共查询到20条相似文献,搜索用时 15 毫秒
1.
The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21 总被引:82,自引:0,他引:82
G A Martin D Viskochil G Bollag P C McCabe W J Crosier H Haubruck L Conroy R Clark P O'Connell R M Cawthon 《Cell》1990,63(4):843-849
The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations. 相似文献
2.
GTPase activating proteins for the smg-21 GTP-binding protein having the same effector domain as the ras proteins in human platelets 总被引:5,自引:0,他引:5
T Ueda A Kikuchi N Ohga J Yamamoto Y Takai 《Biochemical and biophysical research communications》1989,159(3):1411-1419
Two proteins stimulating the GTPase activity of the smg-21 GTP-binding protein (smg p21) having the same effector domain as the ras proteins (ras p21s) are partially purified from the cytosol fraction of human platelets. These proteins, designated as smg p21 GTPase activating protein (GAP) 1 and 2, do not stimulate the GTPase activity of c-Ha-ras p21. The GAP activity for c-Ha-ras p21 is also detected in the cytosol fraction of human platelets. smg p21 GAP1 and 2 are separated from c-Ha-ras p21 GAP by column chromatographies. The activity of smg p21 GAP1 and 2 is killed by tryptic digestion or heat boiling. The Mr values of smg p21 GAP1 and 2 are similar and are estimated to be 2.5-3.5 x 10(5) by gel filtration analysis. These results indicate that there are two GAPs for smg p21 in addition to a GAP for c-Ha-ras p21 in human platelets. 相似文献
3.
Identification of the human platelet GTPase activating protein for the CDC42Hs protein 总被引:9,自引:0,他引:9
M J Hart K Shinjo A Hall T Evans R A Cerione 《The Journal of biological chemistry》1991,266(31):20840-20848
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. 相似文献
4.
Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s 总被引:17,自引:0,他引:17
Y Hata A Kikuchi T Sasaki M D Schaber J B Gibbs Y Takai 《The Journal of biological chemistry》1990,265(13):7104-7107
ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity. 相似文献
5.
B Rubinfeld G Wong E Bekesi A Wood E Heimer F McCormick P Polakis 《International journal of peptide and protein research》1991,38(1):47-53
Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras. 相似文献
6.
A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. 总被引:41,自引:8,他引:33
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M S Marshall W S Hill A S Ng U S Vogel M D Schaber E M Scolnick R A Dixon I S Sigal J B Gibbs 《The EMBO journal》1989,8(4):1105-1110
The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein. 相似文献
7.
S Hattori N Ohmi M Maekawa M Hoshino M Kawakita S Nakamura 《Biochemical and biophysical research communications》1991,177(1):83-89
Cellular fractionation of GTPase activating protein (GAP) activity using bovine cerebral cortex revealed that about half of GAP activity was found in membrane fraction. GAP activity of membrane was not solubilized with 0.5% (v/v) triton X-100 and was immunoprecipitated with antibody against carboxy-terminus of neurofibromatosis type 1 (NF1) gene product. In contrast, soluble GAP activity was precipitated with antibody against GAP but not with anti-NF1. These results suggest that NF1 gene product is a GTPase activating protein toward ras p21 with completely different intracellular distribution from that of GAP. 相似文献
8.
Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins 总被引:30,自引:0,他引:30
The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP. 相似文献
9.
A Kikuchi T Sasaki S Araki Y Hata Y Takai 《The Journal of biological chemistry》1989,264(16):9133-9136
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. 相似文献
10.
Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. 总被引:42,自引:22,他引:20
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M F Moran P Polakis F McCormick T Pawson C Ellis 《Molecular and cellular biology》1991,11(4):1804-1812
The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras. 相似文献
11.
M Hoshijima A Kikuchi M Kawata T Ohmori E Hashimoto H Yamamura Y Takai 《Biochemical and biophysical research communications》1988,157(3):851-860
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. 相似文献
12.
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. 相似文献
13.
14.
Differential regulation of cellular activities by GTPase-activating protein and NF1. 总被引:2,自引:2,他引:0
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
N al-Alawi G Xu R White R Clark F McCormick J R Feramisco 《Molecular and cellular biology》1993,13(4):2497-2503
The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies. 相似文献
15.
16.
We report the development of a quantitative assay for measuring SH2 domain binding in vitro. Using this assay we have analyzed the binding of purified recombinant SH2 domains from ras GTPase activating protein (GAP) and the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) to proteins from epidermal growth factor-stimulated and v-src-transformed cells. The purified recombinant SH2 domains from GAP and p85 bind to the tyrosine phosphorylated epidermal growth factor receptor with nanomolar affinities. Moreover, competition studies suggest that these two proteins bind to equivalent or overlapping sites on this receptor. In v-src-transformed cells the purified recombinant SH2 domains from GAP and p85 bind to distinct but overlapping sets of proteins. 相似文献
17.
Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells. 相似文献
18.
Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1 总被引:29,自引:0,他引:29
B Rubinfeld S Munemitsu R Clark L Conroy K Watt W J Crosier F McCormick P Polakis 《Cell》1991,65(6):1033-1042
The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines. 相似文献
19.
M Hayakawa T Hori S Shibamoto M Tsujimoto N Oku F Ito 《Archives of biochemistry and biophysics》1991,286(2):323-329
Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes. 相似文献
20.
The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae 总被引:49,自引:0,他引:49
Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo. 相似文献