Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b

Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences. A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets. An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from [3H]mevalonic acid in the insect/baculovirus system. This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.     

Isoprenylation of rap2 proteins in platelets and human erythroleukemia cells

The covalent modification of proteins by isoprenoid derivatives of mevalonic acid was investigated in human platelets, cells that lack the ability to synthesize endogenous cholesterol, and human erythroleukemia (HEL) cells, cholesterol-producing cultured cells derived from megakaryocytes. When washed platelets or HEL cells were incubated with [3H]mevalonic acid, the radiolabel was incorporated into a distinct group of proteins with molecular masses between 21,000 and 28,000. We have identified one of these proteins as a ras-related rap2 protein based on its immunoreactivity with a polyclonal antiserum raised against purified recombinant rap2b. This anti-rap2 antiserum was used for two-dimensional immunoblotting analysis and immunoprecipitation of mevalonate-labeled rap2 from platelets and HEL cells. These results suggest that rap2 may undergo a series of carboxyl-terminal modifications similar to the p21ras proteins. In addition, it is shown that non-cholesterol-producing cells are capable of incorporating isoprenyl groups into specific proteins.     

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.     

Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

Activation of human neutrophils involves the degranulation of specific and azurophil granules. This process is GTP-dependent and the presence of small GTP-binding proteins (SGBPs) has been detected in the two granule populations. At present, none of these SGBPs has been definitely identified. In order to characterize some of these proteins and obtain further insights as to their potential role in degranulation processes, we have used specific antibodies directed against the ras-related rap1 and rap2 proteins. By immunoblot analysis, we observed that rap2p is predominantly located in specific granules, whereas rap1p is detected both in specific granules and a fraction enriched in plasma membranes. Neither rap1p nor rap2p was found in the cytosol or in azurophil granules. Similarly, by indirect immunofluorescence, we observed that cytoplasmic granules were stained with anti-rap1p antibodies and anti-rap2p antibodies, and the plasma membrane was labeled with both antibodies but more distinctly with anti-rap1p than with anti-rap2p antibodies. rap1p and rap2p are tightly bound to the membrane of specific granules since they cannot be extracted by high salt or alkaline buffers. In addition, treatment of intact specific granules with pronase induced the degradation of rap proteins suggesting that they are exposed to the cytoplasmic face of the granules. Degranulation of neutrophils consists of the translocation and subsequent fusion of granules with the plasma membrane. Activation of this process induced the accumulation of rap proteins in the plasma membrane as observed by subcellular fractionation and indirect immunofluorescence experiments; this was not associated with the appearance of a soluble form of these proteins, showing that they remain membrane-bound during this process. The identification and subcellular localization of rap1p and rap2p at the surface of specific granules and the observation that they translocate to the plasma membrane upon cell stimulation without appearance of soluble forms constitute an important step toward the understanding of their physiological functions in human neutrophils.     

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.     

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.     

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.     

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.     

Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.     

Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate

Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study. Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-[methyl-3H]adenosylmethionine and platelet cytosol. About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions. Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-[methyl-3H]adenosylmethionine by 25%. These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-[methyl-3H]adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues. Protein methylation did not occur if membranes or cytosol alone was incubated with S-[methyl-3H]adenosylmethionine. Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner. Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins. On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins. The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.     

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.     

Functional expression of soluble ICAM-1 by baculovirus-infected Sf9 cells.

Inflammation, metastasis and ischemia are processes that require lymphocyte or leukocyte cell recognition and adherence to endothelial counter receptors such as ICAM-1. Mapping the sites of interaction of ICAM-1 with LFA-1, the receptor for ICAM-1 on lymphocytes, may lead to the design of novel inhibitors of inflammation or metastasis. To this end, recombinant soluble ICAM-1 cDNA was engineered into the baculovirus expression system, which is capable of expressing large amounts of proteins. These constructs were designed to contain a protein leader sequence so that the transfected insect cells would secrete the recombinant polypeptide into the culture media for ease of isolation. We engineered four constructs of ICAM-1 into the baculovirus system and obtained relatively high expression of two soluble forms of ICAM-1, a two domain and a five domain form. These truncated proteins were isolated and shown to promote adherence of HL-60 cells and Molt-4 cells. These recombinant soluble proteins also inhibited cell adherence to purified intact ICAM-1 isolated from K562 cells.     

Incorporation of a product of mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, non-equilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.

Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.     

Guanine nucleotide binding properties of rap1 purified from human neutrophils.

The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.     

Expression of intermediate filament proteins in TPA-induced MPC-11 and HL-60 cells.

Under normal culture conditions, the tumor cell lines MPC-11 and HL-60 exhibit high rates of proliferation and show a peculiar expression of intermediate filament proteins as they appear to synthesize only lamin B. A 48-h exposure of murine plasmacytomas MPC-11 to the phorbol ester TPA reduces their growth and induces vimentin synthesis without affecting the composition of their nuclear lamina. When applied to human leukemic promyelocytes HL-60, such treatment promotes their maturation into macrophage-like cells: their proliferative ability is suppressed, a differentiated phenotype is developed, and their content in intermediate filament proteins now includes vimentin and a full complement of lamins A, B, and C. In the present study, a kinetic analysis of vimentin and lamin A/C expression in relation to proliferation and differentiation has been performed in these two cellular systems. Proliferation rates of MPC-11 and HL-60 populations were evaluated by monitoring cell growth and measuring thymidine incorporation. Maturation of HL-60 cells was assessed by Giemsa staining and percentage of adherent cells. Expression of vimentin and lamins A/C was analyzed using immunofluorescence and immunoblotting techniques. Our data show that there is a relationship between the level of vimentin expression and the extent of growth inhibition in both systems. They also suggest that the expression of lamins A/C during the TPA-induced maturation of HL-60 promyelocytes might be part of the processes which lock these cells into the macrophage pathway.     

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.     

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

We have made a specific antiserum recognizing both smg p21A (the rap1A/Krev-1 protein) and -B (the rap1B protein), ras p21-like GTP-binding proteins having the same putative effector domain as ras p21s and have used this antiserum to study the tissue and subcellular distributions of smg p21s by immunoblot and immunocytochemical analyses. By immunoblot analysis, smg p21s were detected in various rat tissues and at the highest level in brain. By light microscopic immunocytochemical analysis, smg p21s were also detected in various rat tissues. Particularly, smg p21s in brain were found abundantly in the cytoplasmic region of most types of neuronal cell bodies and moderately in neuropil, whereas c-ras p21s were found more abundantly in neuropil than in the cytoplasmic region of most types of neuronal cell bodies. smg p21s in testis were found in spermatogenic cells, in which c-ras p21s were not significantly detected. By subcellular fractionation analysis of cerebrum, smg p21s were detected in all of the particulate fractions but not in the cytosol fraction. Among the particulate fractions, approximately 70% of smg p21s was recovered with the highest specific content in the fraction containing mainly synaptosomes, mitochondria, and myelin. In further fractionation of this fraction, approximately 40% of smg p21s was recovered in each of the synaptosome fraction and the mitochondrial fraction. This subcellular distribution of smg p21s in cerebrum was partly distinct from that of c-ras p21s, which were mainly recovered in the synaptosome and microsome fractions but present at very low levels in the mitochondrial fraction. These tissue and subcellular distributions of smg p 21s together with the fact that smg p21s have the same putative effector domain as ras p21s exert their own specific actions in addition to the actions similar or antagonistic to those of c-ras p21s.