©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Detection of a Novel Cell Cycle-regulated Kinase Activity That Associates with the Amino Terminus of the Retinoblastoma Protein in G/M Phases (*)

Jacqueline M. Sterner (1)(§), Yoshihiko Murata (1), Hyung Goo Kim (1), Sarah B. Kennett (1), Dennis J. Templeton (2), Jonathan M. Horowitz (1)(¶)

From the (1) Departments of Molecular Cancer Biology and Microbiology, Duke University Medical Center, Durham, North Carolina 27710 and the (2) Institute of Pathology and Program in Cell Biology, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent genetic and functional evidence suggests that the amino terminus of the retinoblastoma (Rb) protein plays an important role in Rb-mediated growth suppression. To explore the mechanism(s) by which this portion of Rb may regulate cell growth, we have sought to characterize cellular proteins that associate with the Rb amino terminus using an in vitro protein-binding assay. Here we report that at least one such protein is a cell cycle-regulated Rb/histone H1 kinase (RbK) whose enzymatic and/or Rb association activity is most prevalent in G/M phases of cells. In contrast to previously characterized cyclin-dependent and Rb-associated kinases, such as cdk1 (cdc2) and cdk2, G/M RbK 1) is not depleted by incubation with p13-beads, 2) is not detected with antisera against several Rb-associated cyclins-cdks, and 3) associates with Rb via the Rb amino terminus, a region that is dispensable for interaction with other Rb-associated kinases. RbK is clearly distinct from previously characterized mitotic cdks since cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 did not associate with the Rb amino terminus. Coprecipitation experiments with Rb antisera confirmed the association of Rb with a RbK-like kinase in metaphase-arrested cells in vivo. Interestingly, G/M RbK did not appreciably associate with an analogous portion of p107, a Rb-related protein. Taken together, these data indicate that the Rb amino terminus specifically associates with a novel cell cycle-regulated kinase in late cell cycle stages.


INTRODUCTION

The retinoblastoma (Rb)() gene, Rb-1, spans a 200-kilobase pair segment of the q14 region of human chromosome 13 (1, 2, 3) . Mutational inactivation of this gene has been clearly implicated in both sporadic and familial forms of retinoblastoma (a rare intraocular tumor) and secondary neoplasms of familial retinoblastoma patients such as osteosarcoma (4) . Functional inactivation of Rb-1 is also associated with the genesis of a variety of human tumors in the general population, including small cell lung, breast, and bladder carcinomas (5, 6, 7, 8, 9, 10, 11, 12) . In addition to its role in human cancer, the Rb protein (p105-Rb) is involved in another pathway to deregulated cell growth and tumorigenesis, that being transformation induced by several families of DNA tumor viruses. Adenovirus E1A, the large-T antigens of SV40 and polyomavirus, and the E7 protein of papillomavirus can cause the outgrowth of tumorigenic cells partly dependent on their formation of stable complexes with p105-Rb (13, 14, 15, 16) .

Rb-1 encodes a set of 105-kDa nuclear phosphoproteins that are distinguished from one another by their extent of post-translational modification (9, 17, 18, 19) . The state of Rb phosphorylation is quite dynamic during the mammalian cell cycle; resting, senescent, or terminally differentiated cells express largely unphosphorylated p105-Rb, whereas in cycling cells Rb is phosphorylated prior to S phase, as well as during S and G, and then is progressively dephosphorylated during late stages of mitosis (20, 21, 22, 23) . Rb is likely to be phosphorylated by more than one kinase in vivo. Rb kinases identified to date have proven to be members of the cyclin-dependent kinase (cdk) family. cdk1 (cdc2), cdk2, and cdk4 phosphorylate Rb in vitro at a subset of sites that are phosphorylated in vivo and cdks 1 and 2 have been found in association with Rb in vivo (19, 24, 25, 26, 27, 28) . The cyclin component of these Rbkinase complexes has yet to be definitively established, although transfection of cyclins A, D, and E can stimulate Rb phosphorylation in vivo (29, 30) . It is believed, but not as yet certain, that Rb function is regulated by these cell cycle-dependent phosphorylation and dephosphorylation reactions.

Reconstitution of Rb-negative tumor cells with a wild-type Rb gene has been shown to block or reduce tumorgenicity and additional parameters of transformation in some, but not all, cell systems (31, 32, 33, 34, 35, 36, 37) . Microinjection of the carboxyl-terminal two-thirds of p105-Rb into certain Rb-negative tumor cells has been shown to block subsequent DNA synthesis and suggested that Rb's growth-limiting function may be restricted to a discrete temporal ``window'' approximately 6 h prior to the initiation of S phase (38) . These results suggested that Rb function may be critically important for the management of cell cycle progression through a G``checkpoint'' subsequent to which a cell is committed to replicating DNA. Consistent with the notion that the Rb carboxyl terminus plays an important role in mediating growth suppression, this portion of Rb has been shown to be a ``hotspot'' for mutations in human tumors (5, 6, 7, 8, 9, 31, 39) . Additionally, it is this portion of p105-Rb that is essential for binding of Rb by viral oncoproteins and for the interaction of Rb with cellular proteins, such as transcription factors ( e.g. E2F-1) and cyclincdk complexes (40, 41, 42, 43, 44, 45, 46) . Precise mapping experiments with Rb mutants created in vitro have corroborated evidence from tumor cells, delineating amino acids within exons 12 through 18 and 19 through 22 (the so called Rb-``pocket'') as being essential for p105-Rb to complex with these viral and cellular proteins (47, 48) . Thus, the carboxyl-terminal two-thirds of p105-Rb are believed to be necessary and perhaps sufficient for at least one Rb function.

However, this simplified view of Rb activity has been challenged by the results of recently reported functional assays. Karantza et al. (49) reported that the induced expression of full-length p105-Rb in synchronously growing S phase cells resulted in the arrest of cell cycle progression in G. Interestingly, an Rb cDNA carrying a mutation within the Rb carboxyl terminus that abrogates the association of Rb with SV40 large-T antigen also led to growth arrest in Gsuggesting that regions outside of the Rb pocket are necessary for growth arrest in this cell cycle compartment (49) . In addition to these observations, a variety of amino-terminal Rb mutations have been shown by Qian et al. (50) to abrogate Rb-mediated growth suppression and to block Rb phosphorylation in vivo despite the retention of E2F-binding activity within the Rb pocket. More importantly, rare instances of mutations within the Rb amino terminus have been detected in retinoblastoma patients, supporting the supposition that lesions within this portion of Rb result in profound loss-of-function mutations (51, 52) . The precise function(s) of the 400 amino acids of Rb that are linked upstream of the Rb pocket domain is(are) unknown. This amino-terminal portion of Rb is well conserved in human, mouse, chicken, and Xenopus Rb proteins, and results of in vitro mutagenesis experiments suggest that this region is also critical for Rb-mediated growth suppression (50, 53, 54, 55) . As such, Rb appears to be modular in structure, possessing a protein-binding domain within the Rb carboxyl terminus and a separable upstream region of as yet unknown function.

As a first step toward the biochemical and functional characterization of the Rb amino terminus, we sought to identify cellular proteins that specifically bound the amino terminus of human and mouse Rb proteins. Here we report the detection of a novel cell cycle-regulated serine-threonine kinase that specifically associates with this portion of Rb and phosphorylates the Rb amino terminus and histone H1 in vitro.


EXPERIMENTAL PROCEDURES

Cell Lines and Cell Culture

A549 and 5637 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD), ML-1 cells were obtained from Dr. Stephen H. Friend (Massachusetts General Cancer Center, Charlestown, MA), and CHO cells were obtained from the Duke Comprehensive Cancer Center shared tissue culture facility. Cells were cultured in Dulbecco's modified minimal essential media (DMEM) or F12 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Inc., Logan, UT) and penicillin/streptomycin under 5% COin a humidified incubator at 37 °C.

Antisera, Immunoprecipitations, and Western Blotting

Anti-Rb ascites fluid was prepared from XZ77 (56) hybridoma cells (a gift of Dr. Nicholas Dyson, Massachusetts General Hospital Cancer Center). To generate polyclonal antisera against human Rb protein, a full-length human Rb cDNA in plasmid pBSKwas used as substrate for the PCR using the exon 1 and exon 11 oligonucleotides indicated below. A resulting 1.1-kilobase pair amino-terminal Rb fragment was inserted in frame into pGEX2TK, a bacterial-fusion protein expression vector (Pharmacia Biotech, Inc.). This portion of the Rb cDNA encodes the amino-terminal 380 amino acids of human Rb protein. Following bacterial transformation, fusion proteins were induced with IPTG, and a 68-kDa GST-Rb fusion protein was purified as described previously (57) . For immunizations, a single New Zealand White rabbit (no. 9300) was sequentially immunized with 150 µg of affinity-purified fusion protein in Freund's complete and incomplete adjuvants. Polyclonal rabbit anti-cyclin A (Ab-1) and PSTAIRE (Ab-1) antibodies were obtained from Oncogene Science (Uniondale, NY), a polyclonal rabbit anti-human cdk1 carboxyl-terminal peptide antiserum was provided by Dr. Paul Nurse (Imperial Cancer Research Fund, Oxford, United Kingdom), and polyclonal rabbit anti-cyclin A and E antisera were obtained from Dr. Steven I. Reed (Scripps Institute, La Jolla, CA). A monoclonal antibody (no. 107) that detects the MAP kinases erk1/erk2 was a kind gift of Dr. Roger J. Davis (University of Massachusetts Medical Center, Worcester, MA). Monoclonal antibodies prepared against p107 (SD2, SD4, SD6, SD9, and SD15) were kindly provided as hybridoma supernatants by Dr. Nicholas Dyson (Massachusetts General Cancer Center). A mixture of these supernatants was prepared and utilized for immunoprecipitations of p107. For immunoprecipitations with XZ77 or anti-p107 antiserum, whole-cell extracts were prepared and treated as described previously (56) . Western blots were performed as previously described (58) and antigen-antibody complexes were detected using an enhanced chemiluminescent system (ECL, Amersham Corp.) and exposure to Hyperfilm (Amersham) at ambient temperature.

Construction of GST Fusion Proteins

A bacterial plasmid containing a human Rb cDNA, pBSK-HRbC (59) , was linearized with BamHI and Rb exons 1 through 11 were amplified by the PCR using Vent polymerase (New England Biolabs, Inc., Beverly, MA), a thermal cycler, and the following oligonucleotides as primers: 5`-GTCATGCCGCCCAAAACCCCCCGAAAA-3` (exon 1) and 5`-TAACTGGAGTGTGTGGAGGAATTATAT-3` (exon 11). Rb oligonucleotides were synthesized with sites for BamHI cleavage at their 5` ends and amino-terminal Rb PCR products were digested with BamHI and ligated with BamHI-digested pGEX2TK (Pharmacia), generating plasmid pGEX2TK-HuRb, such that the Rb reading frame is collinear with that of GST. To generate a similar mouse GST-Rb fusion protein, a bacterial plasmid carrying the mouse Rb cDNA, pBSK115ROX (53) , was digested with NaeI and DraI to release a 1.3-kilobase pair fragment that encodes the amino-terminal 400 amino acids of mouse Rb protein. This fragment was subsequently ligated in frame with SmaI-digested pGEX1N (Pharmacia). A full-length human p107 cDNA (60) , a kind gift of Dr. Mark E. Ewen, Dana Farber Cancer Institute, Boston, MA, was linearized with BamHI and amplified as above using the following oligonucleotides as primers: 5`-ATGTTCGAGGACAAGCC-3`, and 5`-GGAGTAATGACTGCTTC-3`. p107 oligonucleotides were synthesized with BamHI ends, and the amplified product was subsequently cloned in pGEX2TK (Pharmacia), fusing the amino-terminal 386 amino acids of p107 in-frame with GST. The nucleotide sequences at the junctions of each construction were verified by double-stranded DNA sequencing using Sequenase (United States Biochemicals Corp. Cleveland, OH), [S]dATP, and GST or Rb oligonucleotide primers. A carboxyl-terminal GST-Rb fusion protein has been previously described (61) . A control GST fusion construct, pGEX-FH15, encoding a Schistosome surface antigen was a gift of Dr. Mariano Garcia-Blanco (Duke University Medical Center, Durham, NC).

Expression and Purification of GST Fusion Proteins and in Vitro Protein Binding and Kinase Assays

BL21 bacterial cells were transformed with GST fusion plasmids, and fusion proteins were purified following induction with IPTG and collection on glutathione-agarose beads (Sigma). Fusion proteins were harvested from 100 to 1000 ml of post-induction bacterial extracts with glutathione-agarose beads that had been previously equilibrated with NETN (20 m M Tris, pH 8.0, 100 m M NaCl, 1 m M EDTA, 0.5% Nonidet P-40) supplemented with 0.5% powdered milk. Proteins were visualized by staining with Coomassie Brilliant Blue, and protein concentrations were estimated by comparison with bovine serum albumin standards.

GST fusion proteins were used in in vitro protein-binding assays as described previously (61) with the following modifications. Mammalian cell extracts were prepared from cells that were washed twice with phosphate-buffered saline (PBS) and lysed by the addition of 1 ml of EBC buffer (50 m M Tris, pH 8.0, 120 m M NaCl, 0.5% Nonidet P-40, 100 m M NaF, 200 µ M sodium orthovanadate, 1 m M phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin and leupeptin). Following incubation for 30 min on ice, cell lysates were clarified by centrifugation at 10,000 g for 10 min at 4 °C. Aliquots of cell extracts (0.5 ml, 5 10to 1 10cell equivalents) were precleared of proteins with affinity for the GST moiety by incubation with gentle rocking for 1 h at 4 °C with at least 5 µg of GST protein bound to glutathione-agarose beads. GST protein-bound beads were pelleted by brief centrifugation and discarded. Precleared cell extracts were then incubated with 5 µg of bead-bound GST fusion proteins with gentle rocking for 1 h at 4 °C. Following exhaustive washes with NETN, protein-bound beads were resuspended in 30 µl of 50 m M HEPES, pH 7.2, containing 10 or 20 m M MgCl, 1 m M dithiothreitol, 1 m M phenylmethylsulfonyl fluoride, 10 µCi of [P]ATP, and 25 µ M unlabeled ATP. In some experiments 0.1 mg/ml histone H1 (Sigma) or 1 µg of various GST fusion proteins or purified cellular proteins were included as kinase substrates. GST-E2F-1 and purified phosphotyrosine phosphatase 1B (PTP1B) were kind gifts of Drs. Joseph R. Nevins (Duke University Medical Center) and Nicholas K. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). After incubation for 10-20 min at room temperature, samples were boiled in Laemmli sample buffer and resolved on SDS-polyacrylamide gels. Radiolabeled kinase substrates were visualized following exposure to Hyperfilm for 5 h to overnight at -80 °C. Phosphoamino acid analyses were performed as described previously (19, 62) . Protein-binding and in vitro kinase assays that employed extracts from vaccinia virus-infected cells (63, 64) utilized unlabeled whole cell extracts prepared from cells multiply infected with recombinant viruses carrying epitope-tagged cyclin A-cdc2 and cyclin A-cdk2 or cyclin B-cdc2 and cyclin B-cdk2 cDNAs. Each cyclin cDNA was epitope tagged at its amino terminus with a nine amino acid moiety (NH-EEEEYMPME-COH) derived from the medium T antigen of polyoma virus to which a monoclonal antibody was available.

p13Purification and Extract Depletion

BL21 cells were transformed with a bacterial p13expression plasmid, pRKDSUC (a gift of Dr. Katherine I. Swenson, Duke University Medical Center), and p13expression was induced by the addition of IPTG to cultures at an ODof 0.6. After 4 h of growth at 37 °C, cells were harvested, resuspended in p13lysis buffer (50 m M Tris pH 8.0, 5 m M EDTA, 10% glycerol, 1 m M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and pepstatin), sonicated, and clarified by centrifugation at 17,400 g for 30 min. Recovered supernatants (6 ml) were applied to a Sephacryl S-200 column (100 cm 2.6 cm) equilibrated in 0.1 M NaHCO, pH 8.3, and 0.5 M NaCl. Aliquots of column fractions were resolved on 13% SDS-polyacrylamide gels, stained with Coomassie Brilliant Blue, and fractions containing pure p13protein were pooled and coupled to CNBR-Sepharose (5 mg of p13protein/ml of Sepharose) according to the instructions of the manufacturer (Pharmacia). To preclear cell extracts of p13-associated kinases, 0.5 ml of unlabeled cell extracts were incubated with 100 µl of p13-Sepharose beads (containing 500 µg of p13protein) with gentle agitation for 60 min at 4 °C. Precleared cell extracts were then clarified by centrifugation at 4 °C and supernatants examined for in vitro Rb binding activity and/or histone H1 kinase activity.

Cell Cycle Synchronization and Growth Arrest

Exponentially growing A549 cells were arrested in early Gby incubation in methionine-free media supplemented with 10% fetal bovine serum for 48 h. Cells were arrested at the G/S boundary by incubation in DMEM supplemented with 5 µg/ml aphidicolin (Sigma) and 0.5 m M hydroxyurea (Sigma) for 48 h. To obtain an S phase cell population, G/S-arrested cells were washed with serum-free DMEM, refed with complete DMEM and harvested 6 h later. Populations of cells arrested in mitotic metaphase were prepared by incubation of cells in 0.4 µg/ml nocodazole (Sigma) for 16 h. CHO cells were synchronized as described by Seth et al. (65) , and cell cycle status was monitored by propidium iodide staining and flow-cytometric analysis. Approximately 2 10cells were collected by trypsinization or gentle agitation (metaphase-arrested cells), washed with PBS, and fixed in methanol for 10 min at -20 °C. Fixed cells were washed with PBS, resuspended in 10 µg/ml DNase-free RNase (Sigma), and stained with a solution containing 50 µg/ml propidium iodide for 30 min at room temperature. The timing of DNA synthesis was determined by monitoring the incorporation of [H]thymidine into mammalian cell DNA. Briefly, cells were incubated with 5 µCi/ml [H]thymidine in serum-free media, trypsinized, washed with PBS, and precipitated by the addition of 10% trichloroacetic acid. Incorporation of [H]thymidine into cell precipitates was quantified by scintillation counting.


RESULTS

The Amino Terminus of Rb Specifically Associates with a Histone H1/Rb Kinase That Phosphorylates Rb in Vitro

Since previous reports have suggested that the Rb amino terminus is functionally important, we sought to identify and characterize cellular proteins that specifically interact with this portion of Rb. We reasoned that cellular proteins (N-RBPs) that associate with Rb via its amino terminus in vivo might form stable complexes with Rb in vitro and thus might be detected by affinity chromatography. Furthermore, we speculated that one or more N-RBPs might be a cell cycle-regulated protein kinase since Rb is phosphorylated in vivo in a cell cycle-dependent manner.

To prepare amino-terminal Rb reagents for in vitro protein-binding assays, Rb oligonucleotides and the PCR were used to generate a GST fusion protein that encodes the first 380 amino acids of human Rb. In addition, an equivalent GST-Rb fusion protein was prepared from the mouse Rb cDNA. These fusion proteins include coding sequence from the first 11 Rb exons and are comprised of amino acids outside of the region of Rb required for association with viral oncoproteins and cyclincdk complexes (the Rb pocket). Following induction and purification from bacteria, GST-Rb fusion proteins were bound to glutathione-agarose beads and used as affinity reagents in an in vitro protein-binding assay followed by an in vitro kinase assay. A549 human adenocarcinoma (Rb-positive) cells were utilized to prepare unlabeled, cell cycle-staged extracts following growth arrest by methionine deprivation, treatment with hydroxyurea-aphidicolin, or nocodazole. Extracts were incubated with glutathione-agarose beads that were bound to either GST-Rb or a heterologous GST fusion protein (GST-FH15), and beads were exhaustively washed and subsequently examined for kinase activity following the addition of [-P]ATP and histone H1 as exogenous substrate. As shown in Fig. 1 A, extracts incubated with human GST-Rb contained histone H1 kinase activity that was not detected with glutathione-agarose beads bound to GST-FH15. Histone H1 kinase activity (termed RbK) was noted to be low in A549 extracts prepared from cells in G, G/S, and S phases, and maximal in mitotic extracts. Identical results were obtained with an analogous GST-Rb fusion prepared from a mouse Rb cDNA (data not shown). Moreover, as shown in Fig. 1 B the GST-Rb fusion protein utilized as an affinity reagent to recover RbK activity is also an in vitro substrate of mitotic RbK. In contrast to RbK-mediated phosphorylation of histone H1, efficient in vitro phosphorylation of GST-Rb was noted to require a somewhat lower Mgconcentration (Fig. 1 B). Significantly, in the absence of exogenous histone H1, GST-Rb was the only phosphorylated protein detected in in vitro kinase assays following incubation of GST-Rb with mitotic extracts. Neither RbK itself nor additional exogenous substrates we have examined, including Sp1, E2F-1, PTP1B, or GST , were appreciably phosphorylated by RbK in vitro, suggesting that the phosphorylation of GST-Rb and histone H1 by RbK is specific (Fig. 1 B and data not shown). We have also observed that RbK phosphorylation of Rb is largely confined to the amino terminus of human and mouse Rb proteins; GST-Rb fusion proteins carrying the carboxyl-terminal 600 amino acids of Rb are not appreciably phosphorylated by mitotic RbK in vitro (data not shown). To identify the amino acids phosphorylated by RbK, phosphoamino acid analyses of radiolabeled histone H1 and GST-Rb proteins were performed. Results of these experiments showed that serine -threonine residues are substrates of mitotic RbK (data not shown).


Figure 1:In vitro protein binding and kinase assays. Panel A, in vitro histone H1 kinase assay. Whole-cell extracts were prepared from human A549 adenocarcinoma cells arrested in early Gby methionine deprivation (G), at the G/S transition by treatment with hydroxyurea/aphidicolin (G/S), or cells arrested at metaphase by nocodazole addition ( M). In addition, a synchronously growing population of S phase cells prepared 6 h following release from hydroxyurea arrest ( S) was included for analysis. Following preclearing of cell extracts with GST-protein bound to glutathione-agarose, extracts were incubated with GST fusion proteins, and in vitro kinase assays were performed subsequent to the addition of [-P]ATP and histone H1 protein in 20 m M Mg. Radiolabeled proteins were resolved on a SDS-polyacrylamide gel and visualized by autoradiography. Left, histone H1 kinase activity recovered following incubation of cell extracts with human GST-Rb; right, histone H1 kinase activity recovered following incubation of cell extracts with GST-FH15, a heterologous GST fusion protein prepared from a Schistosome surface antigen. Histone H1 is indicated with an arrow on the right, and molecular weight markers are indicated on the left. Panel B, in vitro kinase assay in the presence and absence of histone H1. Metaphase cell extracts were prepared from nocodazole-arrested A549 cells, incubated with human GST-Rb, and analyzed as in panel A except that the in vitro kinase buffer was adjusted to 10 m M Mg. GST-Rb-bound kinase activity was assessed in the presence ( left lane) or absence ( right lane) of exogenous histone H1. Arrows on the right indicate radiolabeled GST-Rb and histone H1 proteins.



Biochemical and Immunochemical Characterization of Mitotic RbK Activity

Since cyclin-cdk activity is cell cycle dependent and members of this kinase family associate with Rb in vivo and in vitro, we reasoned that these were likely RbK candidate proteins. To determine whether previously identified cyclins-cdks account for mitotic RbK activity, three experiments were performed. 1) We assessed whether RbK activity could be depleted by prior incubation of mitotic extracts with an excess of p13-bound Sepharose beads, a regulatory protein that binds with high affinity to a variety of cyclincdk complexes. 2) We examined mitotic N-RBP eluants for reactivity with antisera to human cyclins A and E, cdk1 (cdc2), or PSTAIRE, a conserved sequence motif present in several cdks. 3) We prepared cell extracts overexpressing cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 and assessed whether these mitotic kinases could associate with and/or phosphorylate the Rb amino terminus in vitro.

To determine if excess p13protein could deplete RbK activity in mitotic extracts, we quantified RbK activity in GST-Rb eluants that had been precleared with p13beads. To ensure that p13beads were employed in excess, cdk1 abundance was examined by Western blotting with cdk1 antisera before and after incubation of mitotic extracts with p13beads. As shown in Fig. 2 A, preincubation of mitotic extracts with an excess of p13beads effectively depleted cdk1 protein from mitotic extracts ( left panel). Consistent with previously published evidence (19, 24, 25, 26, 27, 28) that Rb associates with cyclincdk complexes in vivo, Western blots of p13bead-bound proteins using anti-Rb antisera demonstrated that preincubation of mitotic extracts with an excess of p13beads also resulted in the precipitation of Rb-associated, p13-sensitive cyclincdk complexes (Fig. 2 A, right panel). Yet, despite the depletion of p13-sensitive cyclins-cdks, preincubation of mitotic extracts with p13beads had no effect on the recovery of RbK activity (Table I). Moreover, Western blots of mitotic proteins released from GST-Rb beads did not react with anti-cyclin A, anti-cyclin E, anti-cdk1, anti-PSTAIRE, or anti-MAP kinase (erk1/erk2) antisera (data not shown). Finally, to determine if known mitotic cdks could associate with and/or phosphorylate the Rb amino terminus we prepared extracts from cells infected with recombinant vaccinia viruses that overexpress the following epitope-tagged cyclins-cdks: cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2. Levels of cyclincdk complexes in such extracts have previously been estimated to be 100-1000-fold higher than in extracts prepared from uninfected cells (63, 64) . As shown in Fig. 2 B, each extract contained abundant amounts of cyclins A and B ( lanes 4 and 8) and histone H1 kinase activity ( lanes 12 and 15). However, neither cyclin A nor cyclin B kinase complexes bound the Rb amino terminus in vitro ( lanes 6 and 10) and each only marginally phosphorylated GST-Rb ( lanes 13 and 16). Consistent with a previous report (29) , cyclin A and cyclin B kinase complexes also did not associate with GST or a GST-Rb pocket fusion protein in vitro (Fig. 2 B). Taken together, our results indicate that mitotic RbK activity is not due to kinase complexes containing cyclin Acdc2 or cyclin Acdk2 (two previously analyzed Rb-associated kinases), cyclin Bcdc2 or cyclin Bcdk2, closely related cyclinscdks, or erk1-erk2 and therefore suggest that RbK is a novel amino-terminal Rb-associated mitotic kinase activity. Strong support for this conclusion is also provided by recent protein-binding experiments that indicate some cyclincdk complexes interact with Rb via the carboxyl-terminal 600 amino acids of Rb, a region not contained within our amino-terminal GST-Rb fusion proteins (25, 29, 66) . We have not as yet determined whether the N-RBP kinase(s) that we detect in extracts prepared from early cell cycle stages (Gthrough S phase) is(are) antigenically related to cdks; however, at least a portion of this activity appears to be sensitive to depletion with p13beads (see Fig. 5below).


Figure 2: Biochemical and immunochemical characterization of mitotic RbK kinase. Panel A, anti-cdk1 and anti-Rb Western blots of mitotic A549 extracts that were precleared with an excess of p13-Sepharose beads. Left, mitotic A549 extracts were precleared with p13-Sepharose, and supernatants were subsequently equilibrated with GST-Rb. p13-Sepharose and GST-Rb-bound proteins were eluted by boiling in Laemmli sample buffer and assayed for cdc2 immunoreactivity in a Western blot using a polyclonal rabbit antibody prepared against a carboxyl-terminal peptide from human cdc2. S, GST-Rb-bound proteins recovered from mitotic extracts that had been precleared with an excess of p13-Sepharose; P, p13-Sepharose-bound proteins prepared from mitotic extracts. cdc2 is indicated by an arrow on the right, and molecular weight markers are indicated on the left. Right, p13-Sepharose-bound proteins were prepared from mitotic extracts as in the left panel and assayed for Rb immunoreactivity in a Western blot with a polyclonal anti-Rb antibody (9300, 56). Panel B, in vitro protein-binding and kinase assays using recombinant vaccinia virus-infected cell extracts. Top, Coomassie-stained proteins (2 µg/lane) used in binding assays, lane 1, GST; lane 2, GST-Rb amino terminus; lane 3, GST-Rb carboxyl terminus. Center, Western blot analysis of epitope-tagged cyclin A and cyclin B in infected cell extracts ( lanes 4 and 8, respectively) and in eluants from GST beads ( lanes 5 and 9), amino-terminal Rb beads ( lanes 6 and 10), and carboxyl-terminal Rb beads ( lanes 7 and 11). Each protein binding reaction employed extracts from 3 10cells multiply infected with recombinant vaccinia viruses carrying either cyclin A-cdc2 and cyclin A-cdk2 or cyclin B-cdc2 and cyclin B-cdk2. Arrows indicate recombinant cyclin proteins. Bottom, in vitro kinase assay of virus-infected cell extracts. 20 µl (1.5 10cell equivalents) of uninfected ( lane 18) or virus-infected extracts ( lanes 12-17) were employed in each in vitro kinase assay using histone H1 and amino-terminal GST-Rb (indicated by arrows) as substrates as in Fig. 1 B. Whole cell extracts ( lanes 12 and 15), extracts following incubation with amino-terminal GST-Rb ( lanes 13 and 16), or carboxyl-terminal GST-Rb ( lanes 14 and 17) are shown. Levels of histone H1 phosphorylation in each lane were directly quantified with a PhosphorImager as: uninfected cells, 8 10counts/min; infected cells, 9.8 10-2.1 10counts/min.




Figure 5: Cell cycle dependence of recovered RbK activity in a synchronously growing population of CHO cells. CHO cells were synchronized as described previously (65), and unlabeled extracts were prepared at the indicated times. CHO cells were arrested with hydroxyurea ( G/ S) and released from growth arrest following exhaustive cell washes. Log phase CHO extracts were analyzed in parallel for comparison. The abundance of RbK activity was assayed with (+) and without (-) prior incubation of extracts with p13-Sepharose using equivalent amounts of CHO proteins and purified GST-Rb in each lane. [H]Thymidine incorporation and flow cytometry were performed as described (65). The fraction of cells in G/M phases and counts/minute of [H]thymidine incorporated into high molecular weight DNA are indicated at the bottom. Phosphorylated GST-Rb is indicated by an arrow at the right.



As one further test of the specificity of Rb-RbK association, we prepared a GST fusion protein from an analogous region of the amino terminus of p107 (60) , a Rb-related protein, and utilized this construction in in vitro protein binding and kinase assays. p107 has previously been shown to interact with many of the same viral and cellular proteins as Rb and shares several regions of sequence homology with Rb in their respective amino termini (60, 67, 68) . As shown in Fig. 3, RbK kinase activity is readily harvested from mitotic extracts depleted with p13beads using GST-Rb. In contrast, only marginal amounts of H1 kinase activity were detected with a GST-p107 amino-terminal fusion protein. We conclude that despite the structural and functional homology between Rb and p107, the differential association of RbK suggests that their amino termini are biochemically distinct.

A RbK-like Histone H1 Kinase Is Physically Associated with Rb in Metaphase-arrested Cells

The data presented thus far indicate that a potent Rb-histone H1 kinase activity may be found in association with the Rb amino terminus following incubation of mitotic extracts with Rb in vitro. It became, therefore, important to determine whether a mitotic RbK-like activity is associated with Rb in vivo. Extracts were prepared from nocodazole-arrested Rb-positive (A549 and ML-1; 11, 19) and Rb-negative (5637; 5) cells and were again exhaustively precleared with p13beads, immunoprecipitated with a Rb monoclonal antibody, XZ77 (56) , and immunoprecipitates were assayed for Rb abundance by Western blotting and coprecipitating histone H1 kinase activity in an in vitro kinase assay. XZ77 immunoprecipitates of Rb-positive extracts contained abundant amounts of hyperphosphorylated Rb protein when analyzed in Western blots using a polyclonal anti-Rb antiserum (no. 9300) prepared against the human Rb amino terminus (data not shown). As shown in Fig. 4, these immunoprecipitates also contained abundant amounts of coprecipitating histone H1 kinase activity. As would be predicted, Rb-negative mitotic extracts similarly treated showed little or no XZ77-associated histone H1 kinase activity (Fig. 4). Consistent with our in vitro results, only marginal amounts of coprecipitating histone H1 kinase activity were recovered in similar immunoprecipitates prepared with antisera against p107 (data not shown). Thus, wild-type Rb protein may be found in association with at least one mitotic p13-insensitive histone H1 kinase in vivo. Moreover, the association of this Rbkinase complex is likely to be compatible with Rb phosphorylation, since phosphorylated Rb predominates in mammalian mitotic extracts. Interestingly, similar p13-depleted immunoprecipitates of mitotic extracts that were prepared using an anti-Rb monoclonal antibody directed against the Rb amino terminus (C36; 13) did not contain significant amounts of histone H1 kinase activity nor Rb protein (data not shown). Since we and others have shown that C36 precipitates abundant amounts of Rb protein from log phase extracts or mitotic extracts that have not been precleared with p13beads, we presume that the epitope recognized by C36 is masked by one or more N-RBPs complexed with a fraction of Rb molecules in metaphase cells.


Figure 4: Coprecipitation of Rb and RbK-like histone H1 kinase activity from mitotic Rb-positive cell extracts. Mitotic extracts prepared from nocodazole-arrested Rb-positive ( A549 and ML-1; 11, 19) and Rb-negative ( 5637; 5) cells were precleared with p13-Sepharose as in Fig. 2. Supernatants were immunoprecipitated with anti-Rb antisera (XZ77; 56) and assayed for H1 kinase activity as in Fig. 1 B. An arrow on the right indicates histone H1, and molecular weight markers are indicated on the left.



RbK Enzymatic and/or Rb Association Activity Is Maximal in G/M Phases of Synchronously Growing Populations of Cells

The data presented above suggest that RbK enzymatic and/or Rb association activity is(are) most abundant in extracts prepared from metaphase cells (Fig. 1 A). To more precisely determine the abundance of RbK activity in late cell cycle stages and to ensure that the recovery of mitotic RbK activity is not induced by nocodazole arrest, we examined the cell cycle dependence of RbK activity in extracts prepared from a synchronously growing cell population. Thus, CHO cells were arrested at the G/S boundary with hydroxyurea and then released to yield a synchronously dividing cell population. Cell cycle position of synchronously growing CHO cells was monitored by [H]thymidine incorporation and by flow-cytometric analysis following staining with propidium iodide (Fig. 5). To ensure that we quantified the kinetics of recovered RbK activity and not a p13-sensitive cdk, RbK activity was assessed prior to and following preincubation with an excess of p13beads. Consistent with previous results using growth-arrested A549 cell extracts (Fig. 1 A), a low level of kinase activity is detected in Gthru S phases ( Fig. 5and Table II; G/S boundary thru 6 h post-release). The recovery of a portion of this kinase activity (approximately 50-75%) appears to be sensitive to prior incubation with p13beads (). At the transition from a population containing predominantly S phase cells to one containing predominantly G/M cells (9 h post-release from hydroxyurea arrest), the abundance of p13-insensitive kinase activity, as quantified by Rb phosphorylation in vitro, increased nearly 3-fold ( Fig. 5and ). Identical temporal results were obtained in parallel in vitro kinase assays that utilized histone H1 as exogenous substrate (data not shown). We conclude that the Rb amino terminus interacts with one or more Rb-histone H1 kinases that are most prevalent and/or active in G/M phases and that RbK activity is not artificially induced by mitotic drug arrest. These data also support our earlier observation that at least one additional kinase associates with the Rb amino terminus. The enzymatic and Rb association activities of this additional kinase are apparent prior to S phase, and the recovery of this kinase activity is diminished by preincubation with p13beads.


DISCUSSION

The biochemical results presented in this report support and extend previous genetic and functional studies indicating that the amino-terminal 380 amino acids of Rb comprise a domain of Rb function (49, 50, 51, 52) . Using an in vitro protein-binding assay, we have determined that at least one N-RBP is a novel p13-insensitive Rb-histone H1 kinase (RbK) whose enzymatic and/or Rb association activity is maximal in extracts prepared from post-S phase cells. Moreover, using a monoclonal antibody prepared against a carboxyl-terminal Rb epitope, we have shown that endogenous p105-Rb associates with a histone H1 kinase with identical biochemical properties in vivo.

Our in vitro protein-binding assays have identified at least two biochemically distinct RbKs that interact with Rb via its amino terminus. The RbKs differ from one another with respect to their propensity to physically associate with p13beads and in their temporal abundance and/or Rb association activity. The most predominant RbK, in terms of Rb-histone H1 kinase activity, is a p13-insensitive kinase that associates with Rb in vivo and in vitro. The abundance, enzymatic, and/or Rb association activity of this RbK is maximal in extracts prepared from post-S-synchronized cells. That this G/M RbK is likely to be a novel Rb-associated kinase is indicated by the following observations. 1) The abundance of G/M RbK kinase activity in cell extracts is insensitive to prior incubation with an excess of p13beads. 2) Using appropriate antisera, G/M RbK-containing protein eluants of GST-Rb beads are devoid of cyclin-cdk proteins previously shown to interact with Rb. 3) RbK associates with Rb via a region of Rb previously shown to be dispensible for interaction with several cyclincdk complexes. 4) We have shown that previously characterized mitotic cdks, cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2, do not associate with and only marginally phosphorylate the Rb amino terminus in vitro. Taken together, these data strongly support the conclusion that G/M RbK activity is not due to previously studied Rb-associated kinase complexes. In addition, we have also provided evidence for a distinct p13-sensitive kinase activity that associates with the Rb amino terminus in earlier cell cycle stages.

The identities of the RbKs are at present unknown. Our results indicate that two Rb kinases, cdc2 and cdk2, are unlikely to be G/M RbKs, and one additional Rb kinase, cdk4, has been shown not to phosphorylate histone H1. Additionally, based on our results from Western blotting experiments the G/M RbK kinase(s) appears not to be antigenically related to the MAP kinases erk1/erk2. Since Rb is subject to successive waves of phosphorylation as cells progress from late Gto mitosis (18) and only a subset of these sites of phosphorylation coincide with sites phosphorylated by cdk kinases in vitro (19, 27) , RbKs may be responsible for additional Rb phosphorylations in vivo.

Perhaps the most compelling evidence that an intact Rb amino terminus is required for tumor-suppression derives from analyses of mutated Rb alleles in retinoblastoma. Dryja et al. (52) described a large kindred with low penetrance retinoblastoma in which affected members as well as asymptomatic carriers inherited a defective Rb allele with a precise genomic deletion of exon 4. Inheritance of this Rb allele predisposed a fraction of individuals to unilateral or bilateral retinoblastoma. Similarly, Hogg et al. (51) identified an individual unrelated to the previous family with bilateral retinoblastoma associated with a single nucleotide deletion within the splice donor site of exon 4. Through genomic deletion or aberrant splicing, loss of exon 4 sequences is predicted to lead to the biogenesis of an aberrant Rb message with an in-frame fusion of exons 3 and 5. Whether the association of RbK with the Rb amino terminus is necessary for Rb-mediated growth suppression is currently under investigation. However, consistent with this supposition preliminary mapping experiments indicate that amino acids contained within Rb exon 4 are required for the recovery of wild-type levels of RbK binding activity.() In addition to analyses of naturally occurring Rb mutations in tumor cells, in vitro mutagenesis has been effectively exploited to probe Rb-mediated growth suppression. Using a series of internally deleted Rb cDNAs, Qian et al. (50) have previously shown that subtle deletions within the Rb amino terminus often yield proteins that are unphosphorylated in vivo and are functionally defective in an in vitro growth suppression assay. Importantly, such mutated Rb proteins retain the ability to physically associate with at least one target of Rb function, transcription factor E2F. Consistent with our observation that Rb amino acids comprising exon 4 are required for the recovery of wild-type levels of RbK activity, several mutated Rb cDNAs with internal deletions proximal to exon 4 have also been shown to perturb the recovery of G/M RbK activity in vitro.

Given the specific and cell cycle-regulated association of RbK with Rb, we speculate that RbRbK complexes are functional and are required for the regulation of cell cycle events subsequent to the G/S transition. This hypothesis is supported by recent evidence that the conditional expression of full-length Rb in synchronously growing cells can trigger growth arrest in G(49) . Interestingly, the conditional expression of a cDNA suffering a Rb pocket mutation was as active as wild-type Rb at eliciting Ggrowth arrest. This latter observation is consistent with the notion that interactions of Rb with proteins distinct from those that bind the Rb pocket, such as RbK, may be required for growth arrest in G. Although we can only speculate as to the precise functional role of Rb in mediating cell cycle events in G, it is likely that Rb participates in transcriptional regulation perhaps involving protein targets distinct from those in G. However, it is also conceivable that Rb performs functions in Gthat are distinct from those in earlier cell cycle stages. For example, in conjunction with RbK, Rb may function to prevent the reinitiation of DNA replication in Gor perhaps participate in a checkpoint leading to the establishment of post-mitotic or apoptotic cells. Finally, our observation that a p13-insensitive histone H1 kinase activity similar to that of RbK does not appreciably associate with the p107 amino terminus suggests that the amino termini of Rb and p107 may perform dissimilar functions. The cloning and characterization of the RbKs and cellular RbK substrates will undoubtedly provide insight into the viability of these and other possibilities.

  
Table: Abundance of p13-sensitive and -insensitive RbK activity in extracts prepared from A549 metaphase-arrested cells

A mitotic (nocodozole-arrested) A549 extract was precleared with equivalent amounts of either GST-bound glutathione-agarose or p13-Sepharose, and supernatants were subsequently equilibrated with GST-Rb-bound glutathione-agarose beads. All beads were then assayed for bound histone H1 kinase activity as in Fig. 1 B. Following autoradiography, radiolabeled histone H1 was excised from a SDS-polyacrylamide gel and quantified by scintillation counting.


  
Table: Abundance of p13-sensitive and -insensitive kinase activity in synchronously growing populations of CHO cells



FOOTNOTES

*
This work was supported in part by National Cancer Institute, National Institutes of Health Grant CA53248 and by American Cancer Society Faculty Research Award A-73970. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Robert Steel Foundation for Pediatric Cancer Research.

Supported by the Pew Scholars Program in the Biomedical Sciences. To whom correspondence should be addressed: Depts. of Molecular Cancer Biology and Microbiology, Box 3686, Duke University Medical Center, Durham, NC 27710. Tel.: 919-613-8617; Fax: 919-613-8604. E mail: jmh@galactose.mc.duke.edu.

The abbreviations used are: Rb, retinoblastoma; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-- D-galactopyranoside; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RbK, histone H1 kinase; cdk, cyclin-dependent kinase.

J. M. Sterner and J. M. Horowitz, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Joseph R. Nevins, Michael C. Ostrowski, and Katherine I. Swenson for critically reviewing this manuscript and members of the Horowitz laboratory for helpful discussions and support. We also thank Katherine I. Swenson for cyclin-cdk reagents and technical support, Charles Minkoff for help with phosphoamino acid analyses, Dr. Nicholas Dyson for monoclonal antibodies against Rb and p107, Dr. Mark E. Ewen for supplying a human p107 cDNA construct, Dr. Roger J. Davis for erk1/erk2 antisera, Dr. Nicholas K. Tonks for purified PTP1b, and Drs. Paul Nurse and Mariano Garcia-Blanco for cdk1 antisera and GST-FH15, respectively.


REFERENCES
  1. McGee, T. L., Yandell, D. W., and Dryja, T. P. (1989) Gene ( Amst.) 80, 119-128 [CrossRef][Medline] [Order article via Infotrieve]
  2. T'Ang, A., Wu, K.-J., Hashimoto, T., Liu, W.-Y., Takahashi, R., Shi, X.-H., Mihara, K., Zhang, F.-H., Chen, Y. Y., Du, C., Qian, J., Lin, Y.-G., Murphree, A. L., Qiu, W.-R., Thompson, T., Benedict, W. F., and Fung, Y-K. T. (1989) Oncogene 4, 401-407 [Medline] [Order article via Infotrieve]
  3. Hong, F. D., Huang, H.-J. S., To, H., Young, L.-J. S., Oro, A., Bookstein, R., Lee, E. Y.-H. P., and Lee, W.-H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5502-5506 [Abstract]
  4. Weinberg, R. A. (1989) Cancer Res. 49, 3713-3721 [Medline] [Order article via Infotrieve]
  5. Horowitz, J. M., Park, S.-H., Bogenmann, E., Cheng, J.-C., Yandell, D. W., Kaye, F. J., Minna, J. D., Dryja, T. P., and Weinberg, R. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2775-2779 [Abstract]
  6. Shew, J-Y., Lin, B. T-Y., Chen, P-L., Tseng, B. Y., Yang-Feng, T. L., and Lee, W-H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6-10 [Abstract]
  7. Shew, J.-Y., Chen, P.-L., Bookstein, R., Lee, E. Y.-H. P., and Lee, W.-H. (1990) Cell Growth & Differ. 1, 17-25
  8. Kaye, F. J., Kratzke, R. A., Gerster, J. L., and Horowitz, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6922-6926 [Abstract]
  9. Horowitz, J. M., Yandell, D. W., Park, S.-H., Canning, S., Whyte, P., Buchkovich, K. J., Harlow, E., Weinberg, R. A., and Dryja, T. P. (1989) Science 243, 937-940 [Medline] [Order article via Infotrieve]
  10. T'Ang, A., Varley, J. M., Chakraborty, S., Murphree, A. L., and Fung, Y.-K. T. (1988) Science 242, 263-266 [Medline] [Order article via Infotrieve]
  11. Yokota, J. T., Akiyama, T., Fung, Y.-K. T., Benedict, W. F., Namba, Y., Hanaoka, M., Wada, M., Terasaki, T., Shimosato, Y., Sugimura, T., and Terada, M. (1988) Oncogene 3, 471-475 [Medline] [Order article via Infotrieve]
  12. Lee, E. Y.-H. P., To, H., Shew, J.-Y., Bookstein, R., Scully, P., and Lee, W.-H. (1988) Science 241, 218-221 [Medline] [Order article via Infotrieve]
  13. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 334, 124-129 [CrossRef][Medline] [Order article via Infotrieve]
  14. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C.-M., Lee, W.-H., Marsilio, E., Paucha, E., and Livingston, D. M. (1988) Cell 54, 275-282 [Medline] [Order article via Infotrieve]
  15. Dyson, N., Howley, P. M., Munger, K., and Harlow, E. (1989) Science 243, 934-937 [Medline] [Order article via Infotrieve]
  16. Dyson, N., Bernards, R., Friend, S. H., Gooding, L. R., Hassell, J. A., Major, E. O., Pipas, J. M., Vandyke, T., and Harlow, E. (1990) J. Virol. 64, 1353-1356 [Medline] [Order article via Infotrieve]
  17. Lee, W.-H., Shew, J.-Y., Hong, F. D., Sery, T. W., Donoso, L. A., Young, L.-J., Bookstein, R., and Lee, E. Y.-H. P. (1987) Nature 329, 642-645 [CrossRef][Medline] [Order article via Infotrieve]
  18. DeCaprio, J. A., Furukawa, Y., Ajchenbaum, F., Griffin, J. D., and Livingston, D. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1795-1798 [Abstract]
  19. Lees, J. A., Buchkovich, K. J., Marshak, D. R., Anderson, C. W., and Harlow, E. (1991) EMBO J. 10, 4279-4290 [Abstract]
  20. Buchkovich, K., Duffy, L. A., and Harlow, E. (1989) Cell 58, 1097-1105 [Medline] [Order article via Infotrieve]
  21. Chen, P.-L., Scully, P., Shew, J.-Y., Wang, J. Y. J., and Lee, W.-H. (1989) Cell 58, 1193-1198 [Medline] [Order article via Infotrieve]
  22. Mihara, K., Cao, X.-R., Yen, A., Chandler, S., Driscoli, B., Murphree, A. L., T'Ang, A., and Fung, Y.-K. T. (1989) Science 246, 1300-1303 [Medline] [Order article via Infotrieve]
  23. Ludlow, J. W., Glendening, C. L., Livingston, D. M., and DeCaprio, J. A. (1993) Mol. Cell. Biol. 13, 367-372 [Abstract]
  24. Hu, Q., Lees, J. A., Buchkovich, K. J., and Harlow, E. (1992) Mol. Cell. Biol. 12, 971-980 [Abstract]
  25. Kato, J.-Y., Matsushime, H., Hiebert, S. W., Ewen, M. E., and Sherr, C. J. (1993) Genes & Dev. 7, 331-342
  26. Akiyama, T., Ohuchi, T., Sumida, S., Matsumoto, K., and Toyoshima, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7900-7904 [Abstract]
  27. Lin, B. T.-Y., Gruenwald, S., Morla, A. O., Lee, W. H., and Wang, J. Y. J. (1991) EMBO J. 10, 857-864 [Abstract]
  28. Taya, Y., Yasuda, H., Kamijo, M., Nakaya, K., Nakamura, Y., Ohba, Y., and Nishimura, S. (1989) Biochem. Biophys. Res. Commun. 164, 580-586 [Medline] [Order article via Infotrieve]
  29. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J.-Y., and Livingston, D. M. (1993) Cell 73, 487-497 [Medline] [Order article via Infotrieve]
  30. Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Cell 70, 993-1006 [Medline] [Order article via Infotrieve]
  31. Bookstein, R., Shew, J.-Y., Chen, P.-L., Scully, P., and Lee, W.-H. (1990) Science 247, 712-715 [Medline] [Order article via Infotrieve]
  32. Goodrich, D. W., Chen, Y., Scully, P., and Lee, W.-H. (1992) Cancer Res. 52, 1968-1973 [Abstract]
  33. Madreperla, S. A., Whittum-Hudson, J. A., Prendergast, R. A., Chen, P-L., and Lee, W-H. (1991) Cancer Res. 51, 6381-6384 [Abstract]
  34. Takahashi, R., Hashimoto, T., Xu, H-J., Hu, S-X., Matsui, T., Miki, T., Bigo-Marshall, H., Aaronson, S. A., and Benedict, W. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5257-5261 [Abstract]
  35. Huang, H-J. S., Yee, J-K., Shew, J-Y., Chen, P-L., Bookstein, R., Friedmann, T., Lee, E. Y.-H. P., and Lee, W-H. (1988) Science 242, 1563-1566 [Medline] [Order article via Infotrieve]
  36. Muncaster, M. M., Cohen, B. I., Phillips, R. A., and Gallie, B. L. (1992) Cancer Res. 654-661
  37. Xu, H-J., Sumegi, J., Hu, S-X., Banerjee, A., Uzvolgyi, E., Klein, G., and Benedict, W. F. (1991) Cancer Res. 51, 4481-4485 [Abstract]
  38. Goodrich, D. W., Wang, N. P., Qian, Y-W., Lee, E. Y.-H. P., and Lee, W-H. (1991) Cell 67, 293-302 [Medline] [Order article via Infotrieve]
  39. Bignon, Y-J., Shew, J-Y., Rappolee, D., Naylor, S. L., Lee, E. Y-H. P., Schnier, J., and Lee, W-H. (1990) Cell Growth & Differ. 7, 647-651
  40. Nevins, J. R. (1992) Science 258, 424-429 [Medline] [Order article via Infotrieve]
  41. Rustgi, A., Dyson, N., and Bernards, R. (1991) Nature 352, 541-544 [CrossRef][Medline] [Order article via Infotrieve]
  42. Kratzke, R. A., Otterson, G. A., Lin, A. Y., Shimizu, E., Alexandrova, N., Zajac-Kaye, M., Horowitz, J. M., and Kaye, F. J. (1992) J. Biol. Chem. 267, 25998-26003 [Abstract/Free Full Text]
  43. Gu, W., Schneider, J. W., Condorelli, G., Kaushai, S., Mahdavi, V., and Nadal-Ginard, B. (1993) Cell 72, 309-324 [Medline] [Order article via Infotrieve]
  44. Schneider, J. W., Gu, W., Mahdavi, V., and Nadal-Ginard, B. (1994) Science 264, 1467-1471 [Medline] [Order article via Infotrieve]
  45. Kim, S.-J., Wagner, S., Liu, F., O'Reilly, M. A., Robbins, P. D., and Green, M. R. (1992) Nature 358, 331-334 [CrossRef][Medline] [Order article via Infotrieve]
  46. Wang, C.-Y., Petryniak, B., Thompson, C. B., Kaelin, W. G., Jr., and Leiden, J. M. (1993) Science 260, 1330-1334 [Medline] [Order article via Infotrieve]
  47. Hu, Q., Dyson, N., and Harlow, E. (1990) EMBO J. 9, 1147-1155 [Abstract]
  48. Kaelin, W. G., Jr., Ewen, M. E., and Livingston, D. M. (1990) Mol. Cell. Biol. 10, 3761-3769 [Medline] [Order article via Infotrieve]
  49. Karantza, V., Maroo, A., Fay, D., and Sedivy, J. M. (1993) Mol. Cell. Biol. 13, 6640-6652 [Abstract]
  50. Qian, Y., Luckey, C., Horton, L., Esser, M., and Templeton, D. J. (1992) Mol. Cell. Biol. 12, 5363-5372 [Abstract]
  51. Hogg, A., Bia, B., Onadim, Z., and Cowell, J. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7351-7355 [Abstract]
  52. Dryja, T. P., Rapaport, J., McGee, T. L., Nork, T. M., and Schwartz, T. L. (1993) Am. J. Hum. Genet. 52, 1122-1128 [Medline] [Order article via Infotrieve]
  53. Bernards, R., Shackleford, G. M., Gerber, M. R., Horowitz, J. M., Friend, S. H., Schartl, M., Bogenmann, E., Rapaport, J. M., McGee, T., Dryja, T. P., and Weinberg, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6474-6478 [Abstract]
  54. Destree, O. H., Lam, K. T., Peterson-Maduro, L. J., Eizema, K., Diller, L., Gryka, M. A., Frebourg, T., Shibuya, E., and Friend, S. H. (1992) Dev. Biol. 153, 141-149 [Medline] [Order article via Infotrieve]
  55. Boehmelt, G., Ulrich, E., Kurzbauer, R., Mellitzer, G., Bird, A., and Zenke, M. (1994) Cell Growth & Differ. 5, 221-230
  56. Hu, Q., Bautista, C., Edwards, G. M., Defeo-Jones, D., Jones, R. E., and Harlow, E. (1991) Mol. Cell. Biol. 11, 5792-5799 [Medline] [Order article via Infotrieve]
  57. Smith, D. B., and Johnson, K. S. (1988) Gene ( Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  58. Murata, Y., Kim, H. G., Rogers, K. T., Udvadia, A. J., and Horowitz, J. M. (1994) J. Biol. Chem. 269, 20674-20681 [Abstract/Free Full Text]
  59. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T. P. (1986) Nature 323, 643 [Medline] [Order article via Infotrieve]
  60. Ewen, M. E., Xing, Y., Lawrence, J. B., and Livingston, D. M. (1991) Cell 66, 1155-1164 [Medline] [Order article via Infotrieve]
  61. Kaelin, W. G., Jr., Pallas, D. C., DeCaprio, J. A., Kaye, F. J., and Livingston, D. M. (1991) Cell 64, 521-532 [Medline] [Order article via Infotrieve]
  62. Musil, L. S., Cunningham, B. A., Edelman, G. M., and Goodenough, D. A. (1990) J. Cell Biol. 111, 2077-2088 [Abstract]
  63. Swick, A. G., Blake, M. C., Kahn, J. W., and Azizkhan, J. C. (1989) Nucleic Acids Res. 17, 9291-9304 [Abstract]
  64. Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 192-196 [Abstract]
  65. Seth, A., Gupta, S., and Davis, R. J. (1993) Mol. Cell. Biol. 13, 4125-4136 [Abstract]
  66. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499-511 [Medline] [Order article via Infotrieve]
  67. Schwarz, J. K., Devoto, S. H., Smith, E. J., Chellappan, S. P., Jakoi, L., and Nevins, J. R. (1993) EMBO J. 12, 1013-1020 [Abstract]
  68. Zhu, L., van den Heuvel, S., Helin, K., Fattaey, A., Ewen, M., Livingston, D. M., Dyson, N., and Harlow, E. (1993) Genes & Dev. 7, 1111-1125

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