogs playing a critical role in actin cytoskeleton remodeling, cell migration, proliferation and differentiation. De novo missense variants in RAC1 are associated with a rare neurodevelopmental disorder (MRD48) characterized by DD/ID and brain abnormalities coupled with a wide range of additional features. Structural and functional studies have documented either a dominant negative or constitutively active behavior for a subset of mutations. Here, we describe two individuals with previously unreported de novo missense RAC1 variants. We functionally demonstrate their pathogenicity proving a gain-of-function (GoF) effect for both. By reviewing the clinical features of these two individuals and the previously published MRD48 subjects, we further delineate the clinical profile of the disorder, confirming its phenotypic variability. Moreover, we compare the main features of MRD48 with the neurodevelopmental disease caused by GoF variants in the paralog RAC3, highlighting similarities and differences. Finally, we review all previously reported variants in RAC proteins and in the closely related CDC42, providing an updated overview of the spectrum and hotspots of pathogenic variants affecting these functionally related GTPases.

Modeling human mutations to understand TRIO GEF function during development.

In a recent study, Bonnet and colleagues leveraged in silico structure prediction and human genetic data to understand the molecular regulation of the Rac1-activating guanie nucleotide exchange factor (Rac1-GEF) domain of Trio. Their work sheds new light on the role of Trio during axon guidance and explores the mechanism by which Trio GEF function is regulated in health and dysregulated in disease.

In silico identification and characterization of small-molecule inhibitors specific to RhoG/Rac1 signaling pathway.

Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. RhoG is one of the less explored Rho GTPases with significant sequential and structural homology with Rac1. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), maybe an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Z-score of -6.46, and 87.96% of residues had an averaged 3D-1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our results from docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1 based inhibitor, EHop-016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.Communicated by Ramaswamy H. Sarma.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Regulation of local GTP availability controls RAC1 activity and cell invasion.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

A novel anthraquinone­quinazoline hybrid 7B blocks breast cancer metastasis and EMT via targeting EGFR and Rac1.

At present, effective therapeutic drugs for triple­negative breast cancer (TNBC) are lacking due to the absence of identified or available targets. Therefore, the present study aimed to identify key molecular targets and a specific targeted therapeutic drug to aid with the development of novel therapeutic strategies for TNBC. Based on the high expression of EGFR and Rac1 in TNBC and inspired by a novel antitumor strategy termed combi­targeting, novel anthraquinone­quinazoline hybrid 7B was synthesized to simultaneously target EGFR and Rac1. It was hypothesized that hybrid 7B may possess enhanced potency compared with its parent compounds. Breast cancer cell viability was detected by performing MTT assays. Flow cytometry was conducted to detect the effects of hybrid 7B on the cell cycle, apoptosis and the mitochondrial outer membrane potential. Ultrastructural alterations were observed by transmission electron microscopy. Cell invasion and migration were assessed by performing Transwell and wound­healing assays, respectively. The expression levels of epithelial­mesenchymal transition (EMT) markers and metastasis­related proteins were detected by western blotting. Compared with Rhein and gefitinib, hybrid 7B displayed superior antiproliferative activity in MDA­MB­231 cells with an IC50 value of 2.31 µM, which was 14­fold higher compared with the EGFR tyrosine kinase inhibitor gefitinib. Further experiments demonstrated that hybrid 7B significantly reduced the mitochondrial membrane potential, enhanced MDA­MB­231 cell apoptosis and induced cell cycle arrest at the G2/M phase compared with the control group. Typical morphological alterations of apoptotic cells were observed in hybrid 7B­treated MDA­MB­231 and MCF­7 cells. Compared with the control group, hybrid 7B significantly inhibited MDA­MB­231 cell invasion and migration by downregulating Rac1, EGFR, matrix metalloproteinases, snail family transcriptional repressor 1, Vimentin and ß­catenin protein expression levels, and upregulating E­cadherin protein expression levels. The present study demonstrated that hybrid 7B inhibited TNBC cell migration and invasion by reversing EMT and targeting EGFR and Rac1; therefore, hybrid 7B may serve as a promising therapeutic agent for TNBC.

Regulation of the nuclear speckle localization and function of Rac1.

Despite significant evidence that Rac1 is localized to the nucleus, little is known regarding the function and biological significance of nuclear Rac1. Here, we showed that in response to EGF Rac1 was translocated to nuclear speckles and co-localized with the nuclear speckle marker Serine/arginine-rich splicing factor 2 (SRSF2) in Cos-7 cells. We also showed that the nuclear speckle localization of Rac1 was dependent on its T108 phosphorylation and facilitated by Rac1 polybasic region (PBR) that contains a nuclear localization signal and Rac1 GTPase activity. To gain insight into the function of Rac1 in nuclear speckles, we searched for Rac1 binding proteins in the nucleus. We isolated nuclear fraction of HEK 293 cells and incubated with GST-Rac1 and the phosphomimetic GST-Rac1T108E. We identified 463 proteins that were associated with GST-Rac1T108E, but not with GST-Rac1 by LC-MS/MS. Three notable groups of these proteins are: the heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and SRSFs, all of which are involved in pre-mRNA splicing and associated with nuclear speckles. We further showed by co-immunoprecipitation that Rac1 interacts with SRSF2, hnRNPA1, and U2A' in response to EGF. The interaction is dependent on T108 phosphorylation and facilitated by Rac1 PBR and GTPase activity. We showed that hnRNPA1 translocated in and out of nucleus in response to EGF in a similar pattern to Rac1. Rac1 only partially colocalized with U2A' that localizes to the actual splicing sites adjacent to nuclear speckle. Finally, we showed that Rac1 regulated EGF-induced pre-mRNA splicing and this is mediated by T108 phosphorylation. We conclude that in response to EGF, T108 phosphorylated Rac1 is targeted to nuclear speckles, interacts with multiple groups of proteins involved in pre-mRNA splicing, and regulates EGF-induced pre-mRNA splicing.

Reverse Docking for the Identification of Molecular Targets of Anticancer Compounds.

Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.

Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi.

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation.

We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based (i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1-2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function.

Voltage-dependent activation of Rac1 by Nav 1.5 channels promotes cell migration.

Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

The Intrinsic GDP/GTP Exchange Activities of Cdc42 and Rac1 Are Critical Determinants for Their Specific Effects on Mobilization of the Actin Filament System.

The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.

Rac1 GTPase is a critical factor in phagocytosis in the large yellow croaker Larimichthys crocea by interacting with tropomyosin.

The Rho family GTPase Rac1 acts as a molecular switch for signal transduction to regulate various cellular functions. Here, a Rac1 homolog (LcRac1) was identified in large yellow croaker (Larimichthys crocea), one of the most economically important marine fishes. The LcRac1 protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LcRac1). LcRac1 was ubiquitously expressed in all 12 tissues we examined, with the highest expression in heart and blood and the weakest expression in head-kidney and spleen. Moreover, time course analysis revealed that LcRac1 expression was obviously up-regulated in liver, spleen and head-kidney after immunization with Poly I:C, LPS and Vibrio parahemolyticus. On the other hand, on the basis of protein interaction, it was found that the LcRac1 interacted with Tropomyosin, a crucial protein in the process of phagocytosis. Furthermore, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the LcRac1 gene was silenced by sequence-specific siRNA. Fluorescence microscopy assays revealed FITC-labeled V. parahemolyticus were remarkably decreased after LcRac1 was silenced by sequence-specific siRNA at 24 h. These findings implicate the vital role of LcRac1 in innate immunity in the large yellow croaker.

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor.

The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42.

Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR.

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg2+ , a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

Mutations in MAP3K1 that cause 46,XY disorders of sex development disrupt distinct structural domains in the protein.

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.

cDNA cloning, characterization, and expression analysis of the Rac1 and Rac2 genes from Cynoglossus semilaevis.

Rac1 and Rac2, belonging to the small Rho GTPase family, play an important role during the immune responses. In this study, a Rac1 homolog (CsRac1) and a Rac2 homolog (CsRac2) were cloned from the Cynoglossus semilaevis. The full-length of CsRac1 and CsRac2 cDNA was 1219 bp and 1047 bp, respectively. Both CsRac1 and CsRac2 contain a 579 bp open reading frame (ORF) which encoding a 192 amino acids putative protein. The predicted molecular weight of CsRac1 and CsRac2 was 21.41 kDa and 21.35 kDa, and their theoretical pI was 8.50 and 7.91, respectively. Sequence analysis showed that the conserved RHO domain was detected both from amino acid of CsRac1 and CsRac2. Homologous analysis showed that CsRac1 and CsRac2 share high conservation with other counterparts from different species. The CsRac1 and CsRac2 transcript showed wide tissue distribution, in which CsRac1 and CsRac2 exhibit the highest expression level in liver and gill, respectively. The expression level of CsRac1 and CsRac2 fluctuated in the liver and gill tissues at different time points after challenged by Vibrio harveyi. Specifically, CsRac1 and CsRac2 were significantly up-regulated at 48 h and 96 h post injection. Moreover, the knocking down of CsRac1 and CsRac2 in cell line (TSHKC) reduced the expression of CsPAK1, CsIL1-ß and CsTNF-α. The present data suggests that CsRac1 and CsRac2 might play important roles in the innate immunity of half-smooth tongue sole.

An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK.

Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within the 106PNTP109 motif of Rac1 is likely an ERK phosphorylation site and Rac1 also has an ERK docking site 183KKRKRKCLLL192 (D-site) at the C-terminus. Protein phosphorylation could be assayed by many different methods. Here, we describe an in vitro kinase assay we used to assess Rac1 phosphorylation by ERK. Rac1 phosphorylation is detected based on the transfer of a radiolabeled phosphate from ATP to Rac1 by the phosphotransferase activity of the kinase EKR. This in vitro kinase assay uses commercially available purified active ERK. Substrate Rac1 was generated and purified as a glutathione S-transferase (GST) fusion protein. [γ-32P]ATP is used to radiolabel Rac1. Phosphorylation of Rac1 is viewed by autoradiography.