The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

David M Livingston - One of the best experts on this subject based on the ideXlab platform.

  • cyclin a kinase regulation of E2F 1 dna binding function underlies suppression of an s phase checkpoint
    Cell, 1995
    Co-Authors: Wilhelm Krek, David M Livingston
    Abstract:

    Abstract Commitment of mammalian cells to enter S phase enables the Transcription Factor E2F-1 to activate certain genes whose products mediate cell cycle advance. In S phase, E2F-1 forms stable complexes with cyclin A-kinase, which in turn eliminates E2F-1 DNA binding function. Here, we show that suppression of E2F-1 DNA-binding activity by cyclin A-kinase is linked to orderly S phase progression. Disruption of this linkage resulted in S phase delay/arrest followed by regrowth orapoptosis, depending upon whetherthe DNA-bound E2F-1 could transactivate. Hence, the unscheduled presence of E2F-1 on specific DNA sequences during S phase can activate a specific S phase checkpoint, thereby linking Transcription, DNA replication, and cell cycle control.

  • the Transcription Factor E2F 1 is a downstream target of rb action
    Molecular and Cellular Biology, 1995
    Co-Authors: Xiaoqiang Qin, Mark E. Ewen, David M Livingston, Zoltan Arany, William R Sellers, William G Kaelin
    Abstract:

    Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular Transcription Factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress Transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated Transcriptional repression.

  • deregulated Transcription Factor E2F 1 expression leads to s phase entry and p53 mediated apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Xiaoqiang Qin, William G Kaelin, David M Livingston, Peter D Adams
    Abstract:

    Abstract E2F-1 is a Transcription Factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.

  • negative regulation of the growth promoting Transcription Factor E2F 1 by a stably bound cyclin a dependent protein kinase
    Cell, 1994
    Co-Authors: Wilhelm Krek, Mark E. Ewen, William G Kaelin, Suman Shirodkar, Zoltan Arany, David M Livingston
    Abstract:

    Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting Transcription Factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

  • functional interactions of the retinoblastoma protein with mammalian d type cyclins
    Cell, 1993
    Co-Authors: Mark E. Ewen, Hayla K Sluss, Junya Kato, Hitoshi Matsushime, Charles J Sherr, David M Livingston
    Abstract:

    Abstract The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G 1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the Transcription Factor E2F and reversed the Rb G 1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.

Joseph R Nevins - One of the best experts on this subject based on the ideXlab platform.

  • specificity in the activation and control of Transcription Factor E2F dependent apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Timothy C Hallstrom, Joseph R Nevins
    Abstract:

    Previous work has demonstrated a role for the E2F1 gene product in signaling apoptosis, both as a result of the deregulation of the Rb/E2F pathway as well as in response to DNA damage. We now show that the ability of cells to suppress the apoptotic potential of E2F1, as might occur during the course of normal cellular proliferation, requires the action of the Ras–phosphoinositide 3-kinase–Akt signaling pathway. In addition, we also identify a domain within the E2F1 protein, previously termed the marked-box domain, that is essential for the apoptotic activity of E2F1 and that distinguishes the E2F1 protein from E2F3. We also show that the E2F1-marked-box domain is essential for the induction of both p53 and p73 accumulation. Importantly, a role for the marked-box domain in the specificity of E2F1-mediated apoptosis coincides with recent work demonstrating a role for this domain in achieving specificity in the activation of Transcription. We conclude that the unique capacity of E2F1 to trigger apoptosis reflects a specificity of Transcriptional activation potential, and that this role for E2F1 is regulated through the action of the Akt protein kinase.

  • ectopic E2F expression induces s phase and apoptosis in drosophila imaginal discs
    Genes & Development, 1996
    Co-Authors: Maki Asano, Joseph R Nevins, Robin P Wharton
    Abstract:

    Previous experiments suggest that a key event in the commitment of cultured mammalian cells to entering S phase is a rise in activity of the Transcription Factor E2F. In this report, we study the role of Drosophila E2F in imaginal disc cells in vivo, by examining the distribution of the endogenous protein and studying the consequences of ectopic E2F expression. First, we find that endogenous E2F falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated Gj-S-transition in the eye disc. Second, we find that ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Third, we find that ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Although we do not know the fate of these cells, we suspect that ultimately they are killed by ectopic E2F. Taken together, our results show that an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase. Furthermore, they suggest that the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis.

  • a unique role for the rb protein in controlling E2F accumulation during cell growth and differentiation
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Masa-aki Ikeda, Laszlo Jakoi, Joseph R Nevins
    Abstract:

    Abstract Examination of the interactions involving Transcription Factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

  • E2F a link between the rb tumor suppressor protein and viral oncoproteins
    Science, 1992
    Co-Authors: Joseph R Nevins
    Abstract:

    The cellular Transcription Factor E2F, previously identified as a component of early adenovirus Transcription, has now been shown to be important in cell proliferation control. E2F appears to be a functional target for the action of the tumor suppressor protein Rb that is encoded by the retinoblastoma susceptibility gene. The disruption of this E2F-Rb interaction, as well as a complex involving E2F in association with the cell cycle-regulated cyclin A-cdk2 kinase complex, may be a common mechanism of action for the oncoproteins encoded by the DNA tumor viruses.

  • adenovirus e1a simian virus 40 tumor antigen and human papillomavirus e7 protein share the capacity to disrupt the interaction between Transcription Factor E2F and the retinoblastoma gene product
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Srikumar Chellappan, Virginia B Kraus, B Kroger, K Munger, Peter M Howley, W C Phelps, Joseph R Nevins
    Abstract:

    Abstract The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the Transcription Factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.

Mark E. Ewen - One of the best experts on this subject based on the ideXlab platform.

  • the Transcription Factor E2F 1 is a downstream target of rb action
    Molecular and Cellular Biology, 1995
    Co-Authors: Xiaoqiang Qin, Mark E. Ewen, David M Livingston, Zoltan Arany, William R Sellers, William G Kaelin
    Abstract:

    Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular Transcription Factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress Transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated Transcriptional repression.

  • negative regulation of the growth promoting Transcription Factor E2F 1 by a stably bound cyclin a dependent protein kinase
    Cell, 1994
    Co-Authors: Wilhelm Krek, Mark E. Ewen, William G Kaelin, Suman Shirodkar, Zoltan Arany, David M Livingston
    Abstract:

    Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting Transcription Factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

  • the cell cycle and the retinoblastoma protein family
    Cancer and Metastasis Reviews, 1994
    Co-Authors: Mark E. Ewen
    Abstract:

    Tumor formation results from alterations in the control of normal cell proliferation. To further our understanding of the molecular mechanisms underlying the deregulation of cell proliferation much attention, over the past decade, has been focused on the function of proto-oncogenes. Cellular oncogenes are thought to be growth promoting. More recently, a class of genes known as tumor suppressors have come under intense study. Tumor suppressors are largely thought to restrain cell proliferation. The retinoblastoma protein (Rb) is one of a growing list of tumor suppressors. Concurrent with the study of tumor suppressor genes has been a rapid increase in our understanding of the cell cycle at the molecular level. Rb and a related protein p107 are involved in the processes of cell proliferation and differentiation. Each functionally interacts with and affects the activity of the Transcription Factor E2F as well as other Transcription Factors involved in cell proliferation and differentiation. Additionally, Rb and p107 are modified by, and/or form specific complexes with, several elements of the basic cell cycle machinery. Specifically, Rb and p107 interact with and are modified by various cyclins and cyclin dependent kinases (cdk), some of which have been shown to be essential for cell cycle progression and in some cases their deregulation has been implicated in the development of cancer. This review will attempt to convey our current functional and mechanistic understanding of the biological roles Rb and p107 play in proliferation, development and differentiation. A knowledge of the interplay between these positive and negative regulators of cell proliferation and differentiation, noted above, is central to our understanding of human cancer.

  • functional interactions of the retinoblastoma protein with mammalian d type cyclins
    Cell, 1993
    Co-Authors: Mark E. Ewen, Hayla K Sluss, Junya Kato, Hitoshi Matsushime, Charles J Sherr, David M Livingston
    Abstract:

    Abstract The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G 1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the Transcription Factor E2F and reversed the Rb G 1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.

  • direct binding of cyclin d to the retinoblastoma gene product prb and prb phosphorylation by the cyclin d dependent kinase cdk4
    Genes & Development, 1993
    Co-Authors: Junya Kato, Mark E. Ewen, Scott W. Hiebert, Hitoshi Matsushime, Charles J Sherr
    Abstract:

    : The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The Transcription Factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.

Thomas Chittenden - One of the best experts on this subject based on the ideXlab platform.

  • expression cloning of a cdna encoding a retinoblastoma binding protein with E2F like properties
    Cell, 1992
    Co-Authors: William G Kaelin, Thomas Chittenden, Wilhelm Krek, William R Sellers, James A Decaprio, Florence Ajchenbaum, Charles S Fuchs, Yue Li, Peggy J Farnham, Michael A Blanar
    Abstract:

    Abstract An expression vector was modified to permit the rapid synthesis of purified, 32 P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen λgt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB "pocket" is known to bind, directly or indirectly, to the cellular Transcription Factor, E2F, implicated in cell growth control. We have found that RBAP-1 copurifies with E2F, interacts specifically with the adenovirus E4 ORF 67 protein, binds specifically and directly to a known E2F DNA recognition sequence, and contains a functional transactivation domain. Therefore, RBAP-1 is a species of E2F and can bind specifically to the RB pocket.

  • the Transcription Factor E2F interacts with the retinoblastoma product and a p107 cyclin a complex in a cell cycle regulated manner
    Cell, 1992
    Co-Authors: Suman Shirodkar, Mark E. Ewen, David M Livingston, James A Decaprio, Jeffrey R Morgan, Thomas Chittenden
    Abstract:

    Abstract E2F is a Transcription Factor believed to play a role in the activation of genes required for cellular proliferation. Its regulation is likely important for maintenance of G0 and for the initiation of cell growth. The retinoblastoma product (RB) forms a complex with E2F in G1 in primary and established human cells. As these cells enter S, a second E2F-containing complex appears. It contains p107, a nuclear "pocket" protein with similarities in structure and protein-binding properties to RB, and cyclin A, a cyclin believed to play a role in facilitating DNA replication. Hence, the regulation of E2F is carried out differently in G1 or S. The presence of cyclin A and a pocket protein, a possible cell growth regulator, in the same S phase-associated complex suggests a link between the function of E2F and the regulation of the DNA replication process.

  • the Transcription Factor E2F interacts with the retinoblastoma product and a p107 cyclin a complex in a cell cycle regulated manner
    Cell, 1992
    Co-Authors: Suman Shirodkar, Mark E. Ewen, David M Livingston, James A Decaprio, Jeffrey R Morgan, Thomas Chittenden
    Abstract:

    Abstract E2F is a Transcription Factor believed to play a role in the activation of genes required for cellular proliferation. Its regulation is likely important for maintenance of G0 and for the initiation of cell growth. The retinoblastoma product (RB) forms a complex with E2F in G1 in primary and established human cells. As these cells enter S, a second E2F-containing complex appears. It contains p107, a nuclear "pocket" protein with similarities in structure and protein-binding properties to RB, and cyclin A, a cyclin believed to play a role in facilitating DNA replication. Hence, the regulation of E2F is carried out differently in G1 or S. The presence of cyclin A and a pocket protein, a possible cell growth regulator, in the same S phase-associated complex suggests a link between the function of E2F and the regulation of the DNA replication process.

  • the t e1a binding domain of the retinoblastoma product can interact selectively with a sequence specific dna binding protein
    Cell, 1991
    Co-Authors: Thomas Chittenden, David M Livingston, William G Kaelin
    Abstract:

    Abstract A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNA-binding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNA-binding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the Transcription Factor E2F or with a protein having closely related DNA-binding properties.

William G Kaelin - One of the best experts on this subject based on the ideXlab platform.

  • the Transcription Factor E2F 1 is a downstream target of rb action
    Molecular and Cellular Biology, 1995
    Co-Authors: Xiaoqiang Qin, Mark E. Ewen, David M Livingston, Zoltan Arany, William R Sellers, William G Kaelin
    Abstract:

    Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular Transcription Factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress Transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated Transcriptional repression.

  • deregulated Transcription Factor E2F 1 expression leads to s phase entry and p53 mediated apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Xiaoqiang Qin, William G Kaelin, David M Livingston, Peter D Adams
    Abstract:

    Abstract E2F-1 is a Transcription Factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.

  • negative regulation of the growth promoting Transcription Factor E2F 1 by a stably bound cyclin a dependent protein kinase
    Cell, 1994
    Co-Authors: Wilhelm Krek, Mark E. Ewen, William G Kaelin, Suman Shirodkar, Zoltan Arany, David M Livingston
    Abstract:

    Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting Transcription Factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

  • expression cloning of a cdna encoding a retinoblastoma binding protein with E2F like properties
    Cell, 1992
    Co-Authors: William G Kaelin, Thomas Chittenden, Wilhelm Krek, William R Sellers, James A Decaprio, Florence Ajchenbaum, Charles S Fuchs, Yue Li, Peggy J Farnham, Michael A Blanar
    Abstract:

    Abstract An expression vector was modified to permit the rapid synthesis of purified, 32 P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen λgt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB "pocket" is known to bind, directly or indirectly, to the cellular Transcription Factor, E2F, implicated in cell growth control. We have found that RBAP-1 copurifies with E2F, interacts specifically with the adenovirus E4 ORF 67 protein, binds specifically and directly to a known E2F DNA recognition sequence, and contains a functional transactivation domain. Therefore, RBAP-1 is a species of E2F and can bind specifically to the RB pocket.

  • the t e1a binding domain of the retinoblastoma product can interact selectively with a sequence specific dna binding protein
    Cell, 1991
    Co-Authors: Thomas Chittenden, David M Livingston, William G Kaelin
    Abstract:

    Abstract A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNA-binding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNA-binding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the Transcription Factor E2F or with a protein having closely related DNA-binding properties.