Smg GDS Research Articles

A growing family of guanine nucleotide exchange factors is responsible for activation of ras-family GTPases

GTPases of the Ras subfamily regulate a diverse array of cellular-signaling pathways, coupling extracellular signals to the intracellular response machinery. Guanine nucleotide exchange factors (GEFs) are primarily responsible for linking cell-surface receptors to Ras protein activation. They do this by catalyzing the dissociation of GDP from the inactive Ras proteins. GTP can then bind and induce a conformational change that permits interaction with downstream effectors. Over the past 5 years, approximately 20 novel Ras-family GEFs have been identified and characterized. These data indicate that a variety of different signaling mechanisms can be induced to activate Ras, enabling tyrosine kinases, G-protein-coupled receptors, adhesion molecules, second messengers, and various protein-interaction modules to relocate and/or activate GEFs and elevate intracellular Ras-GTP levels. This review discusses the structure and function of the catalytic or CDC25 homology domain common to almost all Ras-family GEFs. It also details our current knowledge about the regulation and function of this rapidly growing family of enzymes that include Sos1 and 2, GRF1 and 2, CalDAG-GEF/GRP1-4, C3G, cAMP-GEF/Epac 1 and 2, PDZ-GEFs, MR-GEF, RalGDS family members, RalGPS, BCAR3, Smg GDS, and phospholipase C(epsilon).

Identification of Guanine Nucleotide Exchange Factors (GEFs) for the Rap1 GTPase: REGULATION OF MR-GEF BY M-Ras-GTP INTERACTION

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

Involvement of a small GTP-binding protein (G protein) regulator, small G protein GDP dissociation stimulator, in antiapoptotic cell survival signaling.

Small GTP-binding protein GDP dissociation stimulator (Smg GDS) regulates GDP/GTP exchange reaction of Ki-Ras and the Rho and Rap1 family members and inhibits their binding to membranes. In fibroblasts, Smg GDS shows mitogenic and transforming activities in cooperation with Ki-Ras. However, the physiological function of Smg GDS remains unknown. Here we show that mice lacking Smg GDS died of heart failure shortly after birth, not resulting from developmental heart defects but from enhanced apoptosis of cardiomyocytes triggered by cardiovascular overload. Furthermore, neonatal thymocytes and developing neuronal cells underwent apoptotic cell death. Smg GDS-/- thymocytes were susceptible to apoptotic inducers, such as etoposide and UV irradiation. Smg GDS-/- thymocytes were protected from etoposide-induced cell death by ex vivo transduction of the Smg GDS cDNA. These phenotypes partly coincide with those observed in Ki-Ras-deficient mice, suggesting that Smg GDS is involved in antiapoptotic cell survival signaling through Ki-Ras.

Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14

We compared the in vivo characteristics of hemagglutinin (HA)-tagged RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14) stably expressed in Chinese hamster ovary (CHO) cells. Proteins co-precipitating with these HA-tagged GTPases were identified by peptide sequencing or by Western blotting. Dominant negative RhoA(Asn-19) co-precipitates with the guanine nucleotide exchange factor (GEF) SmgGDS but does not detectably interact with other expressed GEFs, such as Ost or Dbl. SmgGDS co-precipitates minimally with wild-type RhoA and does not detectably associate with RhoA(Val-14). The guanine nucleotide dissociation inhibitor RhoGDI co-precipitates with RhoA, and to a lesser extent with RhoA(Val-14), but does not detectably co-precipitate with RhoA(Asn-19). Wild-type RhoA is predominantly in the [(32)P]GDP-bound form, RhoA(Val-14) is predominantly in the [(32)P]GTP-bound form, and negligible levels of [(32)P]GDP or [(32)P]GTP are bound to RhoA(Asn-19) in (32)P-labeled cells. Immunofluorescence analyses indicate that HA-RhoA(Asn-19) is excluded from the nucleus and cell junctions. Microinjection of SmgGDS cDNA into CHO cells stably expressing HA-RhoA causes HA-RhoA to be excluded from the nucleus and cell junctions, similar to the distribution of RhoA(Asn-19). Our findings indicate that the expression of RhoA(Asn-19) may specifically inhibit signaling pathways that rely upon the SmgGDS-dependent activation of RhoA.

Complex Formation of SMAP/KAP3, a KIF3A/B ATPase Motor-associated Protein, with a Human Chromosome-associated Polypeptide

We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase. SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport. We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide). Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present. SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP. The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.

SMAP, an Smg GDS-associating Protein Having Arm Repeats and Phosphorylated by Src Tyrosine Kinase

Smg GDS is a regulator having two activities on a group of small G proteins including the Rho and Rap1 family members and Ki-Ras; one is to stimulate their GDP/GTP exchange reactions, and the other is to inhibit their interactions with membranes. Structurally, it has 11 Arm repeats, a protein interaction motif, found in the Drosophila Armadillo protein, a homolog of mammalian beta-catenin. We have isolated here an Smg GDS-interacting protein from a human brain cDNA library by use of the yeast two-hybrid method and named it SMAP (Smg GDS-associated protein). SMAP was a protein with a Mr of 91,189 and 792 amino acids. SMAP had 9 Arm repeats. Recombinant SMAP interacted with recombinant Smg GDS but did not affect the two activities of Smg GDS on RhoA. SMAP was tyrosine phosphorylated by v-Src, and this phosphorylation reduced the affinity of SMAP for Smg GDS. Tissue and subcellular distribution analyses indicated that SMAP was ubiquitously expressed and highly concentrated at the endoplasmic reticulum area. Searches for sequence homology to SMAP revealed that SMAP was significantly homologous to sea urchin SpKAP115, suggesting that SMAP is a mammalian counterpart of SpKAP115 or its related protein. SpKAP115 is an accessory subunit of sea urchin kinesin II, an ATPase motor that transports vesicles along microtubules. These results suggest that SMAP serves as an adaptor for both Smg GDS and kinesin II or its related protein and links them with both the Smg GDS-regulated small G protein and Src tyrosine kinase signalings.

Low Molecular Weight GTP-binding Proteins in HL-60 Granulocytes: ASSESSMENT OF THE ROLE OF ARF AND OF A 50-kDa CYTOSOLIC PROTEIN IN PHOSPHOLIPASE D ACTIVATION

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

The Dbl Oncogene Product as a GDP/GTP Exchange Protein for the Rho Family: Its Properties in Comparison with Those of Smg GDS

The dbl oncogene product (Dbl) showed the GDP/GTP exchange protein (GEP) activity on all the rho family small GTP-binding protein (G protein) members including RhoA and Rac1 as well as mCdc42. Dbl was active on both the lipid-modified and -unmodified forms of these small G proteins, but was much more active on the former form than on the latter form. In the presence of Rho GDI, an inhibitory GEP for the rho family members, the GEP activity of Dbl was markedly reduced. These properties of Dbl were partly different from those of Smg GDS, another GEP which is active not only on the rho family members but also on Ki-Ras and Rap1 and is active only on their lipid-modified form.

Comparison of kinetic properties between two mammalian ras p21 GDP/GTP exchange proteins, ras guanine nucleotide-releasing factor and smg GDP dissociation stimulation.

The mammalian counterpart of the yeast ras p21 GDP/GTP exchange protein CDC25, ras GRF, was expressed in Escherichia coli and purified, and its kinetic properties were compared with those of another mammalian ras p21 GDP/GTP exchange protein, smg GDS. ras GRF was active on Ki- and Ha-ras p21s but inactive on rap1A p21, rhoA p21, rac1 p21, and rab3A p25, whereas smg GDS was active on Ki-ras p21, rap1A p21, rhoA p21, and rac1 p21 but inactive on Ha-ras p21 and rab3A p25. The Kcat values of ras GRF and smg GDS for Ki-ras p21 as a common substrate were calculated to be 1.2 and 0.37 nmol/min/nmol, respectively. The Km values of ras GRF and smg GDS for Ki-ras p21 were 680 and 220 nM, respectively. rasGRF was slightly active on post-translationally unprocessed Ki-ras p21 but much more effective on post-translationally processed Ki-ras p21 than on post-translationally unprocessed Ki-ras p21. smg GDS was active on post-translationally processed Ki-ras p21 but inactive on post-translationally unprocessed Ki-ras p21. Moreover, as described for smg GDS, ras GRF showed a potency to inhibit the binding of Ki-ras p21 to membrane and to induce the dissociation of prebound Ki-ras p21 from the membrane. These results indicate that ras GRF and smg GDS show apparently similar kinetic properties except for the different substrate specificities and the requirement of the post-translational processing of Ki-ras p21.

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25.

The 3'-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3'-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).

The Nucleotide Exchange Rates of Rho and Rac Small GTP-Binding Proteins Are Enhanced to Different Extents by Their Regulatory Protein Smg GDS

Rho p21 and rac p21 small GTP-binding proteins are regulated by the same inhibitory and stimulatory GDP/CTP exchange proteins termed rho GDI and smg GDS, respectively. RhoA p21 and rac1 p21 bind with similar affinities to rho GDI and both proteins also bind with similar affinities to smg GDS. RhoA p21 and rac1 p21 have similar GDP/GTP exchange rates in the absence of rho GDI and smg GDS. The velocity of the GDP/GTP exchange reaction for rhoA p21 was enhanced much more by smg GDS than was the velocity of nucleotide exchange for rac1 p21. In the presence and absence of smg GDS, rho GDI reduced the velocities of these nucleotide exchange reactions of rhoA p21 and rac1 p21 to similar extents. These results suggest that rhoA p21 is activated first followed by rac1 p21 activation in response to extracellular signals.

Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI).

Evidence is accumulating that the rho family, a member of the ras p21-related small GTP-binding protein superfamily, regulates cell morphology, cell motility, and smooth muscle contraction through the actomyosin system. The actomyosin system is also known to be essential for cytoplasmic division of cells (cytokinesis). In this study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein, named rho GDI, its stimulatory GDP/GTP exchange protein, named smg GDS, and botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in the cytoplasmic division using Xenopus embryos. The sperm-induced cytoplasmic division of Xenopus embryos was not affected by microinjection into the embryos of either smg GDS or the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of rhoA p21, one member of the rho family, but completely inhibited by microinjection of rho GDI or C3. Under these conditions, nuclear division occurred normally but the furrow formation, which was induced by the contractile ring consisting of actomyosin just beneath the plasma membrane, was impaired. Comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 prevented the rho GDI action. Moreover, the sperm-induced cytoplasmic division of Xenopus embryos was inhibited by microinjection into the embryos of the rhoA p21 pre-ADP-ribosylated by C3 which might serve as a dominant negative inhibitor of endogenous rho p21. These results indicate that rho p21 together with its regulatory proteins regulates the cytoplasmic division through the actomyosin system.

Translocation of Ki- ras P21 between Membrane and Cytoplasm by smg GDS

We have previously shown that smg GDS forms a complex with Ki- ras 4B p21 and thereby stimulates its GDP/GTP exchange reaction. We have shown here that smg GDS inhibits the binding of Ki- ras 4B p21 to membranes and induces the dissociation of prebound Ki- ras 4B p21 from membranes. Moreover, we have found that cotransfection of the smg GDS and Ki- ras 4B p21 cDNAs into COS7 cells increases the ratio of the cytosolic form of ras p21 to the membrane-bound form and the ratio of the GTP-bound form of ras p21 to the GDP-bound form. These results indicate that smg GDS regulates not only the GDP/GTP exchange reaction of Ki- ras 4B p21 but also its translocation between membrane and cytoplasm.

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.

Post-translational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase.

rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.

Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.

smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins). rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins. We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate. smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP. The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS. The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence. Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells. On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21. This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS. These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS. Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other.

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.