首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras   总被引:4,自引:0,他引:4  
A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.  相似文献   

2.
A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.  相似文献   

3.
Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras x RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.  相似文献   

4.
Substituting alanine for glycine at position 60 in v-H-Ras generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNp-bound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg(2+) ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A.GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.  相似文献   

5.
The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A′. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A′(Q72L) mutant form at 1.9 Å resolution. Unexpectedly, we found that Rab6A′(Q72L) possesses GDP/Mg2+ in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A′ can accommodate GDP/Mg2+ in the open conformation.  相似文献   

6.
The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.  相似文献   

7.
The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.  相似文献   

8.
Despite years of study, the structural or dynamical basis for the differential reactivity and oncogenicity of Ras isoforms and mutants remains unclear. In this study, we investigated the effects of amino acid variations on the structure and dynamics of wild type and oncogenic mutants G12D, G12V, and G13D of H‐ and K‐Ras proteins. Based on data from µs‐scale molecular dynamics simulations, we show that the overall structure of the proteins remains similar but there are important differences in dynamics and interaction networks. We identified differences in residue interaction patterns around the canonical switch and distal loop regions, and persistent sodium ion binding near the GTP particularly in the G13D mutants. Our results also suggest that different Ras variants have distinct local structural features and interactions with the GTP, variations that have the potential to affect GTP release and hydrolysis. Furthermore, we found that H‐Ras proteins and particularly the G12V and G13D variants are significantly more flexible than their K‐Ras counterparts. Finally, while most of the simulated proteins sampled the effector‐interacting state 2 conformational state, G12V and G13D H‐Ras adopted an open switch state 1 conformation that is defective in effector interaction. These differences have implications for Ras GTPase activity, effector or exchange factor binding, dimerization and membrane interaction. Proteins 2017; 85:1618–1632. © 2017 Wiley Periodicals, Inc.  相似文献   

9.
RalGDS is a guanine nucleotide dissociation stimulator for Ral, and one of its homologues is RGL (RalGDS-like). In this study, the effects of mutations of Ras and the Ras-binding domains (RBDs) of RalGDS and RGL on their binding have been systematically examined. The D33A mutation of Ras reduces the abilities to bind RGL-RBD and RalGDS-RBD. To identify the RGL residue interacting with Asp33 of Ras, double-mutant analyses between Ras and RGL-RBD were conducted. For example, the K685A mutation of RGL-RBD has a much smaller effect on the RGL-RBD binding ability of the D33A mutant than on those of other mutants of Ras. Accordingly, it is indicated that the attractive interaction of Asp33 in Ras with Lys685 in RGL-RBD (Lys816 in RalGDS-RBD) contributes to the Ras.RBD association. This interaction is consistent with the crystal structure of the complex of RalGDS-RBD and the E31K Ras mutant [Huang, L., Hofer, F., Martin, G. S., and Kim, S.-H. (1998) Nat. Struct. Biol. 5, 422-426]. This crystal structure exhibits interactions of the mutation-derived Lys31 side chain with three RalGDS residues. Glu31 of Ras discriminates Ras from a Ras-homologue, Rap1, with Lys31, with respect to RalGDS and RGL binding; the E31K mutation of Ras potentiates the abilities to bind RGL-RBD and RalGDS-RBD. To examine the role of Glu31 of the wild-type Ras in the interaction with RGL and RalGDS, double-mutant analyses were conducted. The Ras binding ability of the E689A mutant of RGL-RBD is much stronger than that of the wild-type RGL-RBD, and the E31K mutation of Ras no longer potentiates the Ras binding ability of the E689A mutant. Therefore, the repulsive interaction between Glu31 in Ras and Glu689 in RGL-RBD (Asp820 in RalGDS-RBD) may keep the Ras.RBD association weaker than the Rap1.RBD association, which might be relevant to the regulation of the signaling network.  相似文献   

10.
Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.  相似文献   

11.
Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.  相似文献   

12.
Zeng J  Treutlein HR  Simonson T 《Proteins》1999,35(1):89-100
The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.  相似文献   

13.
Here using structural information and protein design tools we have drawn the network of interactions between 20 Ras subfamily proteins with 50 putative Ras binding domains. To validate this network we have cloned six poorly characterized Ras binding domains (RBD) and two Ras proteins (RERG, DiRas1). These, together with previously described RBD domains, Ras and Rap proteins have been analyzed in 70 pull-down experiments. Comparing our interaction network with these and previous pull-down experiments (total of 150 cases) shows a very high accuracy for distinguishing between binders and non-binders ( approximately 0.80). Bioinformatics information was integrated to distinguish those in vitro interactions that are more likely to be relevant in vivo. We proposed several new interactions between Ras family members and effector domains that are of relevance in understanding the physiological role of these proteins. More broadly our results demonstrate that (domain-domain) interaction specificities between members of protein families can be accurately predicted using structural information.  相似文献   

14.
A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.  相似文献   

15.
Ras genes are evolutionary conserved and codify for a monomeric G protein binding GTP (active form) or GDP (inactive form). The ras genes are ubiquitously expressed although mRNA analysis suggests different level expression in tissue. Mutations in each ras gene frequently were found in different tumors, suggesting their involvement in the development of specific neoplasia. These mutations lead to a constitutive active and potentially oncogenic protein that could cause a deregulation of cell cycle. Ras protein moderates cellular responses at several mitogens and/or differentiation factors and at external stimuli. These stimuli activate a series of signal transduction pathways that either can be independent or interconnected at different points. Recent observations begin to clarify the complex relationship between Ras activation, apoptosis, and cellular proliferation. A greater understanding of these processes would help to identify the factors directly responsible for cell cycle deregulation in several tumors, moreover it would help the design of specific therapeutic strategies, for the control on the proliferation of neoplastic cells. We summarize here current knowledge of ras genes family: structural and functional characteristics of Ras proteins and their links with cell cycle and cancer.  相似文献   

16.
Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.  相似文献   

17.
BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms. RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region. CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.  相似文献   

18.
The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha- and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110gamma. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110gamma in vitro and in Sf9 cells, whereas the interaction with p110alpha is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110gamma and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110gamma.  相似文献   

19.
Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.  相似文献   

20.
The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号