Vol. 12, No. 2

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 435-443 0270-7306/92/020435-09$02.00/0 Copyright X) 1992, American Society for Microbiology

Nuclear Binding of Purified Retinoblastoma Gene Product Is Determined by Cell Cycle-Regulated Phosphorylation DENNIS J. TEMPLETON Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106 Received 12 June 1991/Accepted 29 October 1991

The retinoblastoma tumor suppressor gene-product (pRb) is a nuclear protein subject to cell cycle-regulated hyperphosphorylation. I constructed a recombinant vaccinia virus vector that expresses both the underphosphorylated and hyperphosphorylated forms of pRb and purified the recombinant protein by using immunoaffinity chromatography directed toward a synthetic carboxy-terminal epitope. To investigate the hypothesis that hyperphosphorylation of pRb is a means of controlling its growth-regulating activity, I tested purified pRb for the ability to be reincorporated into pRb-deficient nuclei in vitro. The underphosphorylated form of pRb efficiently reassociated with nuclei, but the hyperphosphorylated form remained soluble in this assay. Nuclear binding of pRb was enhanced by phosphatase treatment and reduced by phosphorylation of pRb effected by using a preparation of the cell cycle-regulatory kinase p34cdc2. Mutant-encoded proteins with altered ElA-binding domains failed to bind to nuclei. Pretreatment of target nuclei with nucleases and high-salt extraction did not alter the specificity of binding for underphosphorylated pRb. These observations demonstrate that hyperphosphorylation of pRb can regulate its interaction with nuclei, supporting the hypothesis that hyperphosphorylation controls the growth-regulatory activities of pRb. Further, at least one target of pRb binding appears to be an integral component of the nuclear envelope.

become hyperphosphorylated. We (41) have interpreted these data as indications that interaction of pRb with cellular proteins via its ElA-binding domain is a prerequisite for the recognition of pRb as a substrate for the cell cycle-regulated kinase. When these mutant proteins are expressed in COS-1 cells, they fail to become tightly associated with the cell nucleus, as similarly expressed wild-type pRb is (41). pRb is also less firmly attached to nuclear structures during phases of the cell cycle in which pRb exhibits maximal phosphorylation (31). Taken together, these findings raise the possibility that phosphorylation may serve to regulate pRb function by altering its ability to interact with nuclei. If true, this could imply that hyperphosphorylation of pRb and subsequent detachment of pRb from its nuclear target is a means of disengaging the inhibitory signal of pRb from the cell division machinery. To test this possibility, I have studied the ability of pRb to bind to cell nuclei. I used purified pRb protein to repopulate pRb-deficient nuclei in vitro and measured the ability of the alternately phosphorylated forms of pRb to associate with nuclei. In support of the hypothesis outlined above, I found that forms of pRb hyperphosphorylated in vivo have a decreased nuclear-binding ability. Further, dephosphorylation of pRb increased nuclear binding, and phosphorylation of purified pRb in vitro by the cell cycle-regulatory kinase p34cdc2 decreased the ability of pRb to bind to nuclei. Treatment of nuclei with nucleases and extraction with high salt reduced nuclear binding of pRb by about one-half but did not abolish pRb binding to the residual nuclear envelopes. Altered forms of pRb expressed from vectors mutated in the ElA-binding domains failed to bind to nuclei, suggesting that these domains of pRb are required for nuclear binding. These findings demonstrate that pRb interaction with its nuclear target is regulated by hyperphosphorylation and suggest that at least one nuclear target of pRb binding could be an integral component of the nuclear envelope.

The protein product of the retinoblastoma susceptibility gene RB-1 (pRb) is absent or altered in tumors of many cell types (18, 25, 40, 46). Much accumulated evidence suggests that inactivation of this gene is at least partially responsible for the etiology of these tumors, yet the mechanism by which expression of normal pRb prevents malignant growth re-

mains unclear. One piece of evidence that pRb functions as an antioncogene is that it forms direct and tight contacts with the protein products encoded by several DNA tumor viruses (9, 11, 43), most notably ElA and T antigen. It has been speculated that these viral oncoproteins have evolved to bind pRb as a means of counteracting the normal mechanism of pRb in regulating malignant growth. pRb is modified by phosphorylation (26). Phosphorylation of pRb varies during the cell cycle (6, 8, 28, 30), with a period of maximal phosphorylation (usually referred to as hyperphosphorylation) being attained during late G1 or S phase and a period of minimal phosphorylation beginning in M phase. Hyperphosphorylation of pRb decreases its electrophoretic mobility, so that the hyperphosphorylated form of pRb is distinguishable from the underphosphorylated form in one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cellular kinase that hyperphosphorylates pRb is suspected of being the cell cycleregulatory kinase p34cdc2 or a closely related protein, since pRb can be phosphorylated in vitro by using immunopurified p34cdc2 (27). A proposed (28, 30) mechanism by which the activity of pRb is regulated by phosphorylation suggests that the underphosphorylated form of pRb blocks passage through the cell cycle (perhaps at the G1/S boundary) and that hyperphosphorylation temporarily relieves this block, allowing cell division to occur. All mutant forms of pRb identified thus far in human tumor cells affect the domain of pRb that is sufficient for oncoprotein association (19, 21, 22). Mutants of this domain fail to associate with viral oncoproteins as predicted and also fail to 435

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MATERIALS AND METHODS

Construction of recombinant virus vectors expressing epitope-tagged pRb. Construction of the hemagglutinin antigen epitope-tagged variant of pRb has been described previously (41). The coding portion of this gene was inserted into pTM1 (12, 32) (provided by B. Moss, National Institutes of Health), using a synthetic oligonucleotide to adapt the NcoI site containing the initiation codon of the vector to the EagI site located 26 nucleotides 3' to the initiation codon of pRb. The adaptor was designed to reconstruct the authentic amino-terminal coding region of pRb into the viral expression vector; this was confirmed by nucleotide sequencing. The resulting plasmid (pRb3HA/TM1) was tested for the ability to encode full-sized pRb after transfection into vTF7.3-infected CV1 cells and was then introduced into wild-type vaccinia virus (WR strain) by recombination using standard methods. Virus was propagated on CV1 monolayer cells or in HeLa S3 cells in suspension culture. Plasmid vectors that expressed mutant pRb cDNAs 567L and A22L were constructed by substituting a DNA fragment containing the mutated region of the pRb coding region (in each case an EcoRI-to-AatII fragment, spanning codons 300 to the carboxy-terminal epitope tag) from plasmids pRb3HA567L/SVE and pRb3HAA22/SVE (41), respectively. These plasmids are termed pRb3HA567L/TM1 and

pRb3HAA22/TM1. Expression and purification of pRb. Approximately 107 CV1 cells grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum were infected with vTF7.3 and vRb2.3 at a multiplicity of infection of 5 to 10 (each virus) in 1.0 ml of medium. After 1 h at 37°C, fresh medium was added to the cultures, and the cultures were incubated at 37°C for an additional 24 h. The cells were labelled with 35S-Translabel (100 ,uCi per plate; ICN) for 6 h, then rinsed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline, and lysed in MLB (25 mM morpholinepropanesulfonic acid [MOPS; pH 7.0], 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40 [NP-40], 1 mM dithiothreitol [DTT], 2 mM sodium pyrophosphate, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 jig of aprotinin [Sigma] per ml, 1 RSg of leupeptin [Sigma] per ml, 50 ,ug of phenylmethylsulfonyl fluoride [PMSF] per ml). After a 30-min extraction period, the lysates were centrifuged at 15,000 x g for 30 min, and the supernatant was withdrawn to a fresh tube. Antibody-bound affinity matrix was prepared in advance by coupling 1 ml of mouse ascites fluid containing 3 mg of 12CA5 anti-hemagglutinin epitope antibody to 1 ml of protein-A Sepharose (Repligen), followed by extensive washing and covalent cross-linking of the bound antibody to the matrix by using dimethylpimelimidate as described by Harlow and Lane (15). Twenty microliters of the 12CA5Sepharose beads was added to the clarified cell lysate and incubated with mixing for 2 h. After washing extensively in MLB, the beads containing bound pRb were washed once in 50 mM Tris-HCl (pH 7.4) containing 1 mM DTT and resuspended in 50 ,ul of the same buffer. Bound pRb was eluted by overnight incubation with 20 p.g of the synthetic peptide (YPYDVPDYA) recognized by the antibody, representing approximately a 40-fold molar excess over the concentration of the antigen-binding domains. Mutant forms of pRb were purified from CV1 cells that were initially transfected with pRb3HA567L/TM1 or pRb3HAA22/TM1 by the liposome transfection protocol (36) and then infected with vTF7.3. After 24 h of incubation,

MOL. CELL. BIOL.

cultures were metabolically labelled and the expressed proteins were purified as described above. Nuclear isolation. Nuclei were prepared from actively growing cultures of Saos-2 cells that express no full-sized pRb polypeptide (39). Cells were trypsinized and washed with ice-cold phosphate-buffered saline, and 107 cells were resuspended in 1 ml of NPB (nuclear prep buffer: 10 mM MOPS [pH 7.0], 10 mM KCl, 2 mM MgCl2, 0.1% NP-40, 1 mM DTT, 1 ,ug of aprotinin per ml, 1 ,ug of leupeptin per ml, 50 jig of PMSF per ml, 1 mM sodium pyrophosphate, 10 mM sodium fluoride). After 30 min on ice, resuspended cells were broken with a small Dounce homogenizer (20 strokes of the A pestle) at 0C, and the nuclei were pelleted by centrifugation at 2,000 x g for 5 min. Recovered nuclei were extracted once in NPB containing 250 mM NaCl (including a 30-min incubation at 0WC) and washed once in NPB. Nuclear preparations were examined for complete cell disruption by phase-contrast microscopy and resuspended in NPB at a concentration of 107 nuclei per ml. Preparation of nuclear envelopes. Nuclear envelopes were prepared by the method of Kaufmann et al. (24) as modified by Fields et al. (13). Briefly, Saos-2 cells were lysed by Dounce homogenization in STM (50 mM Tris-HCl [pH 7.4], 250 mM sucrose, 5 mM MgSO4, 1% 2-mercaptoethanol) containing 0.02% NP-40, and nuclei were collected by centrifugation through a sucrose cushion. Nuclei were washed once in STM (step A; see below), resuspended in STM at 108 nuclei per ml, treated with 100 p.g each of DNase I (Boehringer) and RNase A (Sigma) per ml for 60 min at 37°C, and then washed again in STM (step B). Salt extraction of the nuclei was performed by resuspending nuclei at 5 x 108 per ml in 50 mM Tris-HCl (pH 7.4) and adding dropwise 4 volumes of 50 mM Tris-HCl (pH 7.4)-2 M NaCl-1% 2-mercaptoethanol with gentle vortexing. After 30 min on ice, nuclear envelopes were recovered by centrifugation at 5,000 x g for 30 min and washed once in STM (step C). Aliquots of nuclei were taken at steps A, B, and C (see above), representing intact nuclei, nuclease-treated nuclei, and saltextracted nuclear envelopes, respectively, and then were resuspended in NPB, counted, adjusted to 107 per ml, and used in the pRb-binding assay as described above. Nuclear-binding reaction. Affinity-purified pRb (5 ,ul in 50 mM Tris [pH 7.4], representing the product of 106 infected CV1 cells) was mixed with 106 Saos-2 nuclei in 100 ,ul of NPB and incubated on ice for 30 min. Nuclei were then pelleted by centrifugation at 15,000 x g for 5 min, and soluble proteins were removed carefully with a drawn-out pipette tip. This soluble fraction and the nuclear pellet both were frozen and lyophilized in a Speed-Vac (Savant) before solubilization in SDS-containing sample buffer. In some experiments, mock-binding reactions (NPB without nuclei) were performed to verify that pRb does not bind to the centrifuge tubes. Recovered proteins were fractionated on 6% SDS-polyacrylamide gels and stained with Coomassie blue to detect nuclear proteins in the appropriate fractions. In most experiments, radiolabelled proteins were detected by fluorography after impregnation of the gels with 2,5-

diphenyloxazole. Phosphatase treatment of purified pRb. Purified, eluted pRb in 50 mM Tris (pH 7.4) was treated with a mixture of 66 DEA units of bovine alkaline phosphatase (Sigma) and 0.8 U of potato acid phosphatase (Sigma). Following incubation for 30 min at room temperature, phosphatase inhibitors (100 mM NaF and 10 mM sodium pyrophosphate [final concentrations]) were added. Control reactions were included in which the same phosphatase inhibitors were added before

NUCLEAR BINDING OF THE RETINOBLASTOMA PROTEIN

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200k-

116k-0

-pppRb

97.4k-

66k-

FIG. 1. Schematic diagram depicting expression of epitopetagged pRb by using recombinant vaccinia virus vectors. CV1 cells were doubly infected with vTF7.5 (12), which expresses RNA polymerase from bacteriophage T7, and with vRb 2.3, containing a pRb coding region regulated by a 17 promoter, constructed as described in Materials and Methods. Virus vRb 2.3 encodes a form of human pRb modified by the addition of the peptide sequence recognized by monoclonal antibody 12CA5, derived from the influenza virus hemagglutinin antigen.

incubation with phosphatases. Samples of the phosphatasetreated protein were analyzed directly or used for nuclear binding as described above. In vitro phosphorylation of pRb. Protein complexes containing the kinase p34cdc2 were purified from unsynchronized, actively growing Saos-2 cells (maintained in Dulbecco modified Eagle medium supplemented with 10o fetal calf serum) by using immobilized p13s"c) beads (5) obtained commercially (Oncogene Science). Two 9-cm dishes of Saos-2 cells (ca. 107 cells) were rinsed in HEPES-buffered saline and drained well. Cells were lysed in 2 ml of MLB on ice and clarified by centrifugation at 15,000 x g. The clarified extract was added to 30 ,ul of a 50%o slurry of pl3s"cI beads and mixed for 2 h at 4°C. The beads were washed four times in 1 ml of MLB and once in 1 ml of kinase buffer (10 mM MOPS [pH 7.0], 10 mM KCl, 2 mM MgCl2, 1 mM DTT), then divided equally between three tubes, and drained. Purified pRb (5 ,ul, about 20 to 50 ng) or buffer alone (50 mM Tris 7.4, 1 mM DTT) was added to each tube containing p34-bound beads, then 30 p.J of kinase buffer containing 50 ,uCi of [y-32P]ATP (3,000 Ci/mmol; ICN), was added, and the reaction was allowed to proceed for 30 min at 22°C. The third aliquot of p34cdc2-bound beads was tested for histone Hi kinase activity. The supernatants were withdrawn, boiled in SDS-containing sample buffer, and analyzed on 6% polyacrylamide gels.

RESULTS

Purification of pRb expressed with vaccinia virus vectors. I chose the vaccinia virus system developed in Bernard Moss's laboratory (12, 32) for high-level expression of pRb. This system uses two complementing viruses for high-level expression in mammalian cells. The first virus (vTF7.5) encodes the RNA polymerase from bacteriophage T7, which in turn directs the transcription of genes under the control of a T7 promoter. I constructed a recombinant plasmid (pRb3HAITM1) that encodes the full coding region of the pRb protein under the control of the T7 promoter and incorporated this plasmid by recombination into a vaccinia virus vector (resulting in a viral vector termed vRb2.3). Recombinant, virus-encoded pRb protein is produced by coinfection of cells with vTF7.5 and vRb2.3 (Fig. 1).

FIG. 2. Purification of retinoblastoma protein expressed by recombinant vaccinia virus vector. Unlabelled CV1 cells (grown on five 15-cm plates) were infected with recombinant vaccinia viruses expressing pRb as described in Materials and Methods and mixed with one 9-cm plate of similarly infected cells labelled with 100 .Ci of "5S-Translabel (ICN), and pRb was purified from the mixture as described in Materials and Methods. One-twentieth of this protein was analyzed on a 6% polyacrylamide gel that was stained with Coomassie blue G250 (16), dried, and exposed for autoradiography without fluorography. Lanes: 1, autoradiogram depicting "S-labelled pRb; 2, photograph of the same lane from the stained gel. The amount of pRb applied to the gel is estimated as 50 ng by comparison to the staining of dilutions of phosphorylase b and Escherichia coli 3-galactosidase analyzed in adjacent lanes. Abbreviations for all figures: pRb, underphosphorylated form of the pRb; ppRb, hyperphosphorylated pRb. Positions of size markers are shown at the left.

I used a previously described (41), modified form of the human retinoblastoma gene that expresses a protein containing an additional 10 amino acids at the carboxyl terminus of the native protein. This modification adds an epitope for the monoclonal antibody 12CA5 (which recognizes the influenza virus hemagglutinin antigen [44]), allowing simple purification of the hybrid protein by immunoaffinity chromatography. The hybrid protein preserves all of the known biochemical properties of pRb and retains the ability of the wild-type pRb to inhibit cell growth, using a bioassay of Rb function (41). For simplicity of detection of small amounts of pRb, I radiolabelled virus-infected cells with [35S]methionine or 32p; before purification of pRb. Elution of the immunopurified pRb was achieved by incubation of the affinity matrix (12CA5 antibody covalently coupled to protein A-Sepharose) with an excess of the cognate peptide (YPYDVPDYA, single-letter amino acid code) recognized by the antibody. As shown in Fig. 2, the recovered 35S-labelled pRb demonstrated a spectrum of slowly migrating bands that were visible both by Coomassie blue staining (lane 2) and by autoradiography (lane 1). For the experiment shown in Fig. 2, the pRb product of many more cells (1/20 the product of 108 infected cells) was analyzed than was used in subsequent experiments (1/10 the product of 107 infected cells) in order to visualize the protein in gels by conventional staining

techniques. The slowly migrating pRb bands visible in Fig. 2 represent hyperphosphorylated pRb forms, as shown by the ability of these slowly migrating proteins to be metabolically labelled with 32p (see below) and the similarity of the two-dimensional chymotryptic peptide maps of these proteins to maps of hyperphosphorylated pRb expressed in HL60 cells (unpublished data). The purity of pRb thus prepared was estimated from Coomassie blue-stained gels as greater than 50%. Various individual preparations contain a small amount of a 200-kDa protein that comigrates with myosin, and some

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A 1

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prppRb -' -pRb

pRb--

I .*i -ON ppRb pRb

am _~

FIG. 3. Binding of the underphosphorylated form of pRb to nuclei in vitro. [35S]methionine-labelled pRb was purified as described in Materials and Methods, and the amount produced by 106 vRb2.3 and vTF7.5-infected CV1 cells was mixed with nuclei from 106 Saos-2 cells in 100 RI of low-ionic-strength buffer as described in Materials and Methods. Lanes: 1, proteins that bound to nuclei; 2, an identical aliquot of the total pRb preparation equivalent to that added to nuclei. Proteins were analyzed on 6% polyacrylamide gels containing SDS and detected by autoradiography without fluorography, which preserves the resolution of the several bands. Unbound proteins were not recovered in this experiment but were recovered in all subsequent experiments.

antibody leached from the beads. Another contaminant of these preparations is the eluting peptide, which should serve to prevent binding of the antibody to the eluted pRb. I have tested the influence of both peptide and antibody added to the pRb preparations on the nuclear binding of pRb described below and have seen no effects on the distribution of pRb (data not shown). pRb binds to isolated nuclei. In preliminary tests, I observed that the hyperphosphorylated form of pRb stably expressed in cell lines was found predominantly in the cytosolic fraction of disrupted cells, whereas the bulk of the underphosphorylated form is found in the nuclear fraction (see also reference 31). pRb expressed by the recombinant vaccinia virus vector also demonstrated that the underphosphorylated pRb was preferentially bound to the nuclear fraction of infected cell homogenates (not shown). I next developed an assay to study the interaction of pRb with nuclei in vitro. I prepared radiolabelled pRb and mixed it in low-ionic-strength (

Nuclear binding of purified retinoblastoma gene product is determined by cell cycle-regulated phosphorylation.

The retinoblastoma tumor suppressor gene product (pRb) is a nuclear protein subject to cell cycle-regulated hyperphosphorylation. I constructed a reco...
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