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 359 毫秒
1.
S Huang  N P Wang  B Y Tseng  W H Lee    E H Lee 《The EMBO journal》1990,9(6):1815-1822
The retinoblastoma susceptibility gene (RB) encodes a phosphoprotein of 110 kd (pp110RB) that forms specific complexes with SV40 T antigen and the transforming proteins of several other DNA tumor viruses. Interaction with RB is thought to contribute to transformation by these viruses as demonstrated by genetic analyses. To help understand the function of these interactions, the regions of RB that are involved in binding to T have been mapped. An in vitro protein synthesis system capable of producing full-length RB protein has been developed to facilitate the mapping study. A 5- to 10-fold increase in translational efficiency in the reticulocyte lysate was obtained when the 5' non-coding region of RB mRNA was replaced with that of beta-globin mRNA or a plant viral RNA, alfalfa mosaic virus (AMV) RNA4. A series of mutated RB polypeptides produced from this system were assayed for T binding. Two non-contiguous regions of the RB protein, amino acid residues 394-571 and 649-773, were found to be necessary for binding to T: mutations in either region abolished T-RB complex formation. These results are consistent with the finding that, in all the cases analyzed so far, mutated RB proteins in human tumor cells also failed to bind to T antigen due to deletions including at least one of the two required regions. Thus the regions of RB defined in vitro as necessary for interaction with T might be physiologically relevant as well, and might play a fundamental role in normal RB protein function.  相似文献   

2.
The E2F transcription factor is a cellular target for the RB protein.   总被引:190,自引:0,他引:190  
  相似文献   

3.
The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.  相似文献   

4.
5.
Schmitt PT  Ray G  Schmitt AP 《Journal of virology》2010,84(24):12810-12823
Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.  相似文献   

6.
7.
8.
The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.  相似文献   

9.
The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.  相似文献   

10.
To assess biological roles of the retinoblastoma protein (RB), four independent transgenic mouse lines expressing human RB with different deletions in the N-terminal region (RBdeltaN) were generated and compared with mice expressing identically regulated, full-length RB. Expression of both RB and RBdeltaN caused developmental growth retardation, but the wild-type protein was more potent. In contrast to wild-type RB, the RBdeltaN proteins were unable to rescue Rb-/- mice completely from embryonic lethality. Embryos survived until gestational day 18.5 but displayed defects in the terminal differentiation of erythrocytes, neurons, and skeletal muscle. In Rb+/- mice, expression of the RBdeltaN transgenes failed to prevent pituitary melanotroph tumors but delayed tumor formation or progression. These results strongly suggest that N-terminal regions are crucial for embryonic and postnatal development, tumor suppression, and the functional integrity of the entire RB protein. Furthermore, these transgenic mice provide models that may begin to explain human families with low-penetrance retinoblastoma and mutations in N-terminal regions of RB.  相似文献   

11.
G D Parks 《Journal of virology》1994,68(8):4862-4872
The paramyxovirus large protein (L) and phosphoprotein (P) are both required for viral RNA-dependent RNA polymerase activity. Previous biochemical experiments have shown that L and P can form a complex when expressed from cDNA plasmids in vivo. In this report, L and P proteins of the paramyxovirus simian virus 5 (SV5) were coexpressed in HeLa T4 cells from cDNA plasmids, and L-P complexes were examined. To identify regions of the SV5 L protein that are required for L-P complex formation, 16 deletion mutants were constructed by mutagenesis of an SV5 L cDNA. Following coexpression of these L mutants with cDNA-derived P and radiolabeling with 35S-amino acids, cell lysates were analyzed for stable L-P complexes by a coimmunoprecipitation assay and by sedimentation on 5 to 20% glycerol gradients. Mutant forms of L containing deletions that removed as much as 1,008 residues from the C-terminal half of the full-length 2,255-residue L protein were detected in complexes with P by these two assays. In contrast, large deletions in the N-terminal half of L resulted in proteins that were defective in the formation of stable L-P complexes. Likewise, L mutants containing smaller deletions that individually removed N-terminal regions which are conserved among paramyxovirus and rhabdovirus L proteins (domain I, II, or III) were also defective in stable interactions with P. These results suggest that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions. SV5-infected HeLa T4 cells were pulse-labeled with 35S-amino acids, and cell extracts were examined by gradient sedimentation. Solubilized L protein was detected as an approximately 8 to 10S species, while the P protein was found as both a approximately 4S form (approximately 85%) and a species that cosedimented with L (approximately 15%). These data provide the first biochemical evidence in support of a simple domain structure for an L protein of the nonsegmented negative-sense RNA viruses. The results are discussed in terms of a structural model for the L protein and the interactions of L with the second viral polymerase subunit P.  相似文献   

12.
The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.  相似文献   

13.
14.
15.
Type C RNA viruses have been considered oncogenic because they are found associated with animal tumors and can induce cancers in several animal species. Those viruses that rapidly cause cancer appear to contain an oncogenic gene which resembles genetic sequences present in normal cells. This gene codes for a transforming protein which may be a normal cellular enzyme or a slightly altered cellular product. Its mechanism for transforming a cell is not yet known. Other oncogenic viruses, such as the chronic leukemia viruses, may not produce an oncogenic protein but may affect, by other means, specific target cells so they become malignant. Recent evidence now suggests that the majority of endogenous type C viruses are not transforming agents but inherited in the host to function in other biologic processes. These viruses do not contain transduced cellular genes which are responsible for cancer. Their role probably depends on their expression of other gene products which aid in normal development. These observations suggest that the ultimate control of human cancer may result from the identification of the oncogenic cellular-like genes transduced by some type C viruses even if a true human oncogenic virus is not isolated.  相似文献   

16.
Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinase-dependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.  相似文献   

17.
18.
The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein.  相似文献   

19.
The retinoblastoma susceptibility gene product, p105Rb (RB), is generally believed to be an important regulator in the control of cell growth, differentiation, and apoptosis. Several cellular factors that form complexes with RB and exert their cellular regulatory functions have been identified, such as the newly identified RB:cyclophilin A (CypA) complex. The physical interactions between RB and CypA were demonstrated by glutathione S-transferase affinity matrix binding assays and immunoprecipitation, followed by Western blot analyses. The N-terminal region of CypA mediated the interaction with RB, whereas the region upstream of the A-pocket of RB was required for binding to CypA. Ectopic expression of RB into Jurkat cells partially blocks the function of cyclosporin (CsA) to inhibit nuclear factor for activation of T cell (NFAT) activation by phorbol ester (PMA) plus ionomycin A (IA), suggesting that RB may prevent CsA inhibition of T lymphocyte activation. These results are further evidenced by the effect of RB on both calcineurin (CN) and NFAT binding activity in vitro, suggesting that the interaction of RB with CypA interferes with the CsA:CypA complex and blocks CsA-inhibited CN activity. These data reveal the functional link between RB and CypA and their involvement in T cell activation signaling.  相似文献   

20.
A mutational analysis of the bacteriophage P1 recombinase Cre   总被引:12,自引:0,他引:12  
Bacteriophage P1 encodes a 38,600 Mr site-specific recombinase, Cre, that is responsible for reciprocal recombination between sites on the P1 DNA called loxP. Using in vitro mutagenesis 67 cre mutants representing a total of 37 unique changes have been characterized. The mutations result in a wide variety of phenotypes as judged by the varying ability of each mutant Cre protein to excise a lacZ gene located between two loxP sites in vivo. Although the mutations are found throughout the entire cre gene, almost half are located near the carboxyl terminus of the protein, suggesting a region critical for recombinase function. DNA binding assays using partially purified mutant proteins indicate that mutations in two widely separated regions of the protein each result in loss of heparin-resistant complexes between Cre and a loxP site. These results suggest that Cre may contain two separate domains, both of which are involved in binding to loxP.  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