Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins

Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP.     

Stoichiometric interaction of smg p21 with its GDP/GTP exchange protein and its novel action to regulate the translocation of smg p21 between membrane and cytoplasm

We have previously purified a GDP/GTP exchange protein for smg p21A and -B, members of a ras p21/ras p21-like small GTP-binding protein superfamily. This regulatory protein, named smg p21 GDP dissociation stimulator (GDS), stimulates the dissociation of both GDP and GTP from and the subsequent binding of both GDP and GTP to smg p21s. We show here that smg p21 GDS forms a complex with both the GDP- and GTP-bound forms of smg p21B at a molar ratio of about 1:1. Both the GDP- and GTP-bound forms of smg p21B bound to membranes. smg p21 GDS inhibited this binding and moreover induced the dissociation of the prebound smg p21B from the membranes. These results indicate that smg p21 GDS stoichiometrically interacts with smg p21B and thereby regulates its GDP/GTP exchange reaction and its translocation between membranes and cytoplasm.     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region

smg p21B, a member of the ras p21-like small GTP-binding protein superfamily, undergoes post-translational modifications, which are geranylgeranylation of the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids (QLL) and the subsequent carboxyl methylation of the exposed prenylated cysteine residue. smg p21B has a polybasic region upstream of the prenylated cysteine residue. We have previously proposed that these C-terminal structures of smg p21B are essential for the action of its stimulatory GDP/GTP exchange protein, named GDP dissociation stimulator (GDS). We studied here which structure of the C-terminal region of smg p21B is important for its interaction with smg p21 GDS. For this purpose, we synthesized a peptide according to the C-terminal structure of smg p21B, which was PGKARKKSSC-geranylgeranyl-carboxyl methyl, and its variously modified peptides and examined their ability to interact with smg p21 GDS and to interfere with the smg p21 GDS action to stimulate the GDP/GTP exchange reaction of smg p21B. The results indicate that the phosphorylated form of PGKARKKSSC-geranylgeranyl stoichiometrically interacts with smg p21 GDS, that the presence of the geranylgeranyl moiety is essential for, but not sufficient for, the smg p21 GDS action, and that the presence of the methyl moiety, removal of the three C-terminal amino acids, and the presence of the polybasic amino acids also affect the smg p21 GDS action. It is likely that all the steps of the post-translational processing and presence of the polybasic region in the C-terminal region of smg p21B are related to its interaction with smg p21 GDS.     

Transforming and c-fos promoter/enhancer-stimulating activities of a stimulatory GDP/GTP exchange protein for small GTP-binding proteins.

smg GDP dissociation stimulator (GDS) is a stimulatory GDP/GTP exchange protein for a group of ras p21-like small GTP-binding proteins (G proteins) including c-Ki-ras p21, smg p21A, smg p21B, and rhoA p21. smg GDS converts the GDP-bound inactive form to the GTP-bound active form of each small G protein by stimulating their GDP/GTP exchange reaction in a cell-free system. The point-mutated c-Ki-ras p21 (c-Ki-rasval12 p21) is known to strongly transform NIH/3T3 cells and to markedly stimulate the c-fos promoter/enhancer in this cell line, whereas the normal c-Ki-ras p21 is weak in these activities. In the present study, we examined the effect of smg GDS on these activities to explore its physiological function. Overexpression of both smg GDS and c-Ki-ras p21 strongly transformed NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly transformed the cells. Furthermore, overexpression of both smg GDS and c-Ki-ras p21 markedly stimulated the c-fos promoter/enhancer in NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly stimulated it. These results indicate that smg GDS transforms NIH/3T3 cells and stimulates the c-fos promoter/enhancer in this cell line in cooperation with c-Ki-ras p21.     

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.     

Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein

A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein.     

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.     

Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein.

smg p25A is a ras p21-like small GTP-binding protein which is implicated in the regulated secretory processes. We have recently found that bovine brain smg p25A is geranylgeranylated at its C-terminal region. In this study, we examined the function(s) of the C-terminal region of smg p25A. Limited proteolysis of bovine brain smg p25A with Achromobacter protease I produced an N-terminal fragment and a C-terminal tail. The Mrs of intact smg p25A, the N-terminal fragment, and the C-terminal tail were estimated to be about 24,000, 20,000, and less than 2,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal fragment contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities and showed these activities with kinetic properties similar to those of the intact protein but did not bind to plasma membranes or phosphatidylserine-linked Affigel under conditions in which the intact protein bound to them. The C-terminal tail neither contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities nor bound to plasma membranes or phosphatidylserine-linked Affigel. The GDP/GTP exchange protein specific for smg p25A, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of the intact smg p25A at a molar ratio of 1:1 and thereby inhibited its GDP/GTP exchange reaction but neither made a complex with the N-terminal fragment or the C-terminal tail nor affected the GDP/GTP exchange reaction of the N-terminal fragment. We expressed smg p25A in Escherichia coli and purified it to near homogeneity. This bacterial protein was not geranylgeranylated. Bacterial smg p25A did not bind to plasma membranes or phosphatidylserine-linked Affigel. smg p25A GDI neither made a complex with bacterial smg p25A nor affected its GDP/GTP exchange reaction. These results suggest that the N-terminal region of smg p25A has GDP/GTP-binding and GTPase activities but lacks the ability to interact with membranes and smg p25A GDI, that the C-terminal region of smg p25A plays important roles in its interaction with membranes and smg p25A GDI, and that some modifications of the C-terminal region, such as geranylgeranylation, which are absent in bacterial smg p25A, are important for these interactions.     

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.     

Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions

Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action.     

Both stimulatory and inhibitory GDP/GTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins.

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.     

A small GTP-binding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol

We have recently purified from bovine brain cytosol a novel type of regulatory protein for smg p25A, named smg p25A GDP dissociation inhibitor (GDI), that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. This smg p25A GDI is inactive for other ras p21/ras p21-like small GTP-binding proteins (G proteins) including c-Ha-ras p21, smg p21, rhoA p21 and rhoB p20. In human platelet membranes, smg p25A was not detected but a G protein with an apparent Mr value of 24,000 (24KG) was recognized by smg p25A GDI and the dissociation of GDP from and the binding of GTP to 24KG were inhibited by smg p25A GDI. The doses of smg p25A GDI necessary for these activities for both 24KG and smg p25A were the same. This 24KG was not recognized by an anti-smg p25A monoclonal antibody. The GDI activity for human platelet 24KG and smg p25A was detected in human platelet cytosol. This human platelet GDI was recognized by an anti-smg p25A GDI polyclonal antibody. These results indicate that there is a 24KG-24KG GDI system similar to a smg p25A-smg p25A GDI system in human platelets.     

Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24K G, a ras p21-like GTP-binding protein, with properties similar to those of smg p25A GDI

A regulatory protein for a liver GTP-binding protein (G protein) with a molecular weight value of 24,000 (24K G), which we have recently purified, was purified to near-homogeneity from rat liver cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor for 24K G (24K G GDI), inhibited the dissociation of GDP from and the subsequent binding of GTP to 24K G. 24K G GDI was inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, rhoB p20, smg p21B, and smg p25A. 24K G was, however, recognized by bovine brain smg p25A GDI which regulated the GDP/GTP exchange reaction of smg p25A. By analyses of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), immunoblotting with anti-smg p25A GDI antibody, two-dimensional PAGE, and C4 column chromatography, 24K G GDI showed physical properties very similar to those of smg p25A GDI. The peptide map and the partial amino acid sequences of 24K G GDI were not identical with those of smg p25A GDI. Among the 83 residues, 2 amino acids were different between rat liver 24K G GDI and bovine brain smg p25A GDI. These results indicate that there is a specific regulatory protein for 24K G, 24K G GDI, in rat liver cytosol and that 24K G GDI has close similarity to smg p25A GDI.     

Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of rabbit intestine. This protein, designated as rhoB p20 GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP from rhoB p20. rhoB p20 GDI also inhibited the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. GDI did not affect the GTPase activity of rhoB p20 and by itself showed no GTP gamma S-binding activity. GDI was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The Mr value of GDI was estimated to be about 27,000 from the S value. These results indicate that rabbit intestine contains a novel regulatory protein that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to rhoB p20.     

Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.     

Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s

The smg-21 GTP-binding protein (smg p21) has the same effector domain as the ras proteins (ras p21s) and is identical with the proteins of the rap1A and Krev-1 genes. In this paper, two proteins stimulating the GTPase activity of smg p21 are partially purified from bovine brain cytosol. These proteins, designated as smg p21 GTPase-activating protein (GAP) 1 and 2, are separated from a c-ras p21 GAP described previously by column chromatographies. smg p21 GAP1 and -2 stimulate the GTPase activity of only smg p21 but not that of c-Ha-ras p21 or the rho and smg-25A GTP-binding proteins. smg p21 GAP1 or -2 does not stimulate the dissociation of guanosine 5'-3-O-(thio)triphosphate or GDP from smg p21. smg p21 GAP1 or -2 themselves do not have GTP/GDP binding or GTPase activity. The Mr values of smg p21 GAP1 and -2 are estimated to be 250-400 x 10(3) and 80-100 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. The activity of smg p21 GAP1 and -2 is killed by tryptic digestion or heat boiling. These results indicate that bovine brain contains two smg p21 GAPs in addition to c-ras p21 GAP.     

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.     

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.     

Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for smg p25A by use of its antibody

We have recently purified from bovine brain cytosol to near homogeneity a GDP/GTP exchange protein for smg p25A, named smg p25A GDI, that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A. In the present study, we made an antiserum against smg p25A GDI and studied its tissue distribution in rat and its subcellular distribution in rat cerebrum by use of this antiserum. smg p25A GDI was found in secretory cells with both regulated and constitutive secretion types. Since smg p25A was previously found in only secretory cells with a regulated secretion type, this result suggests that small GTP-binding proteins different from smg p25A but recognized by smg p25A GDI are present in secretory cells with a constitutive secretion type, and that smg p25A GDI is involved in both regulated and constitutive secretory processes. In subcellular fractionation analysis of rat cerebrum, smg p25A GDI was mostly found in the cytosol fraction of neuron body and synaptosome. In synaptosome, it was mainly found in the synaptic cytosol.