RESUMO
STUDY HYPOTHESIS: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE: Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 µM compared with an IC50 of 38.5 µM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 µM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). LIMITATIONS, REASONS FOR CAUTION: The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. WIDER IMPLICATIONS OF THE FINDINGS: This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. LARGE SCALE DATA: None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.
Assuntos
Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Glicólise/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
Monoamine oxidase B (MAO-B) plays a key role in the metabolism of dopamine, a neurotransmitter critical for the maintenance of cognitive function. Consequently, MAO-B is an important therapeutic target for disorders characterized by a decline in dopaminergic neurotransmission, including Parkinson's disease (PD). An emerging strategy in drug discovery is to utilize the biophysical approaches of thermal shift and isothermal titration calorimetry (ITC) to gain insight into binding modality and identify thermodynamically privileged chemical scaffolds. Described here is the development of such approaches for reversible and irreversible small molecule inhibitors of MAO-B. Investigation of soluble recombinant MAO-B revealed mechanism-based differences in the thermal shift and binding thermodynamic profiles of MAO-B inhibitors. Irreversible inhibitors demonstrated biphasic protein melt curves, large enthalpically favorable and entropically unfavorable binding, in contrast to reversible compounds, which were characterized by a dose-dependent increase in thermal stability and enthalpically-driven binding. The biophysical approaches described here aim to facilitate the discovery of next-generation MAO-B inhibitors.
Assuntos
Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Doença de Parkinson/tratamento farmacológico , TermodinâmicaRESUMO
SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Western Blotting , Linhagem Celular , Cromatografia em Gel , Dicroísmo Circular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores do Crescimento/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Bibliotecas de Moléculas Pequenas/análise , Proteína rhoA de Ligação ao GTP/metabolismo , Algoritmos , Polarização de Fluorescência/métodos , Inibidores do Crescimento/farmacologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Humanos , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Fatores de Troca de Nucleotídeo Guanina Rho , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases.
Assuntos
GTP Fosfo-Hidrolases/análise , Proteínas ras/análise , Transferência de Energia , Fluorescência , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Espectrometria de Fluorescência , Proteínas ras/metabolismoRESUMO
PURPOSE: Irofulven (6-hydroxymethylacylfulvene, MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a toxin present in the Omphalotus mushroom. Irofulven has demonstrated activity against a broad range of solid tumors in both xenograft models and human trials. The potential application of administering irofulven in combination with aziridine-containing chemotherapeutic agents was evaluated in this study. METHODS: Human lung carcinoma MV522 cells and BALB/c athymic mice bearing the human lung carcinoma MV522 xenograft were used to evaluate the activity of irofulven in combination with aziridine-containing drugs. RESULTS: Irofulven in combination with either thiotepa or mitomycin C demonstrated a strong synergistic (supraadditive) activity both in vitro and in vivo, that exceeded results obtained with monotherapy at the same or higher doses of these agents. The majority of xenograft-bearing animals that received subtoxic doses of irofulven, and either thiotepa or mitomycin C, demonstrated a complete cure. In contrast, there was no detectable synergistic activity between irofulven and other aziridine-containing drugs, including AZQ and thiotepa metabolites such as TEPA or AZD. CONCLUSIONS: These results indicate that the therapeutic activity of irofulven is enhanced when combined with mitomycin C or thiotepa, and further evaluation of these combinations is therefore warranted.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Mitomicina/uso terapêutico , Sesquiterpenos/uso terapêutico , Tiotepa/uso terapêutico , Animais , Aziridinas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The novel agent Irofulven (HMAF, NSC 683863) has demonstrated significant antitumor activity against solid tumors in various xenograft models and human clinical trials. The antitumor potential of combining irofulven with 72 different anti-metabolite, enzyme inhibiting, and miscellaneous agents was investigated in this study. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with these different agents. Irofulven in combination with select anti-metabolites, notably cytidine or adenine-derived agents, displayed strong synergistic activity in both in vitro and in vivo studies. Agents demonstrating strong synergistic interaction with irofulven included gemcitabine, cyclocytidine, cytarabine, fludarabine phosphate, cladribine, and 5-fluorouracil. Other anti-metabolites, enzyme inhibitors, and a variety of miscellaneous agents failed to interact beneficially when administered in combination with irofulven. The therapeutic activity of irofulven is enhanced considerably when irofulven is combined with select anti-metabolite agents, and further clinical evaluation of these combinations is warranted. The synergistic interaction with these combinations may stem from a variety of actions including inhibition of the nucleotide excision repair (NER) pathway, topoisomerase I activity, and caspase-dependent and independent induction of apoptosis.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/administração & dosagem , Fluoruracila/administração & dosagem , Sesquiterpenos/administração & dosagem , Animais , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
PURPOSE: Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. METHODS: The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. RESULTS: Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. CONCLUSION: These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Altretamine/administração & dosagem , Altretamine/farmacologia , Animais , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/genética , Sinergismo Farmacológico , Feminino , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/farmacologia , Distribuição Aleatória , Sesquiterpenos/administração & dosagem , Sesquiterpenos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Bioquímica/métodos , Biofísica/métodos , Dimerização , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Fatores de Troca de Nucleotídeo Guanina Rho , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.
Assuntos
Apoptose , Azacitidina/análogos & derivados , Metilação de DNA , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Quinases raf/fisiologia , Ágar/química , Alelos , Animais , Proteínas Reguladoras de Apoptose , Azacitidina/farmacologia , Northern Blotting , Western Blotting , Butadienos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , DNA Complementar/metabolismo , Decitabina , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Feminino , Fibroblastos/metabolismo , Flavonoides/farmacologia , Vetores Genéticos , Humanos , Camundongos , Mutação , Células NIH 3T3 , Nitrilas/farmacologia , Ovário/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Regulação para CimaRESUMO
The aim of this study was to determine the antitumor activity of irofulven when administered in combination with a variety of antimitotic agents. Irofulven in combination with either paclitaxel or docetaxel demonstrated synergistic activity in both the in vitro and in vivo studies. The majority of xenograft bearing animals that received suboptimal (< MTD) doses of irofulven and a taxane demonstrated complete cures. In contrast, in vitro studies produced either an additive or an antagonistic effect when irofulven was combined with other antimitotic agents such as vinca alkaloids, rhizoxin, s-trityl cysteine, or allocolchicine. Xenograft studies of irofulven and vinca alkaloids reflected in vitro results, as the tumor response in combination treated animals was less than the response in irofulven (monotherapy) treated animals. These results indicate that the therapeutic activity of irofulven is enhanced when combined with taxanes, and warrant further evaluation of these combinations.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Colchicina/análogos & derivados , Cisteína/análogos & derivados , Sesquiterpenos/farmacologia , Taxoides , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Cisteína/farmacologia , Sinergismo Farmacológico , Feminino , Lactonas/uso terapêutico , Macrolídeos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas , Alcaloides de Vinca/farmacologiaRESUMO
Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.