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1.
Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product 总被引：33，自引：0，他引：33
M Frech J John V Pizon P Chardin A Tavitian R Clark F McCormick A Wittinghofer 《Science (New York, N.Y.)》1990,249(4965):169-171
Krev-1 is known to suppress transformation by ras. However, the mechanism of the suppression is unclear. The protein product of Krev-1, Rap1A-p21, is identical to Ras-p21 proteins in the region where interaction with guanosine triphosphatase (GTPase) activating protein (GAP) is believed to occur. Therefore, the ability of GAP to interact with Rap1A-p21 was tested. Rap1A-p21 was not activated by GAP but bound tightly to GAP and was an effective competitive inhibitor of GAP-mediated Ras-GTPase activity. Binding of GAP to Rap1A-p21 was strictly guanosine triphosphate (GTP)-dependent. The ability of Rap1A-p21 to bind tightly to GAP may account for Krev-1 suppression of transformation by ras. This may occur by preventing interaction of GAP with Ras-p21 or with other cellular proteins necessary for GAP-mediated Ras GTPase activity. 相似文献
2.
Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain 总被引：97，自引：0，他引：97
A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein. 相似文献
3.
The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids 总被引：28，自引：0，他引：28
Bacterially synthesized c-Ha-ras protein (Ras) was incubated with guanosine triphosphatase (GTPase) activating (GA) protein in the presence of various phospholipids. The stimulation of Ras GTPase activity by GA protein was inhibited in some cases. Among the lipids most active in blocking GA protein activity were lipids that show altered metabolism during mitogenic stimulation. These included phosphatidic acid (containing arachidonic acid), phosphatidylinositol phosphates, and arachidonic acid. Other lipids, including phosphatidic acid with long, saturated side chains, diacylglycerols, and many other common phospholipids, were unable to alter GA protein activity. The interaction of lipids with GA protein might be important in the regulation of Ras activity during mitogenic stimulation. 相似文献
4.
Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras. A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase. Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off. With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP. The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid. Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory. These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism. 相似文献
5.
Binding of GAP to activated PDGF receptors 总被引：93，自引：0，他引：93
The ras proto-oncogene products appear to relay intracellular signals via the Ras guanosine triphosphatase (GTPase) activator protein, GAP. In dog epithelial cells expressing human platelet-derived growth factor (PDGF) receptors, binding of PDGF caused approximately one-tenth of the total GAP molecules to complex with the receptor. Studies with mutant PDGF receptors showed that maximum association required both receptor kinase activity and phosphorylatable tyrosine residues at both the identified sites of receptor autophosphorylation. 相似文献
6.
Plexins are cell surface receptors for semaphorin molecules, and their interaction governs cell adhesion and migration in a variety of tissues. We report that the Semaphorin 4D (Sema4D) receptor Plexin-B1 directly stimulates the intrinsic guanosine triphosphatase (GTPase) activity of R-Ras, a member of the Ras superfamily of small GTP-binding proteins that has been implicated in promoting cell adhesion and neurite outgrowth. This activity required the interaction of Plexin-B1 with Rnd1, a small GTP-binding protein of the Rho family. Down-regulation of R-Ras activity by the Plexin-B1-Rnd1 complex was essential for the Sema4D-induced growth cone collapse in hippocampal neurons. Thus, Plexin-B1 mediates Sema4D-induced repulsive axon guidance signaling by acting as a GTPase activating protein for R-Ras. 相似文献
7.
Scholich K Mullenix JB Wittpoth C Poppleton HM Pierre SC Lindorfer MA Garrison JC Patel TB 《Science (New York, N.Y.)》1999,283(5406):1328-1331
The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors. 相似文献
8.
Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1 总被引：13，自引：0，他引：13
Krev-1 is an anti-oncogene that was originally identified by its ability to induce morphologic reversion of ras-transformed cells that continue to express the ras gene. The Krev-1-encoded protein is structurally related to Ras proteins. The biological activities of a series of ras-Krev-1 chimeras were studied to test the hypothesis that Krev-1 may directly interfere with a ras function. The ras-specific and Krev-1-specific amino acids immediately surrounding residues 32 to 44, which are identical between the two proteins, determined whether the protein induced cellular transformation or suppressed ras transformation. Because this region in Ras proteins has been implicated in effector function, the results suggest that Krev-1 suppresses ras-induced transformation by interfering with interaction of Ras with its effector. 相似文献
9.
Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans 总被引：59，自引：0，他引：59
W R Schafer R Kim R Sterne J Thorner S H Kim J Rine 《Science (New York, N.Y.)》1989,245(4916):379-385
The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 相似文献
10.
Inhibition of serum- and ras-stimulated DNA synthesis by antibodies to phospholipase C 总被引：17，自引：0，他引：17
Several immunologically distinct isozymes of inositol phospholipid-specific phospholipase C (PLC) have been purified from bovine brain. Murine NIH 3T3 fibroblasts were found to express PLC-gamma, but the expression of PLC-beta was barely detectable by radioimmunoassay or protein immunoblot. A mixture of monoclonal antibodies was identified that neutralizes the biological activity of both endogenous and injected purified PLC-gamma. When co-injected with oncogenic Ras protein or PLC-gamma, this mixture of antibodies inhibited the induction of DNA synthesis that characteristically results from the injection of these proteins into quiescent 3T3 cells. However, when oncogenic Ras protein or PLC-gamma was co-injected with a neutralizing monoclonal antibody to Ras, only the DNA synthesis induced by the Ras protein was inhibited--that induced by PLC was unaffected. These results suggest that the Ras protein is an upstream effector of PLC activity in phosphoinositide-specific signal transduction and that PLC-gamma activity is necessary for Ras-mediated induction of DNA synthesis. 相似文献
11.
A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants 总被引：143，自引：0，他引：143
The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state. 相似文献
12.
[目的]综述小G蛋白家族的研究进展,为更深入地解析小G蛋白的结构、功能及其分子作用模式提供参考。[方法]介绍了小G蛋白的结构、作用机制以及分类,着重综述了其各个亚家族的生物学功能。[结果]小G蛋白是真核生物的1个超基因家族,其成员超过100个,被分为Ras、Rho、Rab、Sar/Arf和Ran5个亚家族,分别参与细胞骨架组装、基因表达、细胞壁合成、囊泡运输、核质运输、微管形成、酵母出芽、纺锤体组装及细胞极性生长等诸多生命体活动过程。[结论]小G蛋白家族成员庞大,其分子作用机制及其调控的复杂网络有待进一步研究。 相似文献
13.
Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors 总被引：96，自引：0，他引：96
D Anderson C A Koch L Grey C Ellis M F Moran T Pawson 《Science (New York, N.Y.)》1990,250(4983):979-982
Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways. 相似文献
14.
KUTSKY RJ 《Science (New York, N.Y.)》1959,129(3361):1486-1487
A new method has been developed for removing the nucleic acid portion of the nucleoprotein fraction which stimulates growth in tissue culture. Biological activity resides in the purified protein fraction, while the high-polymer nucleic acid fraction is inert. The active protein fraction contains extractable lipids which have no effect on the biological activity. 相似文献
15.
mdm2（murine double minute 2,鼠双微粒体-2）是抑制p53（肿瘤抑制因子）活性的重要分子之一,它们之间相互作用对细胞的生存具有重要功能。克隆得到小鼠Mus musculus mdm2 cDNA,构建其真核表达载体p3xFLAG-CMV-7.1-mdm2,Western blotting验证其在真核细胞中的表达大约为60 kDa的蛋白表达产物,间接免疫荧光技术检测mdm2的表达和亚细胞定位,mdm2主要在细胞核内表达。利用p53蛋白泛素化试验和p53报告基因活性检测试验,验证了FLAG-mdm2的活性,构建了mdm2调节p53泛素化和抑制p53活性的细胞模型,为研究p53活性调控、mdm2与p53相互作用等提供了基础和研究手段。图5参15 相似文献
16.
17.
M Trahey G Wong R Halenbeck B Rubinfeld G A Martin M Ladner C M Long W J Crosier K Watt K Koths 《Science (New York, N.Y.)》1988,242(4886):1697-1700
The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP. The messenger RNA of this GAP was detected in human lung, brain, liver, leukocytes, and placenta. The second type appeared to be generated by a differential splicing mechanism and encoded a novel form of GAP with a predicted molecular mass of 100,400 daltons. This protein lacks the hydrophobic amino terminus characteristic of the larger species, but retains GAP activity. The messenger RNA of this type was abundantly expressed in placenta and in several human cell lines, but not in adult tissues. 相似文献
18.
为明确枯草芽孢杆菌(Bacillus subtilis)G87抗菌蛋白的生物活性,采用菌丝生长、分生孢子萌发和芽管伸长抑制的方法,研究其对稻瘟病菌(Magnaporthe grisea)和稻恶苗病菌(Gibberella fujikuroi)的抑制作用。结果表明:抗菌蛋白能明显抑制2种病菌菌丝生长、分生孢子萌发和芽管伸长。1.20mg·mL-1抗菌蛋白对稻瘟病菌菌落直径和菌丝干重的抑制率分别达60%和80%以上;2.40mg·mL-1抗菌蛋白对稻恶苗病菌分生孢子萌发和芽管伸长的抑制率均达60%以上。抗菌蛋白还能显著破坏2种病菌菌丝形态,使菌丝细胞畸形膨大、原生质浓缩和外渗、细胞壁破损以及菌体崩溃等。因此,枯草芽孢杆菌G87抗菌蛋白具有重要的抗菌生物活性。 相似文献
19.
Cordenonsi M Montagner M Adorno M Zacchigna L Martello G Mamidi A Soligo S Dupont S Piccolo S 《Science (New York, N.Y.)》2007,315(5813):840-843
During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program. 相似文献
20.
为确定树舌多糖在小鼠脾淋巴细胞上的定位,应用胺化还原法在树舌多糖(GAP)的还原性末端连接异硫氰酸荧光素(FITC),用荧光分光光度计测定其荧光取代度,xCELLigence RTCA DP全自动实时细胞分析仪检测GAP荧光标记前后生物学稳定性.流式细胞仪检测小鼠脾T淋巴细胞、B淋巴细胞和NK细胞中的GAP荧光强度,荧光显微镜观察GAP在小鼠脾淋巴细胞中的定位.结果显示:在荧光标记GAP (FITC-GAP)前后的细胞毒活性不变,并且其荧光取代率为0.90％;流式细胞仪检测发现小鼠脾T淋巴细胞、B淋巴细胞和NK细胞上的荧光信号强度与FITC-GAP加入量成正相关,荧光显微照片显示GAP定位于小鼠脾淋巴细胞表面并可转运至细胞核. 相似文献