Am J Physiol Cell Physiol AJP: Heart and Circulatory Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 295: C1151-C1160, 2008. First published September 3, 2008; doi:10.1152/ajpcell.00300.2008
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/C1151    most recent
00300.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gorges, L. L.
Right arrow Articles by Baldassare, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gorges, L. L.
Right arrow Articles by Baldassare, J. J.

GROWTH, DIFFERENTIATION, AND APOPTOSIS

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F

Laura L. Gorges,1 Nathan H. Lents,2 and Joseph J. Baldassare1

1Department of Pharmacological Sciences at Saint Louis University, St. Louis, Missouri; and 2Department of Sciences at John Jay College of Criminal Justice, The City University of New York, New York

Submitted 8 June 2008 ; accepted in final form 27 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The retinoblastoma protein pRb plays a pivotal role in G1- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH2-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb{Delta}CDK (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb{Delta}CDK sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH2-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH2-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either Rb{Delta}CDK+T373 or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH2-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

cyclin; cell cycle

THE RETINOBLASTOMA SUSCEPTIBILITY GENE (RB1) was the first tumor suppressor gene to be identified, and it encodes a 110-kDa nuclear phosphoprotein (20, 53, 62). Inactivation of the retinoblastoma tumor suppressor protein pRb is associated with development of many malignant cancers such as retinoblastomas, osteosarcomas, and small cell lung carcinomas (18, 27, 52, 70, 75). In accordance with its role as a tumor suppressor, introduction of Rb into Rb–/– cells diminishes their capacity for malignant transformation (4, 38). In addition, pRb plays a critical role in DNA replication, cellular senescence, differentiation, and apoptosis, placing pRb at the center of cellular proliferation and tumorigenesis (26, 40, 70).

pRb has been shown to interact with E2F transcription factors through a distinct "pocket" domain, encompassing amino acids 379–772, resulting in inhibition of E2F transcriptional activity (2, 3, 8, 10). Transcriptional repression resulting from this high-affinity interaction directly correlates with the ability of pRb to arrest cells in G1 (65, 66). In addition to the pocket domain, the COOH-terminal domain (amino acids 773-928) plays an equally important role in facilitating E2F binding and is required for full repression of E2F-responsive promoters and growth suppression (6, 63, 74). The retinoblastoma protein inhibits E2F-mediated transcription via two distinct mechanisms. First, pRb binds to the E2F transactivation domain and inhibits the ability of E2F to promote transcriptional activation of E2F-dependent genes (32, 33). Second, pRb actively represses the expression of certain genes by recruiting chromatin remodeling factors such as HDAC, Brg1, and Suv39H1 to E2F-responsive promoters in a cooperative effort to silence E2F-mediated transcription (6, 59, 63, 73, 78).

A large body of evidence supports the notion that pRb maintains transcriptional repression of E2F-responsive genes when it is hypophosphorylated (11, 15, 19, 28, 61, 62, 77). However, the transcriptional inhibitory activity of pRb is attenuated upon hyperphosphorylation by the sequential actions of cyclin-CDK-associated activity (14). Thus pRb exists in a repressive hypophosphorylated form in G0 and early G1 and is converted to an inactive hyperphosphorylated protein by late G1 in a cell cycle-dependent manner (7, 9, 60). Moreover, hyperphosphorylation of pRb is concomitant with its release from E2F (31, 34, 41, 67, 75) and transcriptional initiation of E2F target genes necessary for DNA synthesis and G1/S-phase progression (12, 15, 16).

Amino acid sequences surrounding pRb phosphorylation sites resemble those typically phosphorylated by CDKs (53, 54). There are 16 CDK consensus sites within human pRb: seven are located in the COOH-terminal domain (amino acids 773-928), six are located in the NH2-terminal domain (amino acids 1-378), and three are located in the A/B pocket (amino acids 379-772) (Fig. 1). Identification of an NH2-terminally deleted pRb construct that is capable of arresting cells similar to that of wild-type pRb (24) has led to the supposition that the NH2-terminal region is dispensable for the suppressive functions of pRb. Thus extensive focus has been placed on the pocket region and COOH-terminal domain of pRb, since these are also the regions aforementioned that necessitate critical interactions between pRb and E2F and are rich in potential CDK phosphorylation sites.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 1. Schematic representation of the retinoblastoma protein. The 3 domains of the retinoblastoma protein (pRb) and the known CDK phosphorylation sites. [Adapted from Zarkowska and Mittnacht (77)].

 
Interestingly, patients displaying bilateral and unilateral retinoblastomas exhibit a number of different mutations within the NH2-terminal domain of pRb (35, 36, 76). In addition, reports also show variable truncations of the NH2-terminal domain that result in enhanced tumor suppressive abilities, suggesting that the NH2-terminal region may function to regulate cell cycle progression (79). Interestingly, deletion of the NH2 terminus yields pRb mutant constructs that are very weakly phosphorylated, presumably due to the loss of sequences necessary to attain hyperphosphorylation by cyclin-CDK throughout pRb (65). Finally, a recent study by the Mittnacht group (30) reported the crystallization of an NH2-terminal peptide fragment encompassing amino acids 40-355. The crystallized structure of this NH2-terminal region displayed striking resemblance to the pocket region of pRb in that they both share tandem cyclin folds that provide multiple binding sites for ligands. The report provides further evidence supporting a tight association between the NH2-terminal fragment and the pocket domain, an interaction that can be disrupted by binding of protein ligands within the cyclin folds (30).

Although these surprising results await further confirmation, this report strongly suggests that the NH2-terminal domain does not exist as a free entity. Rather, it likely associates with the pocket domain of pRb, as does the COOH-terminal domain. Moreover, binding of protein ligands to the NH2-terminal cyclin folds can modulate this structural conformation, although the biological consequence of this rearrangement remains to be elucidated. Because 6 of the 16 CDK phosphorylation sites are located within the NH2-terminal region of pRb, this domain likely takes part in critical interactions required for proper cell cycle function, such as those involving E2F. Together, these data suggest that the NH2-terminal domain plays a stronger role in pRb regulation than originally thought, and in contrast to the pocket and COOH-terminal domain of pRb, the NH2-terminal region has undergone comparatively limited investigational analysis. Clearly, the complex interactions between the NH2-terminal, COOH-terminal, and pocket domains require further investigation.

In previous studies, we demonstrated that CDK2-cyclin E activity is sufficient in inactivating pRb in {alpha}-thrombin-stimulated cells lacking CDK4-cyclin D activity (42). Furthermore, in the absence of CDK4-cyclin D1 activity, restoration of four CDK2-cyclin E phosphorylation sites to pRb{Delta}CDK (a dominantly repressive allele in which CDK phosphorylation sites have been mutated to alanine) results in transcriptional activation of E2F response elements (42). Although these data contradict the prevailing notion that inactivation of pRb requires sequential phosphorylation by cyclin-CDK activity, they provide a possible explanation as to why increased cyclin E expression correlates with poor prognosis in several types of cancers (5, 13, 22, 25, 29, 42–49, 51, 56, 58, 68, 69, 71). More recent studies in our laboratory have revealed that pRb inactivation can be mediated by CDK2-cyclin E phosphorylation of a single site, Thr-373 (57). These data underscore the importance of CDK2-cyclin E activity in pRb inactivation and G1/S-phase cell cycle entry and provide a potential contributing mechanism of cyclin E-associated enhanced tumorigenicity.

In addition to extending our earlier results to human cancer cells, human primary cells, and native human promoter sequences, the current study focused on the potential contributions of additional phosphorylation sites in the NH2-terminal domain of pRb in the inactivation of the transcriptional repressive properties of pRb. We also explored the necessity of the intact COOH terminus of pRb for phosphorylation-mediated inactivation. We found that a single phosphorylation event at either Thr-373 or, to a lesser extent, Thr-356 within the NH2-terminal domain of pRb{Delta}CDK results in increased E2F activity, suggesting that pRb inactivation is possible with just one phosphorylation event. Indeed, phosphorestoration of Thr-373 reduces the association of pRb{Delta}CDK with E2F and allows normal S-phase entry in stimulated cells. Provocatively, the intact COOH-terminal domain is required for phosphorylation on Thr-373 of both wild-type pRb and pRb{Delta}CDK+T373. Furthermore, when the COOH-terminal domain is removed from either wild-type pRb or pRb{Delta}CDK+T373, they are rendered immune to inactivation during G1 and remain repressive of E2F activation and S-phase entry. Together, these data provide mechanistic and biological evidence for cooperative interactions between the NH2-terminal and COOH-terminal domains in the regulation of the pRb tumor suppressor protein and compel more detailed studies of pRb inactivation in its full-length context.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and transient transfection. Saos-2 osteosarcoma cells and T98G human glioblastoma cells were obtained from the American Type Cell Collection and maintained as recommended. Primary human diploid fibroblasts (HDF cells) were grown and maintained in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose and 2 mM L-glutamine (BioWhittaker, Walkersville, MD), supplemented with 10% (vol/vol) fetal calf serum (FCS). HDF cells were electroporated or transfected with JetPEI as recommended by AMAXA (www.amaxa.com) and the manufacturer's protocol, respectively. After transfection, subconfluent T98G or HDF cells were growth arrested by being washed once with {alpha}-minimal essential medium without phenol red (Invitrogen) containing 2 mM L-glutamine (BioWhittaker), followed by a 48-h incubation in the same medium. Cells were stimulated with 10% serum following serum arrest conditions. Saos-2 cells were transfected using the Fugene reagent (Roche) according to the manufacturer's protocol or electroporated according to the AMAXA protocol. After transfection, asynchronous Saos-2 cells were harvested and assayed accordingly.

Constructs. 3xE2F-Luc was generously provided by Erik Knudsen (72). Cyclin E-luciferase and the Renilla expression plasmid were obtained from Promega. Vectors encoding human wild-type pRb and Rb{Delta}CDK were generously provided by J. Wade Harper (55), and all mutations were confirmed by sequencing analysis. Restoration mutants of Rb{Delta}CDK were generated by site-directed mutagenesis restoring Ser-230, Ser-249, Thr-252, Thr-356, Thr-373, Thr-608, Thr-612, Ser-795, and Thr-821 individually, following the manufacturer's protocol (QuikChange kit; Stratagene). All mutations were confirmed by automated capillary sequencing following the manufacturer's protocol (Beckman-Coulter).

Luciferase reporter assay. A luciferase reporter assay was conducted as previously described (42, 57). Briefly, HDF cells were transfected with 1 µg/ml 3xE2F-Luc, 100 ng/ml Renilla expression plasmid, and 1 µg/ml pRb construct as indicated. After transfection, HDF cells were rendered quiescent (see above) and stimulated with 10% FCS for 17 h. Cell lysates were then prepared for luciferase assay according to the manufacturer's protocol (Promega, Madison, WI). Renilla activity was measured from the same samples using the Dual-Luciferase Reporter Assay System from Promega. Relative light units were then divided by units of optical density from the Renilla assay to normalize for transfection efficiency according to the manufacturer's instructions (Promega). Saos-2 cells were treated in a similar fashion, except asynchronous cells were harvested 38 h posttransfection. Error bars represent standard deviation for triplicate samples within one experiment and are representative of at least three independent experiments.

Coimmunoprecipitations and Western blotting. Saos-2 or HDF cells were transfected with 2 µg/ml Flag-tagged Rb, Rb{Delta}CDK, and E2F1 as indicated. After transfection, HDF cells were synchronized as described above, whereas asynchronous Saos-2 cells were harvested 40 h posttransfection. Cells were washed twice with PBS and scraped into cold lysis buffer [50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% (vol/vol) Nonidet P-40, 1 mM PMSF, 2 mM sodium vanadate, 20 mM sodium fluoride, 50 µM β-glycerophosphate, and 10 µg/ml aprotinin, leupeptin, and pepstatin]. Lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation. Protein concentration was determined using Coomassie Plus (Pierce) as recommended by the manufacturer. Protein lysates (100–200 µg) were incubated with 2 µg of E2F1 antibody (sc-193) for 2 h at 4°C. Immune complexes were precipitated using a 50:50 mixture of A/G beads (sc-2003) overnight with gentle rocking. The immune complexes were pelleted by microcentrifugation at 3,000 g and washed three times with cold lysis buffer. Immunocomplexes were resuspended in 1x Laemmli buffer, resolved by 8% polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Millipore, Boston, MA) as recommended by the manufacturer. Membranes were probed with {alpha}-Flag monoclonal antibody (Sigma F3165). Phospho-T373 membranes were probed with p-Rb (T-373)-R polyclonal antibody (Santa Cruz 16672-R). Immunoreactive bands were visualized by enhanced chemiluminescence detection (ECL; Amersham Biosciences).

[3H]thymidine incorporation assay. A [3H]thymidine incorporation assay was performed precisely as described previously (21). Error bars represent the standard deviation between triplicate samples (n = 3), and the data are representative of three independent experiments.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Restoration of an individual phosphorylation site to pRb relieves transcriptional repression at the cyclin E promoter. The family of Rb pocket proteins is known to interact with members of the E2F transcription family and regulate transcriptional expression of key genes necessary for S-phase progression. Such genes include cdc6, cdk1, cyclin A, and cyclin E. Using "reverse mutational analysis," we restored CDK phosphorylation sites singly and in combination to the Rb{Delta}CDK mutant construct (57). Rb{Delta}CDK is a dominantly repressive mutant of Rb in which 15 of the 16 potential CDK sites have been mutated to alanine (Fig. 1). (The remaining phosphorylation site, Thr-5, is constitutively phosphorylated in a cell cycle-independent manner and is thus not considered to be a CDK-regulated phosphorylation event.)

We began our study in an effort to extend our earlier observations in rodent cells (57) to human cells. Since Saos-2 osteosarcoma cells do not express functional pRb, we utilized these cells as a "clean" context to determine the direct effect of ectopically expressed pRb constructs on E2F activity. As has been shown in other cell types, we report that Rb{Delta}CDK repressed activation of an artificial 3x-E2F-luciferase reporter in which three tandem copies of the E2F binding site from the Dhfr promoter drive expression of the firefly luciferase open reading frame (ORF). Furthermore, in agreement with our previously published findings (57), we observed that restoration of Thr-373 to Rb{Delta}CDK relieved this repression (Fig. 2A). In fact, also as shown previously, activation of E2F was enhanced in cells that expressed Rb{Delta}CDK+T373, even compared with those that expressed wild-type pRb (Fig. 2A). As discussed extensively in our previous report, this supports the notion that pRb may normally exist in several phosphoisoforms, only some subset of which are phosphorylated on the necessary residues to facilitate release of E2F (57). If Thr-373 phosphorylation alone can mediate activation of E2F, an allele of pRb that contains only this phosphorylation site would be expected to function as an all-or-none switch, explaining the enhanced activation of E2F in the context of Rb{Delta}CDK+T373 expression, observed now in both rodent and human cells.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 2. Restoration of Thr-373 to Rb{Delta}CDK (a dominantly active repressive allele of Rb) allows activation of E2F. Saos-2 cells (A and B) or primary diploid human skin fibroblasts (DHFs; C) were transfected with 3xE2F-Luc (A) or cyclin E-Luc (B and C), the Renilla reporter plasmid, and the indicated wild-type (wtRb) or Rb{Delta}CDK construct. Asynchronous cells were harvested and prepared for the luciferase and Renilla assays 38 h following transfection. Data are represented as fold increase in relative light units (RLU) divided by units of optical density from the Renilla activity over the sample in which Rb{Delta}CDK was expressed. Each sample was prepared in triplicate, and error bars represent 1 unit of standard deviation.

 
Although our previous studies had identified Thr-373 as a key phosphorylation event in pRb responsible for alleviating repression of an artificial E2F response element (42, 57), in the present study we wanted to extend our focus to the activation of a native promoter sequence from a bona fide S-phase gene. Thus we employed a cyclin E reporter plasmid containing sequence from the human cyclin E promoter (–363 to –1007) driving expression of the luciferase ORF when induced (23). To that end, we found that Rb{Delta}CDK also effectively blocked transcriptional activation of the native cyclin E reporter in Saos-2 cells (Fig. 2B). Furthermore, in agreement with previously reported data from rodent cells (57), restoration of Thr-373, alone or in combination with other CDK2-cyclin E sites, was sufficient to relieve transcriptional repression of the cyclin E promoter in human cells (Fig. 2B). Interestingly, restoration of additional phosphorylation sites to Thr-373 attenuated the activation of the cyclin E promoter (Fig. 2, A and B).

Although Saos-2 cells provide the opportunity to measure the direct effects of ectopic pRb expression on cyclin E promoter activity, these cells are oncogenically transformed and difficult to synchronize in G0, presumably due to alterations in signaling pathways associated with the malignant phenotype. To circumvent the potential contribution of altered cell cycle dynamics on cyclin E activity and to demonstrate the affect of Thr-373 phosphorylation on cyclin E promoter activity in noncancerous cells, we utilized primary diploid human fibroblasts (DHFs) isolated from skin. In agreement with data resulting from Saos-2 osteosarcoma cells, primary human skin fibroblasts displayed an increase in cyclin E promoter activity in the presence of restored Thr-373 alone or in combination with other phosphorestorations (Fig. 2C). As in Saos-2 cells, restoration of CDK phosphorylation sites other than Thr-373 had no effect on transcriptional derepression of cyclin E activity, and these constructs behaved in a manner similar to the constitutively repressive Rb mutant construct, Rb{Delta}CDK (Fig. 2C and other data not shown). Together, these data support a role for Thr-373 phosphorylation in E2F activation in both osteosarcoma and primary human fibroblast cells.

Additional reverse mutational analysis of the NH2-terminal domain of pRb. Reports have indicated that NH2-terminally truncated pRb (p56Rb) is sufficient in blocking G1 progression (24), lending credence to the idea that the carboxy terminus is the functional domain of pRb with respect to cell cycle control (37, 39). Thus the p56Rb construct has been integrated into many studies involving Rb, rendering little information about the NH2-terminal domain and how it contributes to the regulation of pRb, E2F, cell cycle progression, or differentiation. Interestingly, residues within the NH2 terminus have been found to be phosphorylated in vivo (54), but further investigation regarding the function of these CDK phosphorylations has been limited. In an effort to understand whether other NH2-terminal CDK phosphorylation sites affect the ability of pRb to repress E2F-dependent transcriptional activation, we restored phosphorylation sites to Rb{Delta}CDK found in the NH2-terminal domain (Fig. 1). To test the effect of these Rb mutant constructs on cyclin E activation, we expressed these constructs in asynchronous Saos-2 cells. Interestingly, the Thr-356 restorative site resulted in a substantial increase in cyclin E-luciferase activity, whereas restoration of Ser-230, Ser-249, or Ser-252 had little effect (Fig. 3A).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Thr-373 is the principal NH2-terminal phosphorylation site for inactivation of Rb{Delta}CDK. Saos-2 cells (A) or primary DHFs (B) were transfected with 3xE2F-Luc (A) or cyclin E-Luc (B), the Renilla reporter plasmid, and the indicated wtRb or Rb{Delta}CDK construct. Asynchronous cells were harvested and prepared for the luciferase and Renilla assays 38 h following transfection. Data are represented as fold increase in RLU divided by Renilla activity over the sample in which Rb{Delta}CDK was expressed. Each sample was prepared in triplicate, and error bars represent 1 unit of standard deviation.

 
To further this analysis of the NH2 terminus and extend it to primary human fibroblasts, we combined multiple phosphorestorations within the NH2-terminal domain of pRb, together with Thr-373, and observed the effect on activation of the cyclin E promoter (Fig. 3B). This analysis confirmed that Thr-373 is indeed the key NH2-terminal phosphorylation site and that addition of further phosphorestorations attenuates E2F activity at the cyclin E promoter, similar to restorations in other domains of pRb (Ref. 86 and Fig. 2).

Phosphorylation of Thr-373 results in diminished binding of E2F1. Both the cyclin E promoter construct and the 3xE2F-Luc reporter contain DNA consensus sequences that are recognized by the E2F (18, 50, 64). The retinoblastoma protein is known to associate with these E2F transcription factors and regulate E2F-responsive genes, many of which are required for G1- to S-phase cell cycle progression, in a phosphorylation-dependent manner. Thus the assumed mechanism by which restoration of Thr-373 to Rb{Delta}CDK facilitates activation of E2F is by the phosphorylation of Thr-373 by cyclin-CDK and the subsequent release of E2F from this mutant construct. However, with the recent availability of a specific antibody that recognizes only Thr-373-phosphorylated pRb, we endeavored to demonstrate this mechanism directly. To specifically asses the cell cycle-dependent Thr-373 phosphorylation of our pRb constructs, we utilized primary human skin fibroblasts (DHFs), which can be easily synchronized in G0 by serum deprivation. As expected, we observed that wild-type pRb became phosphorylated on Thr-373 upon serum stimulation, whereas Rb{Delta}CDK did not (Fig. 4A). Furthermore, we have confirmed, for the first time, that restoration of Ala-373 back to the wild-type threonine in Rb{Delta}CDK does rescue the ability of this construct to be phosphorylated during G1 progression, presumably by cyclin-CDK complexes (Fig. 4A).


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 4. Thr-373 phosphorylation on pRb releases E2F. A: T98G human glioblastoma cells were electroporated with either wtRB, Rb{Delta}CDK, or Rb{Delta}CDK+T373 (all Flag tagged), as indicated, followed by serum starvation for 48 h. Cells were stimulated with 10% serum, lysates were prepared, and pRb complexes were immunoprecipitated with {alpha}-Flag antibody (Sigma F3165). Immunoprecipitates were then probed by Western blotting with {alpha}-phospho-pRb (Thr-373: {alpha}-pRb or p-T373) and {alpha}-Flag. B: Saos-2 cells (top) or DHFs (bottom) were electroporated with E2F1 and the pRb construct indicated (all Flag tagged). Next, {alpha}-E2F1 immunoprecipitations were prepared from 100 µg of protein lysate, and complexes were probed with {alpha}-E2F1 and {alpha}-Flag by Western blotting.

 
Next, to detect the association of our mutant constructs with E2F, we expressed various Flag-tagged pRb constructs in both Saos-2 cells and primary skin fibroblasts and observed the appearance of these constructs in E2F1 immunoprecipitations. [Important for the interpretation of these experiments: when transfected, all pRb mutant constructs express at similar levels (Ref. 57 and data not shown).] Not surprisingly, in asynchronous cell populations, a larger amount of Flag-tagged pRb protein was present when Rb{Delta}CDK was expressed than with wild-type pRb (Fig. 4B). This demonstrates the constitutively active nature of Rb{Delta}CDK, due to its inability to be phosphorylated (and thus inactivated) by cyclin-CDK activity. Interestingly, the association of E2F1 with Rb{Delta}CDK+T373 appeared similar to that of wild-type pRb, supporting the conclusion that phosphorylation of Thr-373 alone can facilitate release of pRb from E2F (Fig. 4B).

Phosphorylation of Rb{Delta}CDK+T373 precedes activation of CDK2 kinase. Both CDK4 and CDK2 are capable of phosphorylating pRb on Thr-373 in vitro. Although our previous work had focused on the role of cyclin E-CDK2 in the phosphorylation of pRb (42, 57), the finding that the cyclin E promoter itself became active when pRb was phosphorylated on Thr-373 (Figs. 2 and 3) left open the possibility that cyclin D-CDK4 might also phosphorylate this residue in vivo. To explore this possibility, we first established the G1 time course of activation of CDK4 and CDK2 in primary human skin fibroblasts, which cycle considerably slower than most established cell lines. We found that cyclin D-CDK4 activity peaked 12–15 h after the readdition of serum to quiescent cells, whereas CDK2 activity reached maximal levels in 18–24 h (Fig. 5A). Interestingly, we found that the phosphorylation of Thr-373 closely mirrored the activation of cyclin D-CDK in DHF cells (Fig. 5B), arguing that, in vivo, CDK4 is the dominant kinase at this residue.


Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 5. Thr-373 phosphorylation is concomitant with activation of CDK4. A: primary DHFs were synchronized in G0 by serum starvation for 48 h, followed by stimulation with 10% FBS for the indicated lengths of time. Cell lysates were prepared, and CDK4 and CDK2 activity was measured using in vitro kinase assays with GST-pRB and histone H1 as substrates, respectively. B: primary DHFs were electroporated with Rb{Delta}CDK+T373 and synchronized in G0 by serum starvation for 48 h. Cells were stimulated with 10% FBS for the indicated lengths of time, lysates were prepared, and pRb complexes were immunoprecipitated with {alpha}-Flag antibody (Sigma F3165). Immunoprecipitates were then probed by Western blotting with {alpha}-phospho-pRb (Thr-373) and {alpha}-Flag.

 
Phosphorylation of pRb on Thr-373 is sufficient to allow S phase entry. Expression of E2F-dependent genes such as cdc6, cyclin E, and polymerase {delta} facilitate cell cycle progression and S-phase entry (17). We have demonstrated that phosphorylation of Thr-373 is sufficient in relieving transcriptional repression of E2F at the cyclin E promoter (Figs. 2 and 3). Thus we next questioned whether primary diploid human fibroblasts that express various mutant pRb constructs could enter S phase and synthesize DNA, as measured by the [3H]thymidine incorporation assay. In agreement with previous results seen in rodent cells (57), we have shown that whereas expression of Rb{Delta}CDK blocked DNA synthesis in diploid human cells, restoration of Thr-373 totally abrogated this effect, and restoration of other residues had little effect (Fig. 6; other point mutants not shown).


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Phosphorylation of Thr-373 is required for DNA synthesis. Primary DHFs were electroporated with the indicated pRb construct and synchronized in G0 by serum starvation for 48 h. Cells were then stimulated with 10% FBS for 17 h (except "basal"), followed by addition of [3H]thymidine for 4 h. DNA synthesis was then measured using the [3H]thymidine incorporation assay, as described in MATERIALS AND METHODS. Numbers represent TCA-precipitated counts per minute (CPM), samples were prepared in triplicate, and error bars represent 1 unit of standard deviation.

 
The COOH-terminal domain of pRb is necessary for activation of E2F. Recent studies have shown that a portion of the COOH-terminal domain of pRb is sufficient in binding to E2F in vitro. Interestingly, this COOH-terminal fragment, which encompasses amino acids 786–874, is able to release E2F by binding to the pocket domain of pRb, thereby destabilizing interactions required for binding of pRb to E2F (67). Although these studies reveal that key intramolecular rearrangements occur upon phosphorylation, the precise amino acid residues required for E2F release were not explored. In addition, these in vitro binding assays were conducted in the absence of the NH2-terminal domain and did not reflect the full complexity of interactions that likely exists in the context of full-length pRb. Because we had shown presently and previously that restoration of Thr-373 in the NH2-terminal domain of pRb is capable of inactivating Rb{Delta}CDK, resulting in activation of E2F and S-phase entry, we hypothesized that interactions between multiple domains of pRb likely contribute to the full complexity of pRb function.

To that end, we sought to determine the necessity for the extreme COOH-terminal domain in the inactivation of pRb and release of E2F by deleting the terminal 53 amino acids (residues 877-928) from our pRb mutant constructs. Strikingly, we found that, in Saos-2 cells, removal of the extreme COOH terminus, which does not remove any known CDK phosphorylation sites, changed Rb{Delta}CDK+T373 into a form (pRb{Delta}C) that is constitutively repressive of cyclin E reporter activity, similar to that of Rb{Delta}CDK (Fig. 7A). This intriguing result is not limited to transformed cells and the cyclin E promoter, because we observed similar results using a generic E2F reporter plasmid in primary human fibroblasts (Fig. 7B). We further reasoned that if the extreme COOH terminus of pRb were necessary for inactivation of Rb{Delta}CDK+T373, it might also be required for the normal inactivation of wild-type pRb. Thus we next deleted the terminal 53 amino acids from wild-type pRb and assessed E2F activation in this context. Dramatically, we found that E2F activation was substantially muted when pRb{Delta}C was expressed, compared with that when full-length wild-type pRb was expressed (Fig. 7C). This finding supports the notion that the extreme COOH terminus of pRb is involved in the CDK-mediated phosphorylation/inactivation or pRb.


Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 7. The COOH-terminal domain of pRb is required for activation of E2F. Saos-2 cells (A and C) or primary DHFs (B) were transfected with cyclin E-Luc (A and C) or 3xE2F-Luc (B), the Renilla reporter plasmid, and the indicated wtRb or Rb{Delta}CDK construct ("{Delta}C" indicates deletion of the COOH-terminal 53 codons). Asynchronous cells were harvested and prepared for the luciferase and Renilla assays 38 h following transfection. Data are represented as fold increases in RLU divided by Renilla activity over the sample in which Rb{Delta}CDK was expressed. Each sample was prepared in triplicate, and error bars represent 1 unit of standard deviation.

 
The COOH-terminal domain of pRb is necessary for phosphorylation of Thr-373. The implication of impaired E2F activation upon expression of an pRb mutant lacking the extreme COOH terminus is that this domain plays a role in the phosphorylation of pRb. Because the current study specifically focused on the phosphorylation of Thr-373, we endeavored to assess the effect of deletion of the COOH terminus on the phosphorylation state of this residue. To that end, we observed that the deletion of the COOH-terminal 53 amino acids abolished the serum-induced phosphorylation of both wild-type pRb and Rb{Delta}CDK+T373 (Fig. 8).


Figure 8
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 8. The COOH-terminal domain of pRb is required for phosphorylation on Thr-373. T98G human glioblastoma cells were electroporated with the indicated pRb construct (all Flag tagged), followed by serum starvation for 48 h. Cell were then stimulated, where indicated, with 10% FBS. pRb complexes were immunoprecipitated from 100 µg of protein lysate, and immunoprecipitates were then probed by Western blotting with {alpha}-phospho-pRb (Thr-373) and {alpha}-Flag.

 
The COOH terminus of pRb is necessary for entry into S phase. To determine the biological significance of the pRb COOH terminus in the regulation of S-phase entry, we examined DNA synthesis in the context of expression of mutants of pRb that lack the 53 COOH-terminal amino acids. Consistent with our previously published observations (42, 57) and those of this study, we found that cells in which E2F activation is diminished by hyperrepressive forms of pRb, entry into S-phase also was impaired, as measured by DNA synthesis (Fig. 9). This dramatic observation indicates that, in human cells, the COOH terminus of pRb is somehow required for the proper inactivation of pRb via phosphorylation of the NH2 terminus. Also, to our knowledge, this is the first demonstration that the extreme COOH terminus of pRb is required for S-phase entry.


Figure 9
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 9. The COOH-terminal domain of pRb is required for S-phase entry. Primary DHFs were electroporated with the indicated pRb construct and synchronized in G0 by serum starvation for 48 h. Cells were then stimulated with 10% FBS for 17 h (except "basal"), followed by addition of [3H]thymidine for 4 h. DNA synthesis was then measured using the [3H]thymidine incorporation assay. Numbers represent TCA-precipitated CPM, samples were prepared in triplicate, and error bars represent 1 unit of standard deviation.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Thr-373 as a critical residue involved in pRb inactivation. Many studies have endeavored to identify precise phosphorylation sites critical for the inactivation of pRb. However, human pRb contains 16 putative phosphorylation sites, making it difficult to determine the effects of single alanine substitutions in the presence of a multiphosphorylated pRb construct. Previous efforts reached the reasonable conclusion that inactivation of pRb occurs via the cumulative effects of hyperphosphorylation. However, the dominant view also held that in cycling cells, countless phosphoisomers exist, even within a single cell at a single point in time, and that regulation of the repressive nature of pRb by cooperative phosphorylations probably resembles a "dial" more than an on/off switch. Using reverse mutational analysis, we have been able to determine the effects of single phosphorylation sites added back to the pRb{Delta}CDK. This affords us the opportunity to examine the effects of discreet phosphorylation events in the "clean" context of an otherwise unphosphorylated pRb protein. For this reason, it is easy to conceive why Thr-373 had been previously overlooked as a critical phosphorylation.

However, although our data support the notion that Thr-373 (and to a lesser extent, Thr-356) may be sufficient for pRb inactivation, we have never maintained that this phosphorylation is required, and at least one group has previously shown that it is not (55). These observations are not in conflict, because our claim that this phosphorylation site can mediate inactivation holds intact the prevailing model that cumulative phosphorylation is how inactivation of pRb normally occurs. Our data presented presently and previously (57) indicate that an accumulation of phosphorylations, even those not including Thr-373, indeed leads to inactivation of pRb, in agreement with the current model. Nonetheless, the notion that Thr-373 phosphorylation alone may be able to mediate pRb inactivation in human cells is intriguing and suggests a potential Achilles heel in the tumor suppressive capacity of RB1.

It is striking that the activation of E2F in the context of pRb phosphorylation on Thr-373 alone is actually higher than under endogenous conditions or expression of wild-type pRb, as shown presently and previously (57). This phenomenon has at least three explanations that are in no way mutually exclusive. First, in the wild-type setting of 16 phosphorylation sites, pRb has been shown to exist in many phosphoisomers, only some of which may be phosphorylated on enough residues (or the correct residues) to induce inactivation. However, an pRb allele that can only be phosphorylated by CDKs on one residue is much more likely to function as an all-or-none switch for pRb inactivation and E2F activity. Second, the observation of enhanced E2F activation also could suggest that some phosphorylation sites may act to functionally inactivate pRb, whereas others act to keep pRb in an active, transcriptionally repressive state. Thus it may be the degree and combination of active and inactive phosphorylation sites that ultimately decides whether or not pRb remains transcriptionally repressive. Third, the physiological levels of E2F activity during G1 progression may normally be the product of only some subset of the thousands of pRb proteins becoming inactivated in any given cell. Thus superphysiological levels of E2F activity would be expected when switching to an all-or-none pRb allele in this context. Intriguingly, by regulating the level of E2F activation, Thr-373 phosphorylation may directly influence how pRb/E2F can respond to some signals (mitogens) by inducing cell proliferation but other signals (oncogene activation) by inducing apoptosis.

In any event, our data argue that the function and physiological relevance of the NH2-terminal domain of pRb warrants reexamination. Interestingly, recent X-ray crystallography data show that the NH2-terminal domain shares striking commonalities with the pocket domain of pRb in that they both contain cyclin folds and are able to bind ligands that alter conformational changes between these domains (30). In addition, the NH2-terminal peptide is able to bind the pocket domain, and this interaction is attenuated upon binding of LxCxE proteins to the pocket domain. These data offer critical insight into the possible binding interactions between the NH2-terminal and pocket domains of pRb. Previous studies have investigated the interactions between the COOH-terminal domain and the pocket domain of pRb in determining the contributing factors in pRb/E2F interactions (1–3). However, data from Mittnacht's group (30) provide evidence that the NH2-terminal domain also participates in these aforementioned interactions. It is simply not understood how the interactions among the NH2-terminal, COOH-terminal, and pocket domains affect the conformational structure of pRb and binding to E2F.

The COOH-terminal domain of pRb is required for inactivation by CDKs. In addition to discovering key CDK phosphorylation sites of pRb in the NH2-terminal region of pRb, we also have discovered that the extreme COOH terminus is necessary for pRb inactivation. Provocatively, there are no known CDK phosphorylation sites in this region. This finding in itself is significant, considering that most detailed molecular analysis of pRb structure-function relationships have focused on limited regions of the protein. Our results call for a new look at the intramolecular interactions present in the full-length molecule.

Many studies have relied on NH2-terminally truncated pRb constructs or fragments of the A/B pocket and COOH-terminal domain in probing pRb binding interactions with E2F. Although this has been useful, it negates the potential contributing factor of the NH2-terminal domain of pRb. In addition, as underscored by the present study, it is crucial to consider the three-dimensional interactions that may exist among the NH2-terminal, A/B pocket, and COOH-terminal domains. There are many conceivable mechanisms for our intriguing observations, the simplest of which is that the COOH-terminal domain of pRb is somehow necessary for the CDKs to recognize and act on pRb. It is possible that other binding partners of pRb play a role in the enzyme-substrate recognition event in a way that requires the COOH-terminal region of pRb. Another possibility is that the removal of the extreme COOH terminus induces a conformational change or a new protein-protein interaction that abrogates recognition or kinase activity by the CDKs. In any event, our prediction is that an intricate interaction among all three domains exists for proper physiological function and that these interactions selectively coordinate the binding and release of E2F, which in turn reflects the phosphorylation state of pRb. Further pRb structure-function studies are required to elucidate these complex molecular interactions.

Our conclusions do not undermine the central role of the A/B pocket in E2F binding but merely suggest that that NH2-terminal domain may play an influential role in determining the activation state of pRb. Although the crystallized large pocket domain of pRb has been extremely beneficial in understanding the participating role of the A/B pocket and COOH-terminal domain in E2F and LxCxE binding, it would be interesting to see how the addition of the NH2-terminal domain affects these interactions and whether it too comes in contact with or is in close proximity to E2F or the E2F binding domain. Only then will the nature and function of the extreme COOH terminus, and its relationship to the phosphorylation status of the NH2 terminus, become clear.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Baldassare, Dept. of Pharmacological Sciences at Saint Louis Univ., St. Louis, MO 63104 (e-mail: baldasjj{at}slu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Bagchi S, Raychaudhuri P, Nevins JR. Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation. Cell 62: 659–669, 1990.[CrossRef][Web of Science][Medline]

2. Bagchi S, Weinmann R, Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell 65: 1063–1072, 1991.[CrossRef][Web of Science][Medline]

3. Bandara LR, La Thangue NB. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature 351: 494–497, 1991.[CrossRef][Web of Science][Medline]

4. Bookstein R, Shew JY, Chen PL, Scully P, Lee WH. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247: 712–715, 1990.[Abstract/Free Full Text]

5. Bortner DM, Rosenberg MP. Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E. Mol Cell Biol 17: 453–459, 1997.[Abstract]

6. Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ, Kouzarides T. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391: 597–601, 1998.[CrossRef][Web of Science][Medline]

7. Buchkovich K, Duffy LA, Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58: 1097–1105, 1989.[CrossRef][Web of Science][Medline]

8. Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR. The E2F transcription factor is a cellular target for the RB protein. Cell 65: 1053–1061, 1991.[CrossRef][Web of Science][Medline]

9. Chen PL, Scully P, Shew JY, Wang JY, Lee WH. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 58: 1193–1198, 1989.[CrossRef][Web of Science][Medline]

10. Chittenden T, Livingston DM, Kaelin WG Jr. The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell 65: 1073–1082, 1991.[CrossRef][Web of Science][Medline]

11. Connell-Crowley L, Harper JW, Goodrich DW. Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell 8: 287–301, 1997.[Abstract]

12. DeGregori J, Kowalik T, Nevins JR. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol 15: 4215–4224, 1995.[Abstract]

13. Dou QP, Pardee AB, Keyomarsi K. Cyclin E—a better prognostic marker for breast cancer than cyclin D? Nat Med 2: 254, 1996.[Web of Science][Medline]

14. Dowdy SF, Hinds PW, Louie K, Reed SI, Arnold A, Weinberg RA. Physical interaction of the retinoblastoma protein with human D cyclins. Cell 73: 499–511, 1993.[CrossRef][Web of Science][Medline]

15. Dynlacht BD, Flores O, Lees JA, Harlow E. Differential regulation of E2F transactivation by cyclin/cdk2 complexes. Genes Dev 8: 1772–1786, 1994.[Abstract/Free Full Text]

16. Dynlacht BD, Moberg K, Lees JA, Harlow E, Zhu L. Specific regulation of E2F family members by cyclin-dependent kinases. Mol Cell Biol 17: 3867–3875, 1997.[Abstract]

17. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev 12: 2245–2262, 1998.[Free Full Text]

18. Dyson N, Howley PM, Munger K, Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243: 934–937, 1989.[Abstract/Free Full Text]

19. Ezhevsky SA, Ho A, Becker-Hapak M, Davis PK, Dowdy SF. Differential regulation of retinoblastoma tumor suppressor protein by G1 cyclin-dependent kinase complexes in vivo. Mol Cell Biol 21: 4773–4784, 2001.[Abstract/Free Full Text]

20. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323: 643–646, 1986.[CrossRef][Web of Science][Medline]

21. Gardner A, Phillips-Mason PJ, Raben DM, Baldassare JJ. A novel role for Gq{alpha} in {alpha}-thrombin-mediated mitogenic signalling pathways. Cell Signal 14: 499–507, 2002.[CrossRef][Web of Science][Medline]

22. Geisen C, Moroy T. The oncogenic activity of cyclin E is not confined to Cdk2 activation alone but relies on several other, distinct functions of the protein. J Biol Chem 277: 39909–39918, 2002.[Abstract/Free Full Text]

23. Geng Y, Eaton EN, Picón M, Roberts JM, Lundberg AS, Gifford A, Sardet C, Weinberg RA. Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. Oncogene 12: 1173–1180, 1996.[Web of Science][Medline]

24. Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 67: 293–302, 1991.[CrossRef][Web of Science][Medline]

25. Gray-Bablin J, Zalvide J, Fox MP, Knickerbocker CJ, DeCaprio JA, Keyomarsi K. Cyclin E, a redundant cyclin in breast cancer. Proc Natl Acad Sci USA 93: 15215–15220, 1996.[Abstract/Free Full Text]

26. Harbour JW, Dean DC. Rb function in cell-cycle regulation and apoptosis. Nat Cell Biol 2: E65–E67, 2000.[CrossRef][Web of Science][Medline]

27. Harbour JW, Lai SL, Whang-Peng J, Gazdar AF, Minna JD, Kaye FJ. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 241: 353–357, 1988.[Abstract/Free Full Text]

28. Harbour JW, Luo RX, Dei SA, Postigo AA, Dean DC. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 98: 859–869, 1999.[CrossRef][Web of Science][Medline]

29. Harwell RM, Porter DC, Danes C, Keyomarsi K. Processing of cyclin E differs between normal and tumor breast cells. Cancer Res 60: 481–489, 2000.[Abstract/Free Full Text]

30. Hassler M, Singh S, Yue WW, Luczynski M, Lakbir R, Sanchez-Sanchez F, Bader T, Pearl LH, Mittnacht S. Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture. Mol Cell 28: 371–385, 2007.[CrossRef][Web of Science][Medline]

31. Hatakeyama M, Brill JA, Fink GR, Weinberg RA. Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev 8: 1759–1771, 1994.[Abstract/Free Full Text]

32. Helin K, Harlow E, Fattaey A. Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein. Mol Cell Biol 13: 6501–6508, 1993.[Abstract/Free Full Text]

33. Hiebert SW, Chellappan SP, Horowitz JM, Nevins JR. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev 6: 177–185, 1992.[Abstract/Free Full Text]

34. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 70: 993–1006, 1992.[CrossRef][Web of Science][Medline]

35. Hogg A, Bia B, Onadim Z, Cowell JK. Molecular mechanisms of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma. Proc Natl Acad Sci USA 90: 7351–7355, 1993.[Abstract/Free Full Text]

36. Horowitz JM, Yandell DW, Park SH, Canning S, Whyte P, Buchkovich K, Harlow E, Weinberg RA, Dryja TP. Point mutational inactivation of the retinoblastoma antioncogene. Science 243: 937–940, 1989.[Abstract/Free Full Text]

37. Hu QJ, Dyson N, Harlow E. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. EMBO J 9: 1147–1155, 1990.[Web of Science][Medline]

38. Huang HJ, Yee JK, Shew JY, Chen PL, Bookstein R, Friedmann T, Lee EY, Lee WH. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242: 1563–1566, 1988.[Abstract/Free Full Text]

39. Huang S, Wang NP, Tseng BY, Lee WH, Lee EH. Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. EMBO J 9: 1815–1822, 1990.[Web of Science][Medline]

40. Kaelin WG Jr. Functions of the retinoblastoma protein. Bioessays 21: 950–958, 1999.[CrossRef][Web of Science][Medline]

41. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev 7: 331–342, 1993.[Free Full Text]

42. Keenan SM, Lents NH, Baldassare JJ. Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression. J Biol Chem 279: 5387–5396, 2004.[Abstract/Free Full Text]

43. Keyomarsi K, Conte D Jr, Toyofuku W, Fox MP. Deregulation of cyclin E in breast cancer. Oncogene 11: 941–950, 1995.[Web of Science][Medline]

44. Keyomarsi K, Herliczek TW. The role of cyclin E in cell proliferation, development and cancer. Prog Cell Cycle Res 3: 171–191, 1997.[Medline]

45. Keyomarsi K, O'Leary N, Molnar G, Lees E, Fingert HJ, Pardee AB. Cyclin E, a potential prognostic marker for breast cancer. Cancer Res 54: 380–385, 1994.[Abstract/Free Full Text]

46. Keyomarsi K, Pardee AB. Redundant cyclin overexpression and gene amplification in breast cancer cells. Proc Natl Acad Sci USA 90: 1112–1116, 1993.[Abstract/Free Full Text]

47. Keyomarsi K, Tucker SL, Bedrosian I. Cyclin E is a more powerful predictor of breast cancer outcome than proliferation. Nat Med 9: 152, 2003.[CrossRef][Web of Science][Medline]

48. Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, Hortobagyi GN, Bedrosian I, Knickerbocker C, Toyofuku W, Lowe M, Herliczek TW, Bacus SS. Cyclin E and survival in patients with breast cancer. N Engl J Med 347: 1566–1575, 2002.[Abstract/Free Full Text]

49. Kitahara K, Yasui W, Kuniyasu H, Yokozaki H, Akama Y, Yunotani S, Hisatsugu T, Tahara E. Concurrent amplification of cyclin E and CDK2 genes in colorectal carcinomas. Int J Cancer 62: 25–28, 1995.[Web of Science][Medline]

50. Knudsen KE, Fribourg AF, Strobeck MW, Blanchard JM, Knudsen ES. Cyclin A is a functional target of retinoblastoma tumor suppressor protein-mediated cell cycle arrest. J Biol Chem 274: 27632–27641, 1999.[Abstract/Free Full Text]

51. Kuhling H, Alm P, Olsson H, Ferno M, Baldetorp B, Parwaresch R, Rudolph P. Expression of cyclins E, A, and B, and prognosis in lymph node-negative breast cancer. J Pathol 199: 424–431, 2003.[CrossRef][Web of Science][Medline]

52. Lee EY, To H, Shew JY, Bookstein R, Scully P, Lee WH. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 241: 218–221, 1988.[Abstract/Free Full Text]

53. Lee WH, Bookstein R, Hong F, Young LJ, Shew JY, Lee EY. Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235: 1394–1399, 1987.[Abstract/Free Full Text]

54. Lees JA, Buchkovich KJ, Marshak DR, Anderson CW, Harlow E. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J 10: 4279–4290, 1991.[Web of Science][Medline]

55. Leng X, Connell-Crowley L, Goodrich D, Harper JW. S-phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation. Curr Biol 7: 709–712, 1997.[CrossRef][Web of Science][Medline]

56. Lents NH, Baldassare JJ. CDK2 and cyclin E knockout mice: lessons from breast cancer. Trends Endocrinol Metab 15: 1–3, 2004.[CrossRef][Web of Science][Medline]

57. Lents NH, Gorges LL, Baldassare JJ. Reverse mutational analysis reveals threonine-373 as a potentially sufficient phosphorylation site for inactivation of the retinoblastoma tumor suppressor protein (pRB). Cell Cycle 5: 1699–1707, 2006.[Web of Science][Medline]

58. Lukas J, Herzinger T, Hansen K, Moroni MC, Resnitzky D, Helin K, Reed SI, Bartek J. Cyclin E-induced S phase without activation of the pRb/E2F pathway. Genes Dev 11: 1479–1492, 1997.[Abstract/Free Full Text]

59. Luo RX, Postigo AA, Dean DC. Rb interacts with histone deacetylase to repress transcription. Cell 92: 463–473, 1998.[CrossRef][Web of Science][Medline]

60. Mihara K, Cao XR, Yen A, Chandler S, Driscoll B, Murphree AL, T'Ang A, Fung YK. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246: 1300–1303, 1989.[Abstract/Free Full Text]

61. Mittnacht S, Lees JA, Desai D, Harlow E, Morgan DO, Weinberg RA. Distinct sub-populations of the retinoblastoma protein show a distinct pattern of phosphorylation. EMBO J 13: 118–127, 1994.[Web of Science][Medline]

62. Mittnacht S, Weinberg RA. G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell 65: 381–393, 1991.[CrossRef][Web of Science][Medline]

63. Nielsen SJ, Schneider R, Bauer UM, Bannister AJ, Morrison A, O'Carroll D, Firestein R, Cleary M, Jenuwein T, Herrera RE, Kouzarides T. Rb targets histone H3 methylation and HP1 to promoters. Nature 412: 561–565, 2001.[CrossRef][Web of Science][Medline]

64. Ohtani K, DeGregori J, Nevins JR. Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci USA 92: 12146–12150, 1995.[Abstract/Free Full Text]

65. Qian Y, Luckey C, Horton L, Esser M, Templeton DJ. Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding. Mol Cell Biol 12: 5363–5372, 1992.[Abstract/Free Full Text]

66. Qin XQ, Chittenden T, Livingston DM, Kaelin WG. Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev 6: 953–964, 1992.[Abstract/Free Full Text]

67. Rubin SM, Gall AL, Zheng N, Pavletich NP. Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release. Cell 123: 1093–1106, 2005.[CrossRef][Medline]

68. Rudolph P, Kuhling H, Alm P, Ferno M, Baldetorp B, Olsson H, Parwaresch R. Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer. Int J Cancer 105: 674–680, 2003.[CrossRef][Web of Science][Medline]

69. Schraml P, Bucher C, Bissig H, Nocito A, Haas P, Wilber K, Seelig S, Kononen J, Mihatsch MJ, Dirnhofer S, Sauter G. Cyclin E overexpression and amplification in human tumours. J Pathol 200: 375–382, 2003.[CrossRef][Web of Science][Medline]

70. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2: 103–112, 2002.[CrossRef][Web of Science][Medline]

71. Span PN, Tjan-Heijnen VC, Manders P, Beex LV, Sweep CG. Cyclin-E is a strong predictor of endocrine therapy failure in human breast cancer. Oncogene 22: 4898–4904, 2003.[CrossRef][Web of Science][Medline]

72. Strobeck MW, Fribourg AF, Puga A, Knudsen ES. Restoration of retinoblastoma mediated signaling to Cdk2 results in cell cycle arrest. Oncogene 19: 1857–1867, 2000.[CrossRef][Web of Science][Medline]

73. Strobeck MW, Knudsen KE, Fribourg AF, DeCristofaro MF, Weissman BE, Imbalzano AN, Knudsen ES. BRG-1 is required for RB-mediated cell cycle arrest. Proc Natl Acad Sci USA 97: 7748–7753, 2000.[Abstract/Free Full Text]

74. Vandel L, Nicolas E, Vaute O, Ferreira R, Ait-Si-Ali S, Trouche D. Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase. Mol Cell Biol 21: 6484–6494, 2001.[Abstract/Free Full Text]

75. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 81: 323–330, 1995.[CrossRef][Web of Science][Medline]

76. Yandell DW, Campbell TA, Dayton SH, Petersen R, Walton D, Little JB, McConkie-Rosell A, Buckley EG, Dryja TP. Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling. N Engl J Med 321: 1689–1695, 1989.[Abstract]

77. Zarkowska T, Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272: 12738–12746, 1997.[Abstract/Free Full Text]

78. Zhang HS, Gavin M, Dahiya A, Postigo AA, Ma D, Luo RX, Harbour JW, Dean DC. Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell 101: 79–89, 2000.[CrossRef][Web of Science][Medline]

79. Zhou Y, Li J, Xu K, Hu S, Benedict WF. Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells. Proc Natl Acad Sci USA 91: 4165–4169, 1994.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/C1151    most recent
00300.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gorges, L. L.
Right arrow Articles by Baldassare, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gorges, L. L.
Right arrow Articles by Baldassare, J. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.