GDP-GTP Exchange Factor Research Articles

GEF-H1 Mediates Tumor Necrosis Factor-α-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-alpha, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-alpha on the Rho pathway in tubular cells (LLC-PK(1) and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-alpha-induced alterations of paracellular permeability. We show that TNF-alpha induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-alpha-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-alpha-induced activation of these proteins. Importantly TNF-alpha enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-alpha-induced paracellular permeability increase was absent in LLC-PK(1) cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-alpha-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.

Kalirin-7 Is Required for Synaptic Structure and Function

Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), the predominant adult splice form of the multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain and localizes to the postsynaptic side of excitatory synapses. In vitro studies demonstrated that overexpression of Kal7 increased dendritic spine density, whereas reduced expression of endogenous Kal7 decreased spine density. To evaluate the role of Kal7 in vivo, mice lacking the terminal exon unique to Kal7 were created. Mice lacking both copies of the Kal7 exon (Kal7(KO)) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7(KO) mice. Behaviorally, Kal7(KO) mice showed decreased anxiety-like behavior in the elevated zero maze and impaired acquisition of a passive avoidance task, but normal behavior in open field, object recognition, and radial arm maze tasks. Kal7(KO) mice were deficient in hippocampal long-term potentiation. Western blot analysis confirmed the absence of Kal7 and revealed compensatory increases in larger Kalirin isoforms. PSDs purified from the cortices of Kal7(KO) mice showed a deficit in Cdk5, a kinase known to phosphorylate Kal7 and play an essential role in synaptic function. The early stages of excitatory synaptic development proceeded normally in cortical neurons prepared from Kal7(KO) mice, with decreased excitatory synapses apparent only after 21 d in vitro. Expression of exogenous Kal7 in Kal7(KO) neurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

The Role of Kalirin9 in p75/Nogo Receptor-mediated RhoA Activation in Cerebellar Granule Neurons

p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation.

Inhibition of a eukaryotic initiation factor (eIF2Bδ,/F11A3.2) during adulthood extends lifespan inCaenorhabditis elegans

The critical role of protein synthesis in regulating lifespan has been evidenced. This study shows that adult-onset RNAi inactivation of eukaryotic initiation factor 2Bdelta (eIF2Bdelta/F11A3.2), a subunit of eIF2B, extends the mean lifespan of Caenorhabditis elegans. eIF2B is a GDP-GTP exchange factor for eIF2--a rate-limiting factor for protein synthesis initiation. (35)S-methionine incorporation assay showed that global protein synthesis is reduced by eIF2Bdelta/F11A3.2 RNAi. Inhibition of eIF2Bdelta/F11A3.2 during adulthood conferred thermal and oxidative stress resistance and reduced the fecundity and fat storage, suggesting the possible trade-offs of resources between reproduction and somatic maintenance. Lifespan extension by adult-onset eIF2Bdelta/F11A3.2 RNAi is suppressed in FOXO transcription factor daf-16 deletion mutants. Adult-onset eIF2Bdelta/F11A3.2 RNAi increases the expression of stress-resistant genes, including hsp-16.2, hsp-70, hsp90, and sod-3, some of which are reported to be targets of DAF-16. Adult-onset eIF2Bdelta/F11A3.2 RNAi in daf-16 mutants reduced fecundity, but did not extend lifespan. Furthermore, adult-onset eIF2Bdelta/F11A3.2 RNAi did not extend the lifespan of germline-defective glp-4 organisms. Thus, it is possible that eIF2Bdelta/F11A3.2 RNAi during adulthood prolongs lifespan via daf-16, which induces stress resistance in organisms. This might be the mechanism, at least in part, for trade-offs of resources between reproduction and somatic maintenance.

Autonomous functions for the Sec14p/spectrin-repeat region of Kalirin

Kalirin is a GDP/GTP exchange factor (GEF) for Rho proteins that modulates the actin cytoskeleton in neurons. Alternative splicing generates Δ-isoforms, which encode the RhoGEF domain, but lack the N-terminal Sec14p domain and first 4 spectrin-like repeats of the full-length isoforms. Splicing has functional consequences, with Kal7 but not ΔKal7 causing formation of dendritic spines. Cells lacking endogenous Kalirin were used to explore differences between these splice variants. Expression of ΔKal7 in this system induces extensive lamellipodial sheets, while expression of Kal7 induces formation of adherent compact, round cells with abundant cortical actin. Based on in vitro and cell-based assays, Kal7 and ΔKal7 are equally active GEFs, suggesting that other domains are involved in controlling cell morphology. Catalytically inactive Kal7 and a Kalirin fragment which includes only Sec14p and spectrin-like domains retain the ability to produce compact, round cells and fractionate as high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PI(3,5)P2 and PI3P, but does not alter cell morphology. We conclude that the Sec14p and N-terminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and Δ-Kalirin proteins.

P204 Protein Is a Novel Modulator of Ras Activity

The murine p200 family protein, p204, modulates cell proliferation and tissue differentiation. Many of its activities are exerted in the nucleus. However, in cardiac myocytes, p204 accumulated in the cytoplasm. A yeast two-hybrid assay revealed a p204-cytoplasmic Ras protein interaction. This was confirmed (i) by coimmunoprecipitation of p204 with Ras in mouse heart extract and with endogenous or ectopic H-Ras and K-Ras in cell lysates as well as (ii) by binding of purified H-Ras-GTP to purified p204 in vitro. p204 inhibited (i) the cleavage of RasGTP to RasGDP by RasGAP; (ii) the binding to RasGTP of Raf-1, phosphatidylinositol 3-kinase, and Ral-GDS, effectors of Ras signaling; and (iii) activation by the Ras pathway of the phosphorylation and thus activation of downstream targets (e.g. MEK, Akt, and p38 MAPK). Oncogenic Ras expression triggered the phosphorylation and translocation of p204 from the nucleus to the cytoplasm. This is expected to increase the interaction between the two proteins. Translocation triggered by Ras oncoprotein was blocked by the LY294002 inhibitor of phosphatidylinositol 3-kinase. Ras did not promote phosphorylation or translocation to the cytoplasm of mutated p204 in which serine 179 was replaced by alanine. p204 overexpression inhibited the anchorage-independent proliferation of cells expressing Ras(Q61L) oncoprotein. Ras oncoprotein triggered in MEF3T3 cells the rearrangement of the actin cytoskeleton and the enhancement of cell migration through a membrane. Overexpression of p204 inhibited both. Ras oncoprotein or activated, wild-type Ras was described to increase Egr-1 transcription factor expression. We report that a sequence in the gene encoding p204 bound Egr-1, and Egr-1 activated p204 expression. Ras oncoprotein or activated wild-type Ras increased the expression in 3T3 cells of p204 together with that of Egr-1. Furthermore, the activation of expression of a single copy of K-ras oncogene in cultured murine embryonic cells induced the expression of a high level of p204 as well as its distribution between the nuclei and the cytoplasm. Thus, p204 may serve as a negative feedback inhibitor of Ras activity.

Differential motility of p190bcr-abl- and p210bcr-abl-expressing cells: respective roles of Vav and Bcr-Abl GEFs

The chimeric oncogene Bcr-Abl is known to induce autonomous motility of leukemic cells. We show here that p210(bcr-abl) responsible for chronic myelogenous leukemia induces an amoeboid type of motility while p190(bcr-abl), associated with acute lymphoid leukemia, induces a rolling type of motility. We previously reported that p210(bcr-abl) activates RhoA and Rac1, while p190(bcr-abl) although devoid of a Dbl-homology (DH) domain activates Rac1, but not RhoA. We investigated the regulation of GDP/GTP exchange factor (GEF) activities in the Bcr-Abl complex. For that purpose, different GEF activity mutants of Vav and of Bcr-Abl were constructed and stably transfected in Ba/F3 cells. Using these mutants, we demonstrate that RhoA is exclusively activated by the DH domain of p210(bcr-abl), while Rac1 activation is mostly due to Vav. Inhibition of Rac1 by Vav GEF mutant leads to immobilization of cells. Vav depletion using shRNA also induces immobilization of cells and suppression of GTP-bound Rac1. RhoA inactivation induces the specific loss of amoeboid movements. These results suggest that Rac1 activation by Vav triggers the motility of Bcr-Abl-expressing Ba/F3 cells, while the specific amoeboid mode of motility induced by p210(bcr-abl) is a consequence of RhoA activation.

Glucose-stimulated cAMP-protein kinase a pathway in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, glucose signals activate the production of cellular cAMP. This signaling pathway is called the cAMP-protein kinase A (PKA) pathway, which plays a major role in the regulation of cell growth, metabolism, and stress resistance. Extensive studies have been carried out to clarify the mechanism of this pathway, and many factors involved in the pathway have been identified such as small G proteins, the GDP-GTP exchange factor, adenylate cyclase, and PKA. Also, additional elements involved in this pathway have been evaluated in the last decade. A heterotrimeric G protein alpha subunit was identified as a mammalian Galpha homologue, and a G-protein-coupled receptor (GPCR), which initiates the signaling pathway in response to glucose addition, was identified. GPCR-Galpha was shown to function in a signaling pathway that acts parallel to small G proteins. These signaling pathways regulate cell growth and differentiation in response to nutrients.

Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth

Neurotrophins, such as NGF and BDNF, induce sustained activation of Rap1 small G protein and ERK, which are essential for neurite outgrowth. We show involvement of a GDP/GTP exchange factor (GEF) for Rap1, PDZ-GEF1, in these processes. PDZ-GEF1 is activated by GTP-Rap1 via a positive feedback mechanism. Upon NGF binding, the TrkA neurotrophin receptor is internalized from the cell surface, passes through early endosomes, and arrives in late endosomes. A tetrameric complex forms between PDZ-GEF1, synaptic scaffolding molecule and ankyrin repeat-rich membrane spanning protein which interacts directly with the TrkA receptor. At late endosomes, the complex induces sustained activation of Rap1 and ERK, resulting in neurite outgrowth. In cultured rat hippocampal neurons, PDZ-GEF1 is recruited to late endosomes in a BDNF-dependent manner involved in BDNF-induced neurite outgrowth. Thus, the interaction of PDZ-GEF1 with an internalized neurotrophin receptor transported to late endosomes induces sustained activation of both Rap1 and ERK and neurite outgrowth.

Regulation of Dendritogenesis via a Lipid-Raft-Associated Ca2+/Calmodulin-Dependent Protein Kinase CLICK-III/CaMKIγ

Ca(2+) signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca(2+)/calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIgamma, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca(2+)-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.

Control of the B Cell-Intrinsic Tolerance Programs by Ubiquitin Ligases Cbl and Cbl-b

B cell receptor (BCR) signaling plays a critical role in B cell tolerance and activation. Here, we show that mice with B cell-specific ablation of both Cbl and Cbl-b (Cbl-/-Cblb-/-) manifested systemic lupus erythematosus (SLE)-like autoimmune disease. The Cbl double deficiency resulted in a substantial increase in marginal zone (MZ) and B1 B cells. The mutant B cells were not hyperresponsive in terms of proliferation and antibody production upon BCR stimulation; however, B cell anergy to protein antigen appeared to be impaired. Concomitantly, BCR-proximal signaling, including tyrosine phosphorylation of Syk tyrosine kinase, Phospholipase C-gamma2 (PLC-gamma2), and Rho-family GTP-GDP exchange factor Vav, and Ca2+ mobilization were enhanced, whereas tyrosine phosphorylation of adaptor protein BLNK was substantially attenuated in the mutant B cells. These results suggested that the loss of coordination between these pathways was responsible for the impaired B cell tolerance induction. Thus, Cbl proteins control B cell-intrinsic checkpoint of immune tolerance, possibly through coordinating multiple BCR-proximal signaling pathways during anergy induction.

Vav3 proto-oncogene deficiency leads to sympathetic hyperactivity and cardiovascular dysfunction

Although much is known about environmental factors that predispose individuals to hypertension and cardiovascular disease, little information is available regarding the genetic and signaling events involved. Indeed, few genes associated with the progression of these pathologies have been discovered despite intensive research in animal models and human populations. Here we identify Vav3, a GDP-GTP exchange factor that stimulates Rho and Rac GTPases, as an essential factor regulating the homeostasis of the cardiovascular system. Vav3-deficient mice exhibited tachycardia, systemic arterial hypertension and extensive cardiovascular remodeling. These mice also showed hyperactivity of sympathetic neurons from the time of birth. The high catecholamine levels associated with this condition led to the activation of the renin-angiotensin system, increased levels of kidney-related hormones and the progressive loss of cardiovascular and renal homeostasis. Pharmacological studies with drugs targeting sympathetic and renin-angiotensin responses confirmed the causative role and hierarchy of these events in the development of the Vav3-null mouse phenotype. These observations uncover the crucial role of Vav3 in the regulation of the sympathetic nervous system (SNS) and cardiovascular physiology, and reveal a signaling pathway that could be involved in the pathophysiology of human disease states involving tachycardia and sympathetic hyperactivity with unknown etiologies.

Direct Binding of Translation Initiation Factor eIF2γ-G Domain to Its GTPase-activating and GDP-GTP Exchange Factors eIF5 and eIF2Bϵ

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

Spatial and temporal regulation of GLUT4 translocation by flotillin‐1 and caveolin‐3 in skeletal muscle cells

Skeletal muscle tissue is one of the main sites where glucose uptake occurs in response to insulin. The glucose transporter type-4 (GLUT4) is primarily responsible for the insulin-stimulated increase in glucose uptake. Upon insulin stimulation, GLUT4 is recruited from intracellular reserves to the plasma membrane. The molecular mechanisms that regulate the translocation of GLUT4 to the sarcolemma remain to be fully identified. Here, we demonstrate that GLUT4 is localized to perinuclear stores that contain flotillin-1, a marker of lipid rafts, in skeletal muscle cells. Stimulation with insulin for 10 min results in the translocation of flotillin-1/GLUT4-containing domains to the plasma membrane in a PI3K- and PKCzeta-dependent manner. We also demonstrate that caveolin-3, a marker of caveolae, is required for the insulin receptor-mediated activation of the PI3K-dependent pathway, which occurs 2 min after insulin stimulation. In fact, we demonstrate that lack of caveolin-3 significantly reduces insulin-stimulated glucose uptake in caveolin-3 null myotubes by inhibiting both PI3K and Akt, as well as the movement of GLUT4 to the plasma membrane. Interestingly, caveolin-3 moves away from the plasma membrane toward the cytoplasm 5 min after insulin stimulation and temporarily interacts with flotillin-1/GLUT4-containing domains before they reach the sarcolemma, with the consequent movement of the insulin receptor from caveolin-3-containing domains to flotillin-1-containing domains. Such translocation temporally matches the insulin-stimulated movement of Cbl and CrkII in flotillin-1/GLUT4-containing domains, as well as the activation of the GDP-GTP exchange factor C3G. Disruption of flotillin-1-based domains prevents the activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake in response to insulin. Thus, the activation of the Cbl/C3G/TC10-dependent pathway, which occurs before flotillin-1/GLUT4-containing domains reach the plasma membrane, is flotillin-1 mediated and follows the activation of the PI3K-mediated signaling. Taken together, these results indicate that flotillin-1 and caveolin-3 may regulate muscle energy metabolism through the spatial and temporal segregation of key components of the insulin signaling.

The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

Amplification of the Ect2 proto-oncogene and over-expression of Ect2 mRNA and protein in nickel compound and methylcholanthrene-transformed 10T1/2 mouse fibroblast cell lines

Occupational exposure of humans to mixtures of insoluble and soluble nickel (Ni) compounds correlates with increased incidences of lung, sinus, and pharyngeal tumors. Specific insoluble Ni compounds are carcinogenic to animals by inhalation and induce morphological and neoplastic transformation of cultured rodent cells. Our objectives were to (1) understand mechanisms of nickel ion-induced cell transformation, hence carcinogenesis and (2) develop biomarkers of nickel ion exposure and nickel ion-induced cell transformation. We isolated mRNAs from green nickel oxide (NiO), crystalline nickel monosulfide (NiS), and 3-methylcholanthrene (MCA) transformed C3H/10T1/2 Cl 8 cell lines, and determined by mRNA differential display that nine mRNA fragments were differentially expressed between Ni transformed and non-transformed 10T1/2 cell lines. Fragment R2-5 was expressed at higher steady-state levels in the transformed cell lines. R2-5 had 100% sequence identity to part of the coding region of Ect2, a mouse proto-oncogene encoding a GDP–GTP exchange factor. The 3.9-kb Ect2 transcript was expressed at 1.6- to 3.6-fold higher steady-state levels in four Ni transformed, and in two MCA-transformed, cell lines. Ect2 protein was expressed at 3.0- to 4.5-fold higher steady-state levels in Ni-transformed and in MCA-transformed cell lines. The Ect2 gene was amplified by 3.5- to 10-fold in Ni transformed, and by 2.5- to 3-fold in MCA transformed cell lines. Binding of nickel ions to enzymes of DNA synthesis likely caused amplification of the Ect2 gene. Ect2 gene amplification and over-expression of Ect2 mRNA and protein can cause microtubule disassembly and cytokinesis, contributing to induction and maintenance of morphological, anchorage-independent, and neoplastic transformation of these cell lines. Over-expression of Ect2 protein is a useful biomarker to detect exposure to nickel compounds and nickel ion-induced morphological and neoplastic cell transformation.

Role of Phosphoinositide 3-Kinase Regulatory Isoforms in Development and Actin Rearrangement

Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.

Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis

COPII coat proteins are required for direct capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum (ER). Cargo and SNARE capture occurs during the formation of a 'prebudding complex' comprising a cargo, Sar1p-GTP and the COPII subunits Sec23/24p. The assembly and disassembly cycle of the prebudding complex on ER membranes is coupled to the Sar1p GTPase cycle. Using FRET to monitor a single round of Sec23/24p binding and dissociation from SNAREs in reconstituted liposomes, we show that Sec23/24p dissociates from v-SNARE and complexed t-SNARE with kinetics slower than Sar1p-GTP hydrolysis. Once Sec23/24p becomes associated with v-SNARE or complexed t-SNARE, the complex remains assembled during multiple rounds of Sar1p-GTP hydrolysis mediated by the GDP-GTP exchange factor Sec12p. These data suggest a model for the maintenance of kinetically stable prebudding complexes during the Sar1p GTPase cycle that regulates cargo sorting into transport vesicles.

Double cross-over gene replacement within the sec 7 domain of a GDP–GTP exchange factor from Plasmodium falciparum allows the generation of a transgenic brefeldin A-resistant parasite line

High molecular weight ADP ribosylation factor GDP-GTP exchange factors (ARF-GEF) play an essential role in the formation of COP I coated transport vesicles and are characterized by a structurally and functionally conserved sec 7 domain. The genome of the malaria parasite Plasmodium falciparum encodes a single ARF-GEF that contains an unusual sec 7 domain. In comparison to the sec 7 domain of other eukaryotes, the plasmodial sec 7 domain is characterized by an insertion sequence of 146 amino acids that disrupt helices essential for the GDP-GTP exchange activity of the protein. In a previous study we have shown a correlation between a methionine to isoleucine exchange in helix H of the sec 7 domain and resistance to brefeldin A in a parasite line generated by drug selection. Here we have transfected brefeldin A sensitive parasites with plasmid constructs containing the sec 7 domain of the resistant line either with or without the insertion sequence. Transfection with sec 7 sequences including the insertion resulted in brefeldin A resistant parasites in which double cross-over recombination had replaced the endogenous sec 7 sequences with the transgenic sequences. Thus, the point mutation in helix H is sufficient to confer brefeldin A resistance in P. falciparum. Transfections using constructs lacking the insertion did not result in resistant parasites. Gene replacement by targeted double cross-over recombination is a rare event in P. falciparum. This approach has taken advantage of the fact that the successful integration of the transgene results in a drug selectable phenotype. We anticipate that the strategy described here will be useful for the identification of mutations within target genes that have the potential to confer increased drug resistance.

Specific Residues of the GDP/GTP Exchange Factor Bud5p Are Involved in Establishment of the Cell Type-specific Budding Pattern in Yeast

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.