Retinoblastoma-related Protein Research Articles

Retinoblastoma-related proteins in plants: homologues or orthologues of their metazoan counterparts?

The mammalian retinoblastoma tumor suppressor protein (pRb) regulates cell division, differentiation and apoptotic pathways in specific cell types. In association with other proteins, pRb acts in part by modulating transcriptional activity. Elements of the pRb regulatory network have been identified in higher plants. Recent findings involving these proteins, which display amino acid sequence homology and biochemical binding properties analogous to their mammalian counterparts, are discussed.

Geminiviruses and the plant cell cycle.

Geminiviruses are plant DNA viruses with a small genome that infect a large variety of plant species. Viral proteins regulate viral DNA replication and transcription. Also they appear to have an impact on cellular gene expression. Cellular proteins directly involved in DNA replication, such as PCNA, have long been known to accumulate in cells expressing Rep tomato golden mosaic geminivirus. This effect can be a direct effect of the viral protein and/or be mediated by interference with the G1/S transition control, namely the pathway controlled by the retinoblastoma-related (RBR) protein, analogous to the human retinoblastoma (RB) tumour suppressor protein. Different geminiviruses seem to have evolved two mechanisms to interact with plant RBR proteins. One is dependent on a LxCxE amino acid motif present in proteins, such as RepA, encoded by members of the Mastrevirus genus, and another seems to be mediated by the viral Rep protein, which lacks the LxCxE motif, encoded by members of the Begomovirus, and perhaps the Curtovirus genus. These and other aspects of the relationships between geminivirus replication and cell cycle control pathways will be discussed.

A Methylation-responsive MDBP/RFX Site Is in the First Exon of the Collagen α2(I) Promoter

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Rapid dephosphorylation of p107 following UV irradiation.

In response to UV irradiation, mouse NIH3T3 fibroblasts transiently arrest predominantly in the G1 phase of the cell cycle. Here, we investigate the role of the retinoblastoma-related pocket proteins in this biological process. We report here that UV induces an increase in p107/E2F complexes, shown previously to be repressors of E2F-dependent transcriptional activity. Several lines of evidence indicate that the increase of p107/E2F complexes following UV irradiation is a consequence of rapid dephosphorylation of p107. First, UV-mediated p107 dephosphorylation could be abolished by pretreatment of NIH3T3 fibroblasts with the serine/threonine phosphatase inhibitors calyculin A and okadaic acid. Second, alteration of protein phosphatase 2A holoenzyme composition by over-expression of specific B subunits interfered with UV-mediated dephosphorylation of p107. Consistent with this, p107 could be dephosphorylated in vitro with PP2A. Moreover, dephosphorylation of p107 was shown to be independent of the activity of p53 and p21, as it occurred also in UV-treated p53-null as well as p21-null mouse fibroblasts. We observed a close correlation between the UV dosages required for G1 cell cycle arrest and p107 dephosphorylation. Our data suggest a model in which UV radiation-induced cell cycle arrest depends, at least in part, on the induction of a PP2A-like phosphatase that acts on p107.

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein.

The proteins of the retinoblastoma family are potent inhibitors of cell cycle progression. It is well documented that their growth-inhibitory activity can be abolished by phosphorylation on serine and threonine residues by cyclin dependent kinases. In contrast, very little is known about the dephosphorylation of retinoblastoma-family proteins. We report here the isolation, by virtue of its ability to associate with p107, of a novel Protein Phosphatase 2A (PP2A) regulatory subunit, named PR59. PR59 shares sequence homology with a known regulatory subunit of PP2A, PR72, but differs from PR72 in its expression pattern and its functional properties. We show that PR59 co-immunoprecipitates with the PP2A catalytic subunit, indicating that PR59 is a genuine component of PP2A holo-enzymes. In vivo, PR59 associates specifically with p107, but not with pRb. Elevated expression of PR59 results in dephosphorylation of p107, but not of pRb, and inhibits cell proliferation by causing cells to accumulate in G1. These data support a model in which the distinct PP2A regulatory subunits act to target the PP2A catalytic subunit to specific substrates and suggest a role for PP2A in regulation of p107.

Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene.

The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(-/-) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(-/-) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(-/-) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.

Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo.

The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.

Retinoblastoma-related protein pRb2/p130 and its binding to theB-myb promoter increase during human neuroblastoma differentiation

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins.

RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

Enterocyte differentiation is compatible with SV40 large T expression and loss of p53 function in human colonic Caco-2 cells: Status of the pRb1 and pRb2 tumor suppressor gene products

Transfer of the SV40 large-T (LT) oncogene into isolated human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40 LT antigen in Caco-2 cells did not alter phenotypic markers of differentiation, including expression of villin, sucrase–isomaltase, brush border and dome formation. As compared to parental cells, the transfected Caco-2 LT9 cells exhibited similar growth curves and no invasive properties in vitro. The major oncogenic function of the SV40 LT antigen in transfected Caco-2 cells is associated with reduced latency times necessary for the manifestation of tumors in athymic nude mice. The Caco-2 cell line contained deleted and mutant p53 alleles (stop codon in position 204) and has no detectable truncated p53 protein by Western blot. Molecular complexes between the SV40 LT antigen and the retinoblastoma-related proteins pRb1 and Rb2 were clearly identified at the different phases of the growth curve. When compared to normal human colonic crypts, Caco-2 cell differentiation is related to partial redistribution of pRb1 into hypophosphorylated, antiproliferative forms. The pRb2 protein is found elevated in a subset of human colorectal tumors and their corresponding liver metastases. We conclude that: (1) Caco-2 cells exert a dominant control against the oncogenic functions of the LT antigen; (2) loss of p53 function is not restrictive for the establishment of polarity and differentiation of the enterocyte lineage; (3) the levels and phosphorylation status of the Rb1 and Rb2 proteins may play important roles in the proliferation and differentiation of normal and neoplastic human colonic mucosa.

A Novel CCAAT-binding Protein Necessary for Adhesion-dependent Cyclin A Transcription at the G1/S Boundary Is Sequestered by a Retinoblastoma-like Protein in G0

Loss of adhesion leads to cell cycle arrest at the G1/S boundary in normal, adhesion-dependent, mesenchymal cells. This arrest is accompanied by the inability to produce cyclin A. Using deletional and mutational analysis of the cyclin A promoter, we have identified a CCAAT element that mediates the adhesion-dependent transcriptional activation of cyclin A in late G1 phase of the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric protein complex, which we have named CBP/cycA, to this CCAAT element was detectable in growing but not in G0-arrested or nonadherent normal rat kidney fibroblasts. During G0 CBP/cycA appears to be present but sequestered by a retinoblastoma family member. These results suggest that expression of cyclin A, which controls cell cycle progression by adhesion at the G1/S boundary, is regulated by CBP/cycA and the phosphorylation status of the retinoblastoma protein or a retinoblastoma-related protein.

P21 Disrupts the interaction between cdk2 and the E2F-p130 complex.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

Functional Analysis of Burkitt's Lymphoma Mutant c-Myc Proteins

The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt's lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived from Burkitt's lymphoma cell lines are resistant to inhibition by p107, thus providing a rationale for the increased oncogenic activity of these mutant c-Myc proteins. It has been suggested that these mutant c-Myc proteins resist down-modulation by p107 because they lack cyclin A-cdk2-dependent phosphorylation. Here, we have examined three different Burkitt's lymphoma mutant c-Myc proteins found in primary Burkitt's lymphomas and one mutant c-Myc protein detected in a Burkitt's lymphoma cell line. All four have an unaltered ability to activate transcription and are sensitive to inhibition of transactivation by p107. Furthermore, we provide evidence that down-modulation of c-Myc transactivation by p107 does not require phosphorylation of the c-Myc transactivation domain by cyclin A-cdk2. Our data indicate that escape from p107-induced suppression is not a general consequence of all Burkitt's lymphoma-associated c-Myc mutations, suggesting that other mechanisms exist to deregulate c-Myc function.

Transforming Growth Factor-β1 Modulates p107 Function in Myeloid Cells: CORRELATION WITH CELL CYCLE PROGRESSION

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of hematopoietic cell growth. Here we report that TGF-beta1 signals inhibition of IL-3-dependent 32D-123 murine myeloid cell growth by modulating the activities of cyclin E and cyclin-dependent kinase 2 (cdk2) proteins and their complex formation in the G1 phase of the cell cycle. Whereas the cyclin E protein was hyperphosphorylated in TGF-beta1 treated cells, TGF-beta1 decreased both the phosphorylation of cdk2 and the kinase activity of the cyclin E-cdk2 complex. Decreased cyclin E-cdk2 kinase activity correlated with decreased phosphorylation of the retinoblastoma-related protein p107. In support of these observations, transient overexpression of p107 inhibited the proliferation of the myeloid cells, and expression of antisense oligodeoxynucleotides to p107 mRNA blocked TGF-beta1 inhibition of myeloid cell growth. Furthermore, as reported previously, in 32D-123 TGF-beta1 treated cells, c-Myc protein expression was decreased. TGF-beta1 increased the binding of p107 to the transcription factor E2F, leading to decreased c-Myc protein levels. p107 inhibited E2F transactivation activity and was also found to bind the c-Myc protein, suggesting p107 negative regulation of c-Myc protein function. These studies demonstrate the modulation of p107 function by TGF-beta1 and suggest a novel mechanism by which TGF-beta1 blocks cell cycle progression in myeloid cells.

Association of p107 with Sp1: Genetically Separable Regions of p107 Are Involved in Regulation of E2F- and Sp1-Dependent Transcription

The retinoblastoma-related protein p107 has been shown to be a regulator of the transcription factor E2F. p107 associates with E2F via its pocket region and represses E2F-dependent transcription. In this study, we provide evidence for a novel interaction between p107 and the transcription factor Sp1. We show that p107 can be found endogenously associated with Sp1 in the extracts of several different cell lines. Moreover, in transient transfection assays, expression of p107 represses Sp1-dependent transcription. This repression of Sp1-dependent transcription does not require the DNA-binding domain of Sp1. Transcription driven by a chimeric protein containing the Ga14 DNA-binding domain and the Sp1 activation domains is inhibited by p107. Interestingly, unlike the repression of E2F-dependent transcription, the repression of Sp1-dependent transcription does not depend on an intact pocket region. We show that distinct regions of p107 are involved in the control of Sp1 and E2F.

Cyclin D1/CDK2 Kinase Is Present in a G1 Phase-Specific Protein Complex YI1 That Binds to the Mouse Thymidine Kinase Gene Promoter

Our studies provide evidence for the presence of cyclin D1 in an early G1 cycle-specific DNA binding complex Yi1. Previously we identified several complexes including Yi and E2F that at different times during G0 to S transition bind to three distinct DNA sequences (MT1, MT2, MT3) located in the mouse thymidine kinase upstream promoter. These various complexes contain DNA binding proteins (Sp1, E2F, p110, p60), cyclins A and E, cyclin-dependent kinase 2 (cdk2), and retinoblastoma-related proteins (pRB, p107). Here we report that Yi1 is different from the E2F complexes. Yi1 contains cyclin D1/cdk2 kinase as shown by using specific antibodies to cyclins, cdks and the Yi1 DNA-binding protein in gel retardation, western blotting, and immunoprecipitation assays. Yi1 binding is specific to a consensus sequence different from that of E2F.