The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5'-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the alpha-subunits of which possess molecular mass values of about 40 kDa.     

Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.     

The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000-17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.     

Identification of the human platelet GTPase activating protein for the CDC42Hs protein

The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP). The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified approximately 3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 25,000. The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP. However, a mutant form of the E. coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-12) which is not stimulated by the CDC42Hs-GAP. The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho. Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e. 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.     

A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1–G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30–34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.     

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.     

The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.     

Presence of small GTP-binding proteins in the peroxisomal membrane.

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.     

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.     

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.     

p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta-repeat domains characteristic of the beta-subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid-rich regions, a putative leucine zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy-terminal RCC1-like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1-like motif located at the amino-terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R-Ras2/TC21. These observations suggest that p619 is a Brefeldin A-sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.     

Small GTP-binding proteins in the nuclei of human placenta.

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [alpha(32)P]-GTP. Prior to this study the number of GTP-binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP-binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [alpha(32)P]-GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [alpha(32)P]-GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP-binding proteins. The GTP-binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP-binding proteins in the development and physiology of the placenta.     

Purification and characterization of a low M(r) GTP-binding protein, ram p25, expressed by baculovirus expression system.

The ram gene was isolated from rat megakaryocyte cDNA library with an oligonucleotide probe which is specific for a low M(r) GTP-binding proteins c25KG purified from human platelets. Its gene product (ram p25) is a monomeric 25-kDa guanine nucleotide-binding protein. The protein was expressed by using baculovirus transfer vector, pAcYM1, which allowed the production at a high level of soluble recombinant ram p25 in Spodoptera frugiperda (Sf9) cells under the control of polyhedrin promoter. The expressed protein in cytosol of Sf9 cells was purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. The purified ram p25 bound approx. 0.8 +/- 0.02 mol of guanosine 5'-O-1-thiotriphosphate (GTP gamma S)/mol of protein with a Kd value of 340 +/- 4.91 nM in a reaction mixture containing 10 microM of free magnesium ions. In the presence of 5 mM Mg2+, [3H]GDP was dissociated from ram p25 at the rate of 0.015 +/- 0.0010 min-1 and the dissociation was greatly enhanced by addition of 250 mM (NH4)2SO4. The rate of [gamma-32P]GTP-hydrolysis for ram p25 was 0.010 +/- 0.0012 min-1. Thus, it was indicated that the GTP-hydrolysis reaction is a rate-limiting step in the guanine nucleotide turnover of ram p25. ram p25 shares 23 and 80% amino-acid homology with the Ha-ras p21 and c25KG protein, respectively, and is similar to them in GTP gamma S binding activity in a time- and dose-dependent manner. But it differs from ras p21 in the rate-limiting step of the guanine nucleotide turnover.     

Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

We have purified, characterized, and identified two GTP-binding proteins with Mr of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the beta gamma subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low Mr GTP-binding proteins already reported. c25KG bound about 1 mol of [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 45 nM. [35S]GTP gamma S-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl beta, gamma-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate Pi with the specific activity of 1.8 mmol of Pi/mol of protein/min, which are different from the activities of the already purified low Mr GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.     

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.     

The Crithidia fasciculata CRK gene encodes a novel cdc2-related protein containing large inserts between highly conserved domains.

A gene (CRK) encoding a cdc2-related protein has been identified in the trypanosomatid Crithidia fasciculata. CRK has a high degree of sequence identity with the human cdc2 gene and contains the sixteen amino acid PSTAIR motif, characteristic of p34cdc2 protein-serine/threonine kinases, with four amino acid substitutions in the motif. In addition, two inserts of more than sixty amino acids have been found between conserved domains of this putative protein-serine/threonine kinase. CRK is a single copy gene and is expressed on a 3.8 kb mRNA. Anti-CRK antibodies detect a 53kDa protein in extracts of C.fasciculata in agreement with the size predicted from the nucleotide sequence of the cloned gene. These antibodies also recognize proteins of 48 and 60 kDa in extracts of the trypanosomatid Leishmania tarentolae. Antibodies against the human PSTAIR peptide detect the p34cdc2 protein in human nuclear extracts but fail to detect a 34 kDa protein in C.fasciculata extracts. These results suggest that novel higher molecular weight forms of the cdc2 protein family may be involved in cell cycle control in trypanosomes.     

Distinct guanine nucleotide binding and release properties of the three Gi proteins

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.     

Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys.

Rab proteins are membrane-bound prenylated GTP-binding proteins required for the targeted movement of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear the COOH-terminal sequence Cys-X-Cys (such as Rab3A) and Cys-Cys (such as Rab1A). This enzyme is designated Rab geranylgeranyl transferase (Rab GG transferase). At high salt concentrations, Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity. We purified Component B to apparent homogeneity and found that it contains two peptides of 60 and 38 kDa. The purified Rab GG transferase did not attach geranylgeranyl to p21H-ras-CVLL, which is prenylated by a GG transferase of the CAAX type that resembles the CAAX farnesyltransferase. Rab GG transferase was strongly inhibited by Zn2+, a cation that is absolutely required by farnesyltransferase. The Rab GG transferase was also inhibited by NaCl concentrations in excess of 100 mM. Together with previous data, the current findings indicate that mammalian cells possess at least three protein prenyltransferases (CAAX farnesyltransferase, CAAX GG transferase, and Rab GG transferase) that are specific for different classes of low molecular weight GTP-binding proteins and other proteins.