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.     

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.     

rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.

Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.     

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.     

Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle.

The GDP/GTP exchange reaction of rho p21, a member of ras p21-related small GTP-binding protein superfamily, is regulated by two stimulatory GDP/GTP exchange proteins (GEPs), named smg GDS and rho GDS, and by one inhibitory GEP, named rho GDI. In bovine aortic smooth muscle, rho GDS and rho GDI were major GEPs for rho p21, and the rho GDI activity on the GDP/GTP exchange reaction of rho p21 was stronger than the rho GDS activity in their simultaneous presence. Moreover, in the crude cytosol, the GDP-bound form of rho p21 was complexed with rho GDI but not with rho GDS. These results, together with our recent finding that rho p21 is involved in the vasoconstrictor-induced Ca2+ sensitization of smooth muscle contraction, suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the stimulatory action of rho GDS, eventually leading to the rho p21 activation, in the signaling pathways of the vasoconstrictor receptors in smooth muscle.     

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.     

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.     

Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.     

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.     

Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.     

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.     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.     

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.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

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.     

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.     

A novel prenyltransferase for a small GTP-binding protein having a C-terminal Cys-Ala-Cys structure

smg p25A/rab3A p25 is a member of the small GTP-binding protein superfamily which is implicated in intracellular vesicle transport. smg p25A has a cDNA-predicted C-terminal structure of Cys-Ala-Cys. The protein purified from bovine brain membranes is geranylgeranylated at both the two cysteine residues and carboxyl-methylated at the C-terminal cysteine residue. Two types of prenyltransferase for small GTP-binding proteins have thus far been reported: ras p21 farnesyltransferase (ras p21 FT) and rhoA p21 geranylgeranyltransferase (rhoA p21 GGT). Neither of them geranylgeranylated smg p25A having a C-terminal Cys-Ala-Cys structure. In this paper, a smg p25A GGT was partially purified from bovine brain cytosol and separated from the ras p21 FT and rhoA p21 GGT by column chromatographies. smg p25A GGT transferred the geranylgeranyl moiety from geranylgeranyl pyrophosphate to both the two cysteine residues in the C-terminal Cys-Ala-Cys structure of smg p25A. smg p25A GGT did not use farnesyl pyrophosphate as a substrate and was also inactive on c-Ha-ras p21 and rhoA p21 with either farnesyl pyrophosphate or geranylgeranyl pyrophosphate as a substrate. These results indicate that there are at least three types of prenyltransferase for small GTP-binding proteins in mammalian tissues.     

Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products

We have purified to near homogeneity a Mr 22,000 GTP-binding protein from human platelet membranes and identified it as the smg-21 gene product (smg p21), having the same putative effector domain as the ras gene products, which we have purified to near homogeneity from bovine brain membranes and characterized. This purified human platelet smg p21 was phosphorylated by cyclic AMP-dependent protein kinase. About one mol of phosphate was maximally incorporated into one mol of the protein. Only serine residue was phosphorylated. Both the guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S)-bound and GDP-bound forms were phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affected neither its GTP gamma S-binding nor GTPase activity. Human platelet smg p21 was not phosphorylated by protein kinase C. A Mr 24,000 GTP-binding protein partially purified from human platelet membranes was not phosphorylated by cyclic AMP-dependent protein kinase or protein kinase C.