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Results for “Retinoblastoma Protein”

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At least 19 recordsLinked to original sources
PubMed1999

Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein.

PURPOSE: To identify proteins that physically interact with Pax-6, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development. METHODS: Protein-protein interactions involving Pax-6, TATA-box-binding protein (TPB), and retinoblastoma protein were studied using affinity chromatography with Pax-6 as ligand, glutathione-S-transferase (GST) pull-down assays, and immunoprecipitations. RESULTS: The authors have shown that Pax-6 is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others have shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblastoma protein. In the present study it was shown that Pax-6 interacted with the TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-6 HD, with a substantial role for its N-terminal arm and first two alpha-helices. The experiments also indicated a binding role for the C-terminal-activation domain of the protein. In addition, the present study showed that the HD of Pax-6 interacted with retinoblastoma protein. Immunoprecipitation experiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts. CONCLUSIONS: Blending the present results with those in the literature suggests that Pax-6 and retinoblastoma protein participate in overlapping regulatory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.

Amino Acid Sequence↗
PubMed1992

Proposed interaction between insulin and retinoblastoma protein.

Retinoblastoma protein (RB) is a tumor suppressor gene product involved in embryogenesis and cell cycle progression. One of the major mechanisms leading to RB dysfunction is complex formation with viral oncoproteins using the common RB binding motif Leu X Cys X Glu (LXCXE) which has also been identified in cellular ligands, e.g., RBP-1 and RBP-2. p107, a cellular protein with RB sequence homology, has been shown to bind to the same viral oncoproteins associating with RB and is therefore thought to contribute to cell cycle regulation. It has recently been suggested that insulin stimulates gene transcription through direct association with an, as yet, unidentified intracellular transcription factor. Due to the central roles of RB and p107 in coupling external growth signals with the progression of the cell cycle clock, we have hypothesized that these two proteins might be candidates for mediating the effects of insulin on DNA. We report here the identification of a region in the B-chain of human insulin that has the sequence LXCXE. Based on this finding we predict that the insulin B-chain may interact with RB and/or p107. Since we have also identified sequences hydropathically related to LXCXE in insulin-like growth factor I (IGF-I) and II (IGF-II), but not in relaxin, nerve growth factor, epidermal growth factor, glucagon or beta-endorphin, we further propose that both IGF-I and -II may assemble with RB and/or p107, too. Moreover, binding sites on RB and p107 identical with those suggested for viral oncoproteins and cellular ligands are predicted for insulin/IGF-I/IGF-II by using the hydropathic complementarity approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Hyperphosphorylation of retinoblastoma protein and p53 by okadaic acid, a tumor promoter.

A potent tumor promoter, okadaic acid, induced hyperphosphorylation of tumor suppressor proteins, retinoblastoma protein and p53, by in vitro incubation with nuclei isolated from rat regenerating liver as well as by incubation with primary human fibroblasts. Most of the retinoblastoma protein migrated to a hyperphosphorylated position in electrophoresis. The phosphorylation of p53 was increased at a rate 8 times that in non-treated primary human fibroblasts. Hyperphosphorylation of tumor suppressor proteins, mediated through inhibition of protein phosphatases 1 and 2A, is involved in tumor promotion by okadaic acid. The significance of hyperphosphorylation of the retinoblastoma protein and p53 is discussed in relation to the regulation of the cell cycle.

Animals↗
PubMed1998

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases.

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.

Adenomatous Polyposis Coli Protein↗
PubMed1996

A melanoma cell line sensitive to expression of a fusion protein binding the retinoblastoma protein family pocket domain.

An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta-galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells.

Amino Acid Sequence↗
PubMed2002

Analysis of putative interactions between potyviral replication proteins and plant retinoblastoma proteins.

Sequence comparisons suggest that the RNA-dependent RNA polymerase (NIb) of potyviruses and bymoviruses, as well as the viral polymerase of potexviruses may contain a putative retinoblastoma protein (pRb) binding motif. The possibility that the potyviral NIb may function in the nucleus through interactions with plant pRb-related (RBR) proteins, and the modifications of the cell cycle was investigated by a combination of mutagenesis of the NIb and yeast two-hybrid system (YTHS). Mutation of a highly conserved glutamic acid residue in the putative pRb-binding motif of the NIb had no detectable phenotypic effect on replication of Potato virus A (PVA). Furthermore, the NIb proteins from Potato virus V and PVA failed to interact with maize or tobacco RBR proteins in yeast. Although the conservation of the motif for pRb interaction in plant RNA viruses is intriguing, these proteins from plant RNA viruses appear not to interact with plant RBR proteins.

DNA-Directed RNA Polymerases↗
PubMed1999

Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity.

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase alpha. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase alpha synergistically. B23 exists in two isoforms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase alpha from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase alpha activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase alpha/retinoblastoma protein/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase alpha in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein.

Animals↗
PubMed2001

Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells.

Phosphorylation of the tumor suppressor protein, retinoblastoma (pRb), regulates the progression of the cell cycle. Previous work from this laboratory had shown that estradiol (E(2)) regulates tumor suppressor proteins, p53 and retinoblastoma in breast cancer cells. In the present study, we have examined the phosphorylation of pRB in T47D breast cancer cells following treatments with R5020 and antiprogestins. In growth medium containing serum depleted of endogenous steroids by charcoal treatment, pRb appeared mainly in its hypophosphorylated form. Addition of 10 nM R5020 to the culture medium caused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated form of pRb began to accumulate after 72 h. Upon prolonged R5020 treatment (72-96 h), pRb was detected exclusively in its hypophosphorylated form. While treatment of cells with R5020 caused a transient increase in the level of cyclin D1, E(2) addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation. Antagonists of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E(2) and R5020-induced pRb phosphorylation, respectively. These results suggest that R5020 induces pRb phosphorylation via a transient increased expression of cyclin D1, whereas E(2) treatment results in sustained expression of cyclin D1 and increased pRb phosphorylation. Furthermore, R5020 effects on pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phosphorylation was observed: the R5020 effects were blocked by RU486 and ZK98299, but not by the pure ER antagonist, ICI 182, 780 (ICI).

Breast Neoplasms↗
PubMed1999

Functions of the retinoblastoma protein.

The retinoblastoma protein (pRB) can both positively and negatively regulate transcription. The former correlates with its ability to promote differentiation and the latter with its ability to regulate entry into S-phase. pRB negatively regulates transcription by forming complexes with members of the E2F transcription factor family. These complexes, when bound to E2F sites within certain target genes, actively repress transcription through a variety of mechanisms including physical interaction with adjacent transcriptional activation domains and recruitment of proteins that directly, or indirectly, lead to histone deacetylation. pRB function is, in turn, modulated by phosphorylation mediated by cyclin-dependent kinases. Emerging data suggest that combinatorial control of pRB function may be achieved through the use of different phosphoacceptor sites, different cyclin/cdk docking sites, and different cyclin/cdk complexes. The untimely activation of E2F responsive genes can induce apoptosis. This comes about at least partly through the induction of ARF, which leads to the stabilization and activation of p53. BioEssays 1999;21:950-958.

Cell Cycle↗
PubMed1997

Formation of the early-region-2 transcription-factor-1-retinoblastoma-protein (E2F-1-RB) transrepressor and release of the retinoblastoma protein from nuclear complexes containing cyclin A is induced by interferon alpha in U937V cells but not in interferon-alpha-resistant U937VR cells.

We have analysed the different regulation of cell-cycle-relevant proteins by interferon alpha (IFN alpha) in IFN alpha-sensitive and resistant U937 leukemic cell lines. In contrast to the INF alpha-sensitive U937 variant cell line U937V, the IFN alpha-resistant derivative (U937VR) is insensitive to the antiproliferative activity of IFN alpha. As we found no differences between these cell lines concerning the induction by IFN alpha of the pathway involving tyrosine-protein kinases and the signal transducer and activator of transcription (Jak-Stat), we examined whether cell-cycle-regulating proteins are differently affected by IFN alpha in U937VR and U937VR cells. In U937V cells IFN alpha induced the formation of the complex between early-region-2 transcription factor 1 (E2F-1) and retinoblastoma protein (RB) which is known to repress transcription of E2F-1-inducible genes, necessary for cell cycle progression. Formation of this complex was not inducible by IFN alpha in U937VR cells, although the suitable binding partners (E2F-1 and under-phosphorylated RB) were present. Interestingly, treatment of nuclear extracts from logarithmically growing U937V and U937VR cells with an antiserum against cyclin A that disrupts cyclin-A-containing complexes, led to the formation of the E2F-1-RB complex, suggesting the presence of under-phosphorylated (active) RB, trapped in nuclear complexes that contain cyclin A. This suggestion was supported by combined immunoprecipitation/western blot experiments that revealed a physical interaction between phosphorylated as well as under-phosphorylated forms of RB and cyclin A complex(es) in U937V and U937VR cells. RB, especially the under-phosphorylated form, was released by treatment with IFN alpha from this complex(es) in the case of U937V cells but not U937VR cells. We conclude that the missing induction of the E2F-1-RB transrepressor by IFN alpha and the failure to release RB from cyclin-A-containing complexes might contribute to the resistance of U937VR cells to the antiproliferative effects of IFN alpha.

Antibodies↗
PubMedDate not supplied

Decreased retinoblastoma protein expression in gamma-irradiated mouse ovarian follicles.

We immunohistochemically examined the effect of gamma-radiation on immature mouse ovarian follicles. Mice were Jirradiated with a dose of LD80 (8.3 Gy) in KAERI. At 0 h, 6 h, 12 h, 1 d, 2 d, 4 d and 8 d postirradiation, the ovaries were excised and fixed in neutral buffered formalin. We performed immunohistochemistry for protein retinoblastoma (pRb), terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and routine hematoxylin-eosin staining in the largest cross sections. Radiation-induced follicular degeneration increased before 6 h, and most irradiated ovarian follicles became acutely atretic. The immunohistochemical staining for pRb was strong in the nuclei of granulosa cells of normal follicles and weak in atretic ones which were, conversely, strong for TUNEL staining. It was shown that pRb expression became lower with the degeneration of the ovarian follicles, which was inhibited by gamma-radiation. In the present study, pRb immunohistochemistry was proven to be a useful tool for the identification of follicular status.

Animals↗
PubMed1993

Transcriptional inhibition by the retinoblastoma protein.

The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The underphosphorylated form is able to interact with the E2F transcription factor. Recently, we have cloned a cDNA for E2F-1. By using this clone and a series of non-pRB binding mutants, we have been able to show that the binding of pRB to E2F-1 causes inhibition of E2F-mediated transactivation. pRB's inhibition of E2F-mediated transcription would be lost by mutation in the retinoblastoma gene in human tumours, by pRB's interaction with DNA tumour virus oncoproteins, or by phosphorylation during the cell cycle.

Adenoviruses, Human↗
PubMed1994

Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein.

The human cytomegalovirus major immediate-early (IE) proteins play an indispensable role in regulating viral gene expression. One of these gene products, the IE2 86-kDa protein (IE2 86), is a potent activator of both homologous and heterologous promoters and can form a complex with a component of the basal transcription apparatus, the TATA box-binding protein (TBP). In this report, we show that when IE2 86 is expressed as a glutathione S-transferase (GST)-IE2 86 fusion protein, there are three independent regions that can interact with TBP and with another important cellular regulatory protein, the retinoblastoma gene product (RB). One of these three regions, as well as a domain at the carboxy terminus, contain consensus sites for casein kinase phosphorylation and negatively regulate binding of in vitro-translated IE2 86 to GST-TBP or GST-RB. The dimerization domain of IE2 86 must be present for the interaction of the in vitro-translated protein with GST-TBP and GST-RB. Analysis of IE2 86 mutants in vivo demonstrates that one of the strong binding regions is required for the protein to function as a transactivator. Our results also indicate that domains other than those that interact with TBP and RB are required for the activation function of this protein.

Astrocytoma↗
PubMed1993

Inhibition of histone H1 kinase expression, retinoblastoma protein phosphorylation, and cell proliferation by the phosphatase inhibitor okadaic acid.

Phosphorylation events are major regulatory mechanisms of signal transduction pathways that regulate gene expression and cell growth. To study the potential involvement of serine-threonine specific phosphatases in these processes we used okadaic acid (OA), an inhibitor of type 1 and type 2A protein phosphatases. Here we present evidence that OA arrests cells at defined points in the cell cycle. Concomitantly, expression and associated histone H1 kinase activity of cdc2 and cyclin A, two cell cycle regulatory proteins, are repressed by this agent. Furthermore, phosphorylation of the tumor suppressor protein retinoblastoma, an event thought to be necessary in order to permit cells to proliferate, is inhibited when OA is present. These effects are fully reversible since removal of OA restores cdc2 and cyclin A expression as well as histone H1 kinase activity, and the cells resume growth. Since cdc2 and cyclin A have previously been shown to be absolutely required for cell cycle progression it is likely that blockage of synthesis of these components contributes to the cytostatic effects of OA. Furthermore, our results suggest a positive role for OA sensitive protein phosphatases in the regulation of expression of these cell cycle regulatory proteins.

3T3 Cells↗
PubMed1995

The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein.

The retinoblastoma protein (Rb) is a target of viral oncoproteins. To explore the hypothesis that viral proteins may be structural mimics of cellular proteins, we have searched cDNA libraries for Rb-binding proteins. We report here the cloning of a cDNA for the protein RIZ from rat and human cells. RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs. It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A. A domain is conserved between RIZ and the PRDI-BF1/Blimp-1 differentiation factor. Other motifs of RIZ include putative GTPase and SH3 (src homology domain 3) domains. RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues. It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined. While the function of RIZ is not yet clear, its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma.

Adenovirus E1A Proteins↗
PubMed1997

A thyroid hormone receptor coactivator negatively regulated by the retinoblastoma protein.

The retinoblastoma protein (Rb) plays a critical role in cell proliferation, differentiation, and development. To decipher the mechanism of Rb function at the molecular level, we have systematically characterized a number of Rb-interacting proteins, among which is the clone C5 described here, which encodes a protein of 1,978 amino acids with an estimated molecular mass of 230 kDa. The corresponding gene was assigned to chromosome 14q31, the same region where genetic alterations have been associated with several abnormalities of thyroid hormone response. The protein uses two distinct regions to bind Rb and thyroid hormone receptor (TR), respectively, and thus was named Trip230. Trip230 binds to Rb independently of thyroid hormone while it forms a complex with TR in a thyroid hormone-dependent manner. Ectopic expression of the protein Trip230 in cells, but not a mutant form that does not bind to TR, enhances specifically TR-dependent transcriptional activity. Coexpression of wild-type Rb, but not mutant Rb that fails to bind to Trip230, inhibits such activity. These results not only identify a coactivator molecule that modulates TR activity, but also uncover a role for Rb in a pathway that responds to thyroid hormone.

Amino Acid Sequence↗
PubMed2000

RBP95, a novel leucine zipper protein, binds to the retinoblastoma protein.

We recently identified a novel cDNA encoding a retinoblastoma protein (pRb)-associated protein. It was named RBP95, which was composed of 838 amino acid residues with a calculated molecular size of 94,789 Da. Northern blot analysis showed a single mRNA of about 4. 5 kb ubiquitously expressed in human tissues. RH mapping results showed that RBP95 is mapped to chromosome region 16p11.2-11.1. Sequence analysis indicated that RBP95 contains a conserved pRb-binding motif LXCXE. Interaction between pRb and RBP95 was confirmed in vivo and in vitro. This interaction requires the LXCXE motif of RBP95 and the entire pocket region of pRb. Each point-mutant of the conserved amino acid residues in pRb-binding motif of RBP95 would destroy its interaction with pRb. RBP95 also contains a basic region leucine zipper and could homodimerize through its leucine zipper region. RBP95 was located in the nucleus with a special pattern when expressed as a GFP fusion in HeLa cells. All these findings suggested that RBP95, a new member of pRb-associated protein, may function as a regulation factor in the process of RNA polymerase II-mediated transcription and/or transcriptional processing.

Amino Acid Sequence↗
PubMed1996

Regulation of cyclin and cyclin-dependent kinase gene expression during lens differentiation requires the retinoblastoma protein.

The retinoblastoma protein (pRb) functions as a negative regulator of the cell cycle and is essential to maintain certain cell types in a post-mitotic state during terminal differentiation. In the ocular lens, inactivation of this protein is sufficient to cause lens fiber cells, which are normally post-mitotic, to enter the cell cycle. The current studies address whether regulation of the cell cycle during lens fiber differentiation in normal lenses or in lenses in which pRB has been inactivated is accompanied by changes in expression of cyclin and cyclin-dependent kinase genes. In the normal lens, our experiments using in-situ hybridization reveal that the expression of cyclin A, cyclin B1, cdc2 and cdk2 is restricted to the proliferative epithelial cells, with no expression in the differentiating fiber cells. Cyclins D1 and D2 and cdk4 show a less restrictive pattern and are expressed in some of the post-mitotic cells. Lenses from RB-deficient embryos, in contrast, show inappropriate expression in the fiber cells of cyclins A, B1 and E, as well as cdc2 and cdk2. The lens fiber cells in these embryos express protein markers for differentiation, such as beta- and gamma-crystallins, even though the cells do not withdraw from the cell cycle. These results indicate that the regulated expression of multiple cell cycle regulatory genes during lens fiber cell differentiation requires the presence of pRb.

Animals↗