©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of Human Pur with the Retinoblastoma Protein, Rb, Regulates Binding to the Single-stranded DNA Pur Recognition Element (*)

(Received for publication, July 5, 1995)

Edward M. Johnson (1)(§) Phang-Lang Chen (2) Chavdar P. Krachmarov (1) Sharon M. Barr (1) Mechael Kanovsky (1) Zhi-Wei Ma (1) Wen-Hwa Lee (2)

From the  (1)Department of Pathology and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029 and the (2)University of Texas Health Science Center, Institute of Biotechnology, San Antonio, Texas 78245

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Puralpha, a sequence-specific single-stranded DNA-binding protein implicated in control of gene transcription and DNA replication. These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Puralpha or Rb. The PuralphabulletRb complexes contain a form of Puralpha with extensive post-synthetic modification, as demonstrated following expression of Puralpha cDNA fused to a 9-amino acid epitope tag. Human Puralpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Puralpha binds to p56, an NH(2)-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Puralpha recognition element disrupts these complexes. Conversely, high concentrations of p56 prevent Puralpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Puralpha is localized to a series of modular amino acid repeats. Rb binding involves a Puralpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Puralpha to p56, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.


INTRODUCTION

The retinoblastoma protein, Rb, (^1)product of the rb tumor suppressor gene, is modified by phosphorylation in late G1 phase of the cell cycle(1, 2) . This phosphorylation alters the activity of Rb, causing it to release several cellular proteins with which it is associated and allowing the cell to progress from G1 to S-phase. (^2)Through such associations Rb controls a link between DNA synthesis, required for cell proliferation, and expression of specific genes, required for cell differentiation. Hypophosphorylated Rb associates with members of the D cyclin family(4, 5) . It has been reported that at least one member of this family, cyclin D1, together with cyclin E, participates in the phosphorylation of Rb.^2 In non-transformed cells the D cyclins reportedly form complexes which include the DNA polymerase processivity factor proliferating cell nuclear antigen(6) . Hypophosphorylated Rb also binds to the SV40 large T-antigen(7, 8) , the adenoviral protein E1A(9) , and human papilloma virus protein E7(10) , each of which plays a role in both viral gene transcription and DNA replication. Hypophosphorylated Rb also associates directly with cellular transcription factor E2F, originally identified as a protein binding to sites in the adenovirus E2A promoter (11) . E2F, which binds the same region of Rb as do the D cyclins, T-antigen, and E1A, activates many genes, including several involved in aspects of DNA replication(12, 13, 14, 15, 16) . The mammalian cellular protein Puralpha has recently been implicated in control of JC viral DNA gene transcription and DNA replication. Because of homologies between Puralpha and certain Rb-binding proteins, we have examined the interaction of Puralpha with the Rb protein.

Puralpha binds both single-stranded and double-stranded DNA in a sequence-specific fashion, but it has a 10-fold preference for the purine-rich single strand of its recognition element, which consists of repeats containing the sequence (GGN), where N is not G(17) . Two members of a Pur protein family have been identified, Puralpha and Purbeta, and the complete sequence and DNA-binding properties of Puralpha have been reported(18) . The protein consists of 322 amino acids in human and 321 in mouse(19) . Human and mouse Puralpha proteins differ by only 2 amino acids, a very high degree of conservation. Puralpha mRNA is detected in every mammalian tissue thus far examined (17) .

Evidence from a variety of sources indicates that Puralpha is a transcriptional transactivator. Puralpha has been implicated in control of transcription of genes activated by two different retroviruses. In avian fibroblasts infected with Rous sarcoma virus v-src, a PUR element functions as an enhancer for the clusterin gene. Positive regulation of clusterin gene transcription is mediated by an avian Pur protein closely related to human Puralpha(20) . In human glial cells Puralpha mediates activation of the late promoter of neurotropic virus JC by the HIV-1 Tat protein(21, 22) . Puralpha interacts with the cellular protein YB-1, which binds to the opposite strand of the JCV Puralpha recognition element. The binding of Puralpha to its element is altered by association of YB-1 with the JC viral T-antigen, possibly playing a role in the shift from early to late viral gene transcription(22) . Puralpha, encoded by a transfected expression vector, stimulates transcription of a CAT reporter gene linked to the myelin basic protein gene promoter, and a Puralpha recognition site in this promoter is required for stimulation.^2 Transactivation may be mediated by a glutamine-rich domain, including Q(7), located near the COOH terminus of Puralpha(18) .

Puralpha binds a series of recognition elements, located approximately 1.6 kilobases upstream of the P1 promoter of the human c-myc gene near the center of a recently mapped zone of initiation of DNA replication(23) . The Puralpha binding repeats are positioned at an intrinsic bend in the DNA near an extensive AT-rich region(17) . Both DNA bending and AT-rich elements are correlated with origins of replication(24, 25) . The position of PUR elements in the c-myc locus, and in other reported replication initiation zones(24, 26, 27, 28) , is reminiscent of their position in JCV. In the viral DNA PUR elements in the late-region promoter overlap the origin of replication and are juxtaposed with AT-rich regions. Puralpha recognition elements in such a configuration are present in two 98-base pair repeats located on the late side of the central palindrome of the core origin, and these elements are essential for T-antigen-mediated replication(29) . These PUR elements are necessary for helical alterations in the adjacent AT-rich region effected by T-antigen helicase activity(30) . While Puralpha and JCV T-antigen may not directly associate they mutually interact with cellular protein YB-1(22) . In this paper we show that Puralpha and SV40 T-antigen mutually interact with Rb.

Puralpha has a region of limited homology to T-antigen which may be involved in protein-protein interaction(19) . The homology of Puralpha to SV40 large T-antigen involves a region in each protein beginning with PTY and ending in SEEM (19) (aa's 251-278 in Puralpha). Allowing for a 2-amino acid gap, there are 9 identities in 26 residues. Although this homology is limited, it includes several residues conserved not only in initiator proteins of DNA tumor viruses, but also in certain cellular proteins potentially involved in initiation, including MCM2 of yeast and BM28 of human cells(19) . BM28 is a human homolog of MCM2 reportedly required for entry of cultured cells into S phase of the cell cycle(31) . Most intriguingly, the homology of Puralpha to T-antigen spans a region necessary for binding of T-antigen to Rb, the product of the human Rb tumor suppressor gene(7, 32) . In this regard it became of interest to determine whether or not Puralpha binds the Rb protein and whether or not this motif is involved. We show here that this motif is included in the region of Puralpha involved in binding to the hypophosphorylated form of Rb. Furthermore, Rb modulates the binding of Puralpha to its single-stranded recognition element, and presence of this element affects the binding of Rb to Puralpha.


EXPERIMENTAL PROCEDURES

Expression Plasmids and Mutants of Puralpha and Rb

The EcoRI insert of Puralpha cDNA from gt11 clone AB6 (18) was excised and cloned in the EcoRI site of plasmid pGEX-1T (Pharmacia Biotech Inc.), fusing the protein in frame with Escherichia coli glutathione S-transferase (GST). The resulting plasmid, denoted pGPUR4, was propagated in E. coli strain BL21 grown as a -phage DE3 lysogen and containing the pLysS plasmid(33) . This strain, denoted BL21-LysS, synthesizes lysozyme, is protease deficient, and is advantageous for isolation of insoluble or readily degraded proteins. GST-Puralpha was purified from transfected BL21 LysS using glutathione-agarose beads (Pharmacia) as described by Smith and Johnson(34) . For experiments on Rb binding, GST-T, containing the amino-terminal 273 aa's of SV40 large T-antigen (35) , was prepared in the same fashion. Puralpha deletion mutants were produced either by restriction cleavage of pGPUR4 (1-215, 1-314, D55-314, 216-322, 274-322) or by subcloning of polymerase chain reaction-generated puralpha segments in pGEX-1T (85-322, 167-322). Resulting GST fusion proteins were purified as described above. To construct Rb expression plasmid pCEP4-RB, the plasmid pRB(44-2) (36) was digested with BamHI, and the 2.8-kilobase insert fragment, containing the coding sequence of p110, was subcloned into the BamHI site of pCEP4 (Invitrogen). The Rb mutant p56 is the primary translation product of RNA transcribed from p110 cDNA in E. coli using the pETRbc plasmid. The p56 protein was purified as described previously(37) . It is the truncated COOH-terminal portion of Rb containing the protein regions involved in SV40 large T-antigen binding but lacking sequences responsible for Rb oligomerization(37) .

Binding of Rb Protein in Cell Extracts to GST Fusion Proteins Immobilized on Glutathione-agarose Beads

The amounts of different proteins bound to glutathione-agarose beads were quantified by Coomassie Blue staining after SDS-gel electrophoresis. Extracts made from 2 times 10^6 WR2E3 cells were incubated with beads containing 2-3 mg of GST or GST fusion proteins in lysis 150 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5.0 mM EDTA, 0.1% Nonidet P-40, 50 mM sodium fluoride, 1.0 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) for 30 min at room temperature. Complexes were washed extensively with lysis 150 buffer, boiled in loading buffer, and subjected to electrophoresis in 7.5% SDS-polyacrylamide gels. Gels were transferred to Immobilon membranes (Millipore) and immunoblotted with anti-Rb monoclonal antibody 11D7 (13) . Following addition of an alkaline phosphatase-conjugated secondary antibody, bound Rb protein was visualized with 5-bromo-4-chloro-3-indolylphosphate toluidinium and nitro blue tetrazolium (Promega, Madison, WI).

Construction of a Vector for Expression of Puralpha Fused to a 9-Amino Acid Epitope Tag and Detection of Tagged Puralpha

Restriction endonuclease RsrII cleaves Puralpha cDNA plasmid pPUR6 once just downstream of NH(2)-terminal Met-Ala codons. RsrII and EcoRI were used to excise the truncated Puralpha coding sequence, containing codons 3-322 plus termination and polyadenylation signals. To the RsrII terminus of this DNA fragment was ligated the following oligonucleotide encoding a 9-aa influenza virus hemagglutinin epitope tag(38) , Ala to replace Ala^2 of Puralpha and an NH(2)-terminal Met with a good initiation configuration(39) :

CTAGC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GCG

G TAC ATG GGT ATG CTA CAA GGT CTA ATG CGA CGC CTG

M Y P Y D V P D Y A A

The resulting fragment was then ligated into the NheI and EcoRI sites of eukaryotic expression vector pBK-CMV (Stratagene) downstream of the cytomegalovirus (CMV) promoter. The resulting HA-Puralpha expression vector is termed pHAPur1. This vector was transfected into cells using a modified calcium phosphate procedure (40) either alone or together with plasmid pCEP4-Rb, expressing a full-length Rb protein under control of the CMV promoter. 5 µg of each vector were transfected per monolayer dish of 10^6 cells. Cells were harvested, lysed as described above for WR2E3 cells, and subjected to immunoextraction procedures as described in the legends to the figures. Monoclonal antibody 12CA5 (38) was used for immunodetection of the HA epitope.

Monoclonal Antibodies to Puralpha

GST-Puralpha, purified from BL21-LysS as described above, was injected into BALB/c mice. Hybridomas were prepared by fusing spleen cells with cells of myeloma line SP/O using standard procedures(41) . Radioimmunoassay and Western blotting were performed to insure that each monoclonal antibody selected reacts exclusively with Puralpha and not with GST. At least three different Puralpha epitopes are recognized by the antibodies prepared. A detailed characterization of the epitope specificities of antibodies designated 16A2, 12A4, 9C12, 5B11, and 2B3 will be presented elsewhere.

Immunoprecipitation of PuralphabulletRb Complexes from Lysates of CV-1 Cells

Monkey CV-1 cells were cultured in 75-cm^2 flasks in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Flasks were washed two times with 0.15 M NaCl and placed on ice. Lysis procedures were carried out quickly to avoid aggregation of Rb and to minimize proteolysis. Extracts of 10^7 cells, either non-transfected or transfected with vector pHAPur1 and cloned as described in the previous section, were prepared by lysis at 0 °C in 4.5 ml of lysis 150 buffer supplemented to 250 mM NaCl. For indicated experiments monolayers of cells were preincubated for 24 h with 100 µCi of [S]Met in 15 ml of Met-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) in 75-cm^2 flasks. After 2 min in lysis buffer, extracts were clarified by centrifugation at 14,000 times g for 4 min in an Eppendorf centrifuge. After addition of 1.5 ml of lysis 150 buffer with no NaCl, monoclonal antibodies were added to 1.0-ml aliquots of extract. In one case, 10 µl of monoclonal anti-Rb antibody 11D7 (35) containing 2.0 µg of antibody was added. In the other case, aliquots of hybridoma supernatant (10-100 µl) containing approximately 2 µg of each of several monoclonal anti-Puralpha antibodies were added. After incubation at 0 °C for 30 min, magnetic beads coupled to sheep anti-mouse antibody (Dynal; 500 µg of beads coupled to approximately 5 µg of antibody) were added to each 1-ml aliquot. After 2 h at 4 °C with gentle shaking, beads were collected by magnetism and washed 5 times with 1 ml of 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, containing 1 mM EDTA. As an alternative to magnetic beads, after addition of antibodies to the lysate, protein A-agarose beads (25 mg of beads/ml of extract; Sigma) were added, and immunoprecipitation was carried out by 10 successive centrifugation (14,000 times g for 5 min) and washing (1.0 ml of lysis 150 buffer with aprotinin and leupeptin omitted per Eppendorf tube) steps, all at 4 °C. Proteins attached either to sheep anti-mouse magnetic beads or to protein A-agarose beads were eluted in 40 µl of SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis on 10% gels.

An Electrophoretic Method for Analysis of Puralpha-Rb Interaction in the Absence or Presence of the Single-stranded DNA Puralpha Recognition Element

This method is based on the ability of Puralpha to bind its recognition element while attached to a nitrocellulose filter. Samples of purified GST-Puralpha and p56 (0.01-5 µg) were incubated, either singly or in combination in lysis 150 buffer, with leupeptin and aprotinin omitted, for 1 h at 30 °C. To assay effects of DNA binding by Puralpha upon the GST-Puralpha interaction with p56, unlabeled MFO677, the single-stranded DNA Puralpha 24-mer recognition element(17) , was either omitted or included at 10 ng/ml in the incubation. Samples were loaded onto a gel of 3% polyacrylamide (acrylamide: bis-acrylamide = 25:1) in running buffer (40 mM Tris acetate, pH 8.0, 20 mM sodium acetate, 2 mM EDTA) essentially as described by Morrow and Haigh, Jr. (42) and subjected to electrophoresis at 40 V for 36 h. Proteins thus separated were electroblotted to a nitrocellulose filter. Each filter was then blocked with 5% Carnation powdered milk in TNE buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA) for 12 h. Filters were then incubated with P-end-labeled MFO677 (60 Ci/mmol; 200 ng/ml) in TNE containing 1% powdered milk and poly(dI-dC) (2.0 µg/ml) for 3 h at 22 °C. Binding of labeled MFO677 indicates the position of GST-Puralpha on the filter. Filters were washed 8 times with TNE buffer, air dried, and autoradiographed on DuPont Reflection film.


RESULTS

Puralpha Specifically Binds the Hypophosphorylated Form of p110

Since Puralpha has homologies to Rb-binding regions of SV40 large T-antigen and to certain other transforming proteins of DNA tumor viruses, we sought to determine whether or not Puralpha binds to the Rb protein. The cDNA of Puralpha was cloned in pGEX-1T, which allows expression of proteins as fusion proteins with GST in E. coli. As such fusion products, proteins can be isolated from bacterial extracts based on affinity of the GST portion of the fusion protein for glutathione coupled to agarose beads(34) . Both GST-Puralpha and GST-T, containing the Rb-binding 273 amino-terminal aa of SV40 large T-antigen(35) , were isolated from bacterial extracts on glutathione-agarose beads. Such beads containing equimolar amounts of each protein, in vast molar excess over calculated amounts of Rb to be applied, were then used as affinity columns to isolate Rb from extracts of WR2E3 cells. WR2E3 is an Rb-reconstituted cell line which synthesizes large quantities of normal Rb protein(43) . Western blots were used to detect Rb protein bound to each column. In this fashion the binding of different GST fusion proteins to Rb proteins could be quantitatively compared. Columns containing GST alone were used as controls. Fig. 1shows that binding of Rb to GST-Puralpha and GST-T is approximately equal. In most experiments binding of Rb to GST-Puralpha was slightly greater than to GST-T. In proliferating cells the Rb protein exists in several states of phosphorylation, the hypophosphorylated state, p110, migrating most rapidly on SDS gels(1) . Fig. 1shows that both GST-Puralpha and GST-T bind exclusively to the hypophosphorylated form of Rb, and not to phosphorylated forms pp110. In contrast, binding of Rb to control GST alone is nil.


Figure 1: Puralpha binds the hypophosphorylated form of p110. GST-Puralpha and GST-T, a mutant form of SV40 large T-antigen, were synthesized in E. coli and bound to glutathione-agarose beads as described under ``Experimental Procedures.'' Unfused GST was prepared in the same manner for use as a control. Beads containing equivalent amounts of each protein were used for each lane. The amounts used represent approximately 40 pmol of GST-Puralpha or GST-T. Beads were collected, washed, and bound proteins subjected to electrophoresis on 7.5% polyacrylamide SDS gels as described previously(35) . After electrophoresis, proteins were blotted to Immobilon membranes and probed with anti-Rb monoclonal antibody 11D7 followed by alkaline phosphatase-conjugated secondary antibody, and Rb protein bands visualized as described under ``Experimental Procedures.'' IPP represents Rb immunoprecipitated from WR2E3 cells using rabbit polyclonal anti-Rb antibody 0.47.



A PuralphabulletRb Complex Is Immunoprecipitated from Lysates of Cultured Cells by Anti-Puralpha Monoclonal Antibodies

We employed monkey CV-1 cells to examine the interaction of cellular Puralpha and Rb. CV-1 cells have a prominent G1 phase in growth culture and possess Rb in its hypophosphorylated form(44) . CV-1 cells make significant quantities of both p110 and Puralpha, and they are not known to harbor any transforming viral genome, as do HeLa cells, for example. Extracts were prepared from cells, pulsed in log phase with [S]Met, under conditions expected to release Puralpha from the nucleus and to minimize aggregation of both Rb and Puralpha. Fig. 2shows that each of several monoclonal antibodies precipitates a prominent band of approximately 47 kDa together with an array of less prominent associated proteins. The filter autoradiographed to assay [S]Met incorporation could be subsequently Western blotted using the DuPont NEN Renaissance system, a procedure relying on emission of visible light, since exposure of the film for the latter procedure is very rapid. It can be seen that each antibody precipitating a band at 47 kDa also precipitates a band at approximately 110 kDa that reacts with the anti-Rb monoclonal antibody 11D7. A control lane at the right demonstrates that 11D7 reacts highly specifically with the Rb protein in total CV-1 cell lysates. An irrelevant antibody or protein A-agarose beads alone do not precipitate either p110 or the 47-kDa protein. An additional control demonstrates that the band at 110 kDa is not generated artifactually by a monoclonal antibody (lane mab 5B11 alone). Further controls addressing this point are presented in Fig. 3. We conclude that the band recognized by the anti-Rb monoclonal antibody at 110 kDa is Rb. The amounts of Rb precipitated by each anti-Puralpha antibody in Fig. 2correlate with the intensities of the 47-kDa band. While the intensity of S labeling of the Rb band is not as high as that of the putative Puralpha band at 47 kDa, conclusions about the stoichiometry of Puralpha-Rb interaction are difficult to draw since little is known about turnover rates of these proteins and since both proteins are apparently post-synthetically modified. A band at 47 kDa is larger than expected for Puralpha since the bacterially expressed protein migrates at 38 kDa. (An even more rapidly migrating band was originally observed for HeLa Puralpha UV-coupled to a single-stranded nucleic acid(17) .) In Fig. 3we demonstrate that Puralpha synthesized in CV-1 cells is post-synthetically modified to migrate at 47 kDa.


Figure 2: Immunoprecipitation of a PuralphabulletRb complex from lysates of monkey CV-1 cells using monoclonal antibodies to Puralpha. Monkey CV-1 cells, not transfected with any vector, were cultured as monolayers, pulsed with [S]Met and lysed as described under ``Experimental Procedures.'' Monoclonal antibodies were added to aliquots of lysate, and antibody-antigen complexes extracted with protein A-agarose beads as described. Lane labels refer to the precipitating antibody as follows. prot A, protein A-agarose beads alone were added to the lysate with no prior antibody; mab's 16A2, 12A4, 9C12, 5B11, and 2B3, anti-Puralpha monoclonal antibodies; anti-HA, monoclonal antibody 12CA5, to influenza virus coat protein hemagglutinin (irrelevant antibody). Immunoprecipitates were washed, proteins eluted and subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. Bottomleftpanel, [S]Met incorporation into immunoprecipitated proteins as revealed by autoradiography of the gel. Exposure was 72 h. Markers, not visible, are the Sigma high-MW prestained standards. Topleftpanel, anti-Rb Western blot of the gel autoradiographed at bottom. The gel was electroblotted and probed with anti-Rb monoclonal antibody 11D7 using the Renaissance system (DuPont NEN). Segments of the lanes corresponding to 110 kDa are shown. Exposure of the filter was for 1.5 h. Bands from the first antibody, detected by the Renaissance second antibody, are below 80 kDa and are not shown. As a control for possible 110-kDa bands generated by the first antibody, mab 5B11 was run alone in the lane at right of this panel. Rightlane, specificity of reaction of anti-Rb mab 11D7 with a CV-1 cell lysate. A single lane containing the cell lysate was run, blotted, excised from the filter and subjected to Western blot analysis with anti-Rb mab 11D7 as described for the top panel. Additional controls for the specificity of anti-Puralpha mab and the position of the Puralpha bands in cell lysates are presented in Fig. 3.




Figure 3: Immunoprecipitation, using an anti-Rb monoclonal antibody, of a PuralphabulletRb complex from lysates of CV-1 cells transfected to overexpress Puralpha and Rb. CV-1, HeLa, and mouse NIH-3T3 cells were transfected with vector pHAPur1, and G418-resistant clones were selected as described under ``Experimental Procedures.'' pHAPur 1 expresses Puralpha mRNA, from the CMV promoter, fused to a sequence encoding the 9-amino acid HA epitope tag (38) . Monolayers of selected clones were then transiently transfected with vector pCEP4, which expresses a full-length Rb mRNA from the CMV promoter. Lysates of cells were prepared as described and subjected to immunoprecipitation using various mouse antibodies followed by Dynal magnetic beads coupled to sheep anti-mouse antibody as described under ``Experimental Procedures.'' Immunoprecipitates were washed, proteins eluted and subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. Following blotting of the gel to a membrane filter, the filter was cut so that parallel lanes could be reacted with one of two different antibodies. Western blots were performed according to the Renaissance protocol supplied by DuPont NEN. Left panel, Western blot with anti-Puralpha mouse monoclonal antibody 12A4 followed by peroxidase-conjugated goat anti-mouse antibody (Sigma). Lysate lanes indicate total lysates from CV-1, HeLa, or NIH-3T3 cells as described. Imm. ext. lanes indicate immunoextractions prepared with the following antibodies. No 1st ab, magnetic beads alone with no first antibody; Anti-Rb 11D7, monoclonal anti-Rb antibody 11D7. GST-Pur times thrombin is a lane with bacterially expressed GST-Puralpha treated with the protease thrombin, releasing Puralpha from GST, to demonstrate that the antibody reacts with Puralpha but not with GST (27.5 kDa). Right panel, Western blot from parallel lanes to those at left, but reacted with anti-HA rabbit polyclonal antibody (BabCo) followed by peroxidase-conjugated donkey anti-rabbit antibody (Amersham). No 1st ab, immunoprecipitation of the transfected CV-1 lysate with magnetic beads but no first antibody; Anti-Rb 11D7, immunprecipitation of the lysate with anti-Rb mouse monoclonal antibody 11D7 and magnetic beads; mab 9C12, mouse anti-Puralpha antibody 9C12, 20 ng; Rabbit ab 0.47, anti-Rb rabbit polyclonal antibody 0.47, 20 ng.



An Anti-Rb Monoclonal Antibody Immunoprecipitates a Post-synthetically Modified Form of Puralpha from Lysates of Transfected CV-1 Cells

We sought to determine whether, as a reciprocal to the result obtained in Fig. 2, anti-Rb antibodies could co-immunoprecipitate Puralpha from cell lysates. Since there is a family of Pur proteins in mammalian cells(17) , it is necessary to determine whether the protein co-immunoprecipitated with Rb is actually Puralpha. The experiment of Fig. 3was performed with CV-1 cells transfected with both a vector for expression of p110 and a vector for expression of Puralpha fused to a 9-amino acid epitope tag. Anti-Rb antibody 11D7 was used to immunoextract Rb and associated proteins from lysates by a procedure employing magnetic beads coupled to sheep anti-mouse second antibody. Our intention was to use dual antibody detection to determine whether any Puralpha bands observed had been, in fact, expressed from the transfected Puralpha cDNA. The panel at left in Fig. 3shows a Western blot performed with anti-Puralpha monoclonal antibody 12A4. A control lane at the right (GST-Pur times thrombin) shows that 12A4 reacts highly specifically with the bacterially expressed Puralpha and not with GST. This lane also shows that the bacterially expressed Puralpha migrates, as expected, at 38 kDa. The anti-Rb antibody extracts a band reacting with the anti-Puralpha antibody (lane Anti-Rb 11D7), and this band migrates at approximately 47 kDa, as do the S-labeled Puralpha bands of Fig. 2. The PuralphabulletRb complex is not immunoextracted with magnetic beads when no first antibody (lane No 1st ab) is added to the lysate. The band at 47 kDa is a compound band with components migrating at 45-51 kDa. This band may include the mouse first antibody heavy chain, which migrates in our gels at approximately 50 kDa. Controls in the panel at the right were performed to rule out confusion of the Puralpha band with any antibody bands. We used a rabbit polyclonal anti-HA antibody, followed by a donkey anti-rabbit second antibody, to probe the filter in the right panel of Fig. 3. Lane mab 9C12 shows that the donkey anti-rabbit antibody has virtually no reaction with the mouse monoclonal antibody run alone as a control. The adjacent lane, Rabbit ab 0.47, contains a sample of rabbit antibody alone, at the same concentration as the adjacent mouse antibody, demonstrating that the rabbit antibody does react with the peroxidase-conjugated second antibody. The panel at the right shows that the 47-kDa band reacts with the antibody for the 9-amino acid HA epitope tag (lane Anti-Rb 11D7). Note that in this lane, where the mouse antibody is not seen, no separate band can be seen at 50 kDa, and the band present, representing Puralpha, appears to comprise the more rapidly migrating component of the compound band seen in the left panel. It can be concluded that the prominent band at 47 kDa in the right panel, immunoextracted with the anti-Rb mouse antibody, represents Puralpha fused to the HA epitope. Thus the 47-kDa Puralpha band observed was expressed from the transfected Puralpha cDNA, exactly the same cDNA sequence, minus the epitope tag, that produces the 38-kDa band in E. coli (left panel, lane GST-Pur times thrombin). Therefore, the anti-Rb monoclonal antibody immunoextracts a post-synthetically modified form of Puralpha. Rb does not associate exclusively with the modified Puralpha since we have demonstrated in Fig. 1that Rb will bind to the bacterially produced, i.e. unmodified, Puralpha. The left panel of Fig. 3includes lanes with total lysates of 3 transiently transfected cell types, CV-1, HeLa, and NIH-3T3. In each case prominent Puralpha bands are seen at 45-47 kDa while only a slight band is present at 38 kDa. We conclude that most Puralpha synthesized in these cells is rapidly post-synthetically modified to migrate at higher molecular weight. It is of interest to know whether the modified form of Puralpha interacts specifically with the hypophosphorylated form of Rb. While gel electrophoretic analyses such as in Fig. 2are consistent with complex formation involving hypophosphorylated Rb, a definitive answer must involve a detailed characterization of the post-synthetic modifications of Puralpha and their effects on Rb binding.

PuralphabulletRb Complexes, Formed in a Manner Dependent on Rb Concentration, Are Dissociated in the Presence of the Single-stranded DNA Puralpha Recognition Element

We sought to determine whether Puralpha and Rb association in the absence of DNA is affected by Puralpha DNA binding. Standard gel shift conditions are not suitable for this since electrophoresis under such conditions always involves migration of protein-DNA complexes. We therefore devised a new electrophoresis and blotting method to detect protein-protein interactions in either the presence or absence of DNA, provided one of the proteins, in our case Puralpha, is a DNA-binding protein. GST-Puralpha and p56 were incubated either together or separately and subjected to nondenaturing gel electrophoresis as described under ``Experimental Procedures.'' After electrophoresis, protein bands were electroblotted to nitrocellulose and incubated with labeled oligonucleotide MFO677, the Puralpha single-stranded DNA binding element, under DNA binding conditions. After washing the filter as described, the positions of GST-Puralpha bands were discerned by autoradiography. As indicated in Fig. 4, the interactions of Puralpha with both its single-stranded DNA recognition element and with Rb itself are dependent upon Rb concentration. One interesting aspect of this experiment is that the presence of the recognition element during gel electrophoresis enhances the binding of GST-Puralpha to the labeled recognition element once on the nitrocellulose filter. The reason for this enhancement is not known but may reflect a cooperative effect of DNA concentration on binding by Puralpha. Several conclusions can be drawn from this experiment. First, in the absence of DNA p56 shifts GST-Puralpha to a diffuse, slower migrating position (lanes P+R1 through P+R100). Since both Rb and Puralpha can form homomultimeric aggregates, smearing may be expected as a consequence of their interaction in this system, as is seen when the proteins are subjected to electrophoresis together. GST alone does not bind to p56. Therefore, Puralpha and p56 associate in the absence of DNA. This association exhibits a biphasic dependence on Rb concentration: it is more pronounced at 5-fold molar excess of p56 than at 20-fold excess or higher. Second, binding to this truncated form of Rb at low Rb:Puralpha molar ratios greatly enhances the binding of Puralpha to its recognition element (lanes P+R1 and P+R5). (Note that when this DNA binding is observed, i.e. after the two proteins have been blotted to the nitrocellulose filter, they may no longer be in contact.) In most experiments the enhancement by p56 of GST-Puralpha band intensity due to bound, labeled oligonucleotide was 3-5-fold at a 5-fold molar excess of the Rb protein, as seen by comparing lane P+R5 to GST-Pur. Finally, a mutual exclusivity is evident in the interactions between Puralpha, Rb, and the single-stranded Puralpha recognition element. At 20-100-fold molar excess, Rb inhibits Puralpha binding to DNA. Conversely, the presence of the Puralpha binding element, in unlabeled form during gel electrophoresis (lanes labeled +PUR R.E.), eliminates the ability of Rb to shift Puralpha to multimeric complexes. For example, at 5-fold molar excess of p56 in the presence of MFO677 (lane P+R5, +PUR R.E.), virtually all of GST-Puralpha is seen at the position of the GSTbulletPuralphabulletDNA complex in the absence of Rb (lane GST-Pur, +PUR R.E.) rather than in the slower migrating bands seen in the absence of MFO677 (lane P+R5). We know from many experiments not presented here that the binding element does not block the ability of Puralpha to form homomultimeric aggregates. Thus the bands disrupted by the presence of the single-stranded DNA are not likely to represent multimers of Puralpha alone, but most likely represent PuralphabulletRb complexes.


Figure 4: Formation of complexes between GST-Puralpha and p56 is dependent on p56 concentration and is altered by single-stranded Puralpha recognition element. Proteins were allowed to interact, subjected to nondenaturing gel electrophoresis, blotted to a nitrocellulose membrane, and the membrane incubated with labeled MFO677 single-stranded oligonucleotide as described under ``Experimental Procedures.'' An autoradiograph is shown. p56Rb, purified p56, used at 320 ng in lane1 in absence of other proteins or DNA; GST-Pur, purified GST-Puralpha, used at 80 ng in absence of other proteins or DNA (lane2) or in the presence of 100 ng unlabeled oligonucleotide MFO677 (lane8); P + R1, etc., 80 ng of GST-Puralpha incubated with a 1-, 5-, 20-, 50-, or 100-fold molar excess of p56, respectively. Samples for the seven lanes at the left were incubated and loaded for electrophoresis in the absence of any DNA. For the six lanes at the right (+Pur R.E.), proteins were incubated and loaded for electrophoresis in the presence of 100 ng of unlabeled single-stranded Puralpha recognition element, MFO677. After electrophoresis, gel lanes were electroblotted to a nitrocellulose membrane, and the membrane was incubated with P-labeled single-stranded MFO677, washed, and subjected to autoradiography as described under ``Experimental Procedures.''



Mutational Analysis Localizes Specific DNA Binding to a Puralpha Region of Repeat Modules

A series of deletion mutants of GST-Puralpha was constructed to determine protein domains involved in binding to the Puralpha recognition element and to the Rb protein. A diagram of these mutants, together with a map of Puralpha domains, is shown in Fig. 5A. Fig. 5B shows results of a gel band-shift study using several of these mutants to assay for binding to the Puralpha single-stranded DNA recognition element. All binding assays were performed with a vast excess of unlabeled poly(dI-dC) to eliminate nonspecific DNA binding. The results localize specificity of DNA binding to the central repeat region of Puralpha. This region contains three repeats of a 23-amino acid basic module (thickline in Fig. 5A) and two repeats of a 26-amino acid acidic module (thinline in Fig. 5A). Some inhibition of binding is seen upon removing the COOH terminus of Puralpha (lanes1-314 and 1-215), but specific specific single-stranded DNA binding is still present. DNA binding by mutant 1-215 occurs primarily in multimeric aggregates. Mutant 167-322, which has three repeat modules removed, binds only slightly to DNA, albeit specifically, and primarily as a dimeric protein form. Mutant 216-322, which has an additional acidic module removed, does not bind to DNA. Mutant 85-322, not shown in Fig. 5B, showed reduced DNA binding relative to GST-Puralpha, although difficulties with aggregation were experienced with this mutant. The data indicate that the entire repeat region is involved in DNA binding and that one copy of the acidic module is essential.


Figure 5: Mutational analysis of specific single-stranded DNA-binding regions of the Puralpha protein. Deletion mutants of the Puralpha cDNA, cloned as a fusion gene in vector pGEX-1T, were constructed as described under ``Experimental Procedures'' and subjected to standard gel band-shift analysis (17) using labeled single-stranded 24-mer oligonucleotide MFO677, representing the c-myc PUR element. A, depiction of deletion mutants with respect to structural features of the Puralpha protein. The NH(2)-terminal GST portion of each fusion protein is omitted from this diagram. The * after 1-314 is to note that whereas 8 amino acids have been deleted, 14 other amino acids have been added. B, autoradiograph of a gel band-shift analysis carried out on a 6% polyacrylamide gel(17) . - and + lanes refer to the absence or presence of a 30-fold excess of unlabeled competitor MFO677 DNA. GST-Pur lanes refer to a binding reaction carried out with full-length GST-Puralpha. Other lanes refer to GST-Puralpha deletion mutants described in panelA.



Localization of the Puralpha Rb-binding Domain

Several of the GST-Puralpha deletion mutants, shown in Fig. 5A, were employed in experiments to localize the Puralpha domains involved in binding to p110. Proteins were prepared for each of these deletion mutants, and these were used to construct affinity columns over which extracts of WR2E3 cells were passed, as described for Fig. 1. As for Fig. 1, columns contained equimolar amounts of each protein to facilitate comparison of binding activity. Fig. 6A depicts the 4 GST-Puralpha deletion mutants used. Fig. 6B shows a Western blot of proteins eluted from the affinity columns and subjected to SDS-gel electrophoresis. GST alone and GST-T were used as negative and positive controls, respectively, and an immunoprecipitate of Rb proteins was included to indicate positions of hypo- and hyperphosphorylated states. As in Fig. 1, Puralpha is at least as effective as T-antigen at binding Rb, and GST alone does not bind Rb at all. Removal of the amino-terminal 85 amino acids of Puralpha actually enhances Rb binding slightly (lane GST-Pur(85-322)). Deletion of up to 216 amino acids from the amino terminus does not completely suppress Rb binding. However, deletion of residues between 216 and 274 does suppress Rb binding (compare lanes GST-Pur(216-322) and GST-Pur(274-322)). This region includes the ``psycho'' motif, previously shown to be present in several Rb-binding proteins including SV40 large T-antigen, certain viral transforming proteins, and other proteins involved in the initiation of DNA replication(19) . Other regions of Puralpha are most likely also involved, although not essentially, in Rb binding. An additional mutant, Puralpha(1-314), omits 8 amino acids at the COOH terminus, 7 of which are Glu or Asp residues, while substituting 14 other amino acids. Although this mutant binds Rb (data not shown), the efficiency is reduced, suggesting that the acidic residues cooperate in the association.


Figure 6: Binding to p110 requires a region of the Puralpha protein comprising amino acids 216-274. Binding of Rb in extracts of WR2E3 cells to affinity columns containing GST, GST-T, GST-Puralpha, or GST-Puralpha deletion mutants was carried out exactly as described for Fig. 1. A, description of GST-Puralpha deletion mutants employed in panel B. Refer to Fig. 5A for structural features of Puralpha. B, proteins bound to columns were subjected to SDS-gel electrophoresis, transferred to an Immobilon membrane, reacted with anti-Rb monoclonal antibody 11D7, and visualized as described for Fig. 1. IPP refers to an immunoprecipitate of Rb protein from WR2E3 cells. Each other lane consists of protein bound to the particular affinity column indicated.



Puralpha Binds the COOH-terminal Portion of Rb, and This Binding Is Inhibited by a Peptide Containing the T-antigen Rb-binding Site

We sought to determine which domain of Rb is involved in binding the Puralpha protein. For this we employed the p56 mutant, which is essentially the COOH-terminal half of Rb. Binding to Puralpha was assayed by the GST-fusion protein affinity method of Fig. 1and Fig. 6. Glutathione-agarose beads were coupled either to GST alone or to GST-Puralpha. Beads were incubated with aliquots of purified p56 under various conditions, washed, and bound proteins eluted with SDS gel sample buffer. It can be seen in Fig. 7that p56 does not bind to GST but that it binds strongly to GST-Puralpha. When a 20-amino acid synthetic peptide containing the T-antigen Rb-binding domain is added to the incubation mixture, the binding of p56 to Puralpha is inhibited. Note that these protein interaction reactions are all performed in the presence of a vast excess of bovine serum albumin (1 mg/ml) to block nonspecific binding and to minimize nonspecific protein concentration effects. Results indicate that the T-antigen peptide competes with Puralpha for binding to Rb, suggesting that the peptide and Puralpha bind to the same domain of Rb, i.e. the T/E1A domain. One must be cautious in interpreting these results since any study of protein-protein interactions involving truncated proteins or fusion proteins may be subject to highly complex effects of these structural alterations upon protein conformation.


Figure 7: Binding of Puralpha to p56 and inhibition by a synthetic peptide containing the Rb-binding motif of SV40 large T-antigen. The mutant p56 was incubated with either Sepharose-GST beads (GST; 5-µl bead volume; Pharmacia) or Sepharose-GST-Puralpha beads (GST-Pur; 5-µl bead volume) in lysis 150 buffer with bovine serum albumin (1.0 mg/ml), with or without 33 pmol of p56, in the presence or absence of synthetic peptide (T-Ag pept), 2X, 10X, or 100X molar excess over p56. The sequence of the peptide is: KKENLFCSEEMPSSDDEATA, all but the first two lysines were derived from the T-antigen sequence, with the LXCXE motif printed in bold. After incubation at 22 °C for 45 min, beads were collected by centrifugation, washed, and proteins eluted with SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. Production of Sepharose-GST-Puralpha beads was as described for agarose-GST-Puralpha beads, and washing was conducted with lysis 150 buffer as described under ``Experimental Procedures.'' Proteins were blotted to an Immobilon membrane and probed with anti-Rb monoclonal antibody 11D7 using the Renaissance system as described in the legend to Fig. 3. The lane at the right (CV-1 lysate) contains 50 µg of total protein from a CV-1 cell lysate to indicate the position of full-length Rb at approximately 110 kDa. Markers, not visible, were the Sigma high-MW prestained markers.




DISCUSSION

Several cellular and viral proteins have now been identified which bind to Rb. For certain of these proteins interaction has been demonstrated by co-immunoprecipitation from cell extracts or through use of yeast hybrid-protein interaction systems, evidence taken by many investigators to indicate in vivo interaction (see Goodrich and Lee (45) for a review). The results of this study demonstrate that Puralpha binds specifically to the hypophosphorylated form of the Rb protein, p110 ( Fig. 1and Fig. 6), and that the PuralphabulletRb complex is present in CV-1 cell extracts ( Fig. 2and Fig. 3). Although several different proteins bind specifically to the hypophosphorylated form of Rb, a variety of binding mechanisms are implicated. Hypophosphorylated Rb binds to three known viral proteins, SV40 large T-antigen(7, 8) , adenoviral protein E1A(9) , and human papilloma viral protein E7(10) . Each of these proteins possesses a region of 23-26 aa's, with limited homology, containing the motif LxCxE(19) . The three viral proteins each bind to a T/E1A domain located in the COOH-terminal portion of Rb. Most naturally occurring mutations in rb in cancers are found in this T/E1A domain(46, 47) . In mammalian cells the Ets-related transcription factor Elf-1 (32) and the cell cycle regulatory proteins, cyclins D1, D2, and D3(4, 5) , also possess the LxCxE motif in their regions of Rb binding. In contrast, the Rb-binding domain of the transcriptional regulatory protein E2F possesses neither the viral protein homology nor the LxCxE motif, but E2F also binds the hypophosphorylated form of Rb(12, 13, 14, 15) . A domain near the COOH terminus of E2F containing approximately 33% acidic residues may be involved in Rb binding(13, 14, 15) . Although E2F does not bind Rb through an LxCxE motif, E2F does bind to the COOH-terminal portion of Rb, as do the viral proteins. Recently a nuclear matrix Rb-binding protein, p84, has been identified using the yeast two-hybrid system(44) . This protein binds specifically to the amino-terminal portion of Rb. As do the other proteins cited here, p84 binds to the hypophosphorylated form of Rb. Thus evidence points to a complex array of protein interaction mechanisms involving Rb, all subject to control by phosphorylation. The region of Puralpha essential for Rb-binding, delineated in Fig. 6, includes a region of 28 aa's, the psycho motif, with limited homology to the T-antigen Rb-binding motif but lacking the LxCxE configuration(19) . At the COOH terminus of Puralpha lies a segment of 18 aa's with 39% Asp and Glu residues. Disruption of this region strongly inhibits Rb binding but does not eliminate it. This mutant was not included in Fig. 6because the disruption was not technically a deletion mutant but a combination of 8-aa deletion and 14-aa insertion. Binding of Puralpha to Rb may thus be complex in that disparate Puralpha domains influence the interaction. Nonetheless, the region of Rb bound by Puralpha includes or overlaps the region bound by SV40 large T-antigen. This binding domain is retained in p56, and its binding to Puralpha is blocked by a 20-aa peptide containing the T-antigen Rb-binding motif (Fig. 7).

The interaction of Puralpha and Rb in cells is highly modulated. Puralpha binds only to a hypophosphorylated form of Rb, and Rb binds to a post-synthetically altered form of Puralpha. Both of these proteins are capable of forming large multi-protein aggregates. Rb is a nuclear protein, and Puralpha is a nuclear protein which can also be extracted from cytoplasm. Little is known about the cellular compartmentalization of modified forms of either Rb or Puralpha. It is conceivable that within a particular cell compartment, e.g. the nucleus, a hypophosphorylated form of Rb could sequester, through binding and aggregation, most or all Puralpha molecules even though the latter may be present in molar excess. Such sequestering would be subject to additional modulation if only a specifically modified form of Puralpha were the target of Rb binding. It is equally likely that Puralpha could bind, and potentially alter activity, of most or all hypophosphorylated Rb molecules within a given cell compartment. Studies on the cellular distribution of modified forms of Puralpha and Rb will help elucidate the functional significance of their interaction at particular cell locations.

Association of Puralpha and Rb affects the interaction of Puralpha with its single-strand DNA recognition element in a dynamic fashion. When Rb is hypophosphorylated, as it is in early G1, it forms filamentous aggregates which are tethered to the inner regions of the nuclear membrane, possibly through associations with lamins A and C (48) or to the nuclear matrix(44) . In this state Rb is associated with Puralpha in cells, and this would limit access of Puralpha to many, perhaps all, of its recognition elements in chromosomes in a manner yet to be detailed. We show here that Puralpha also forms multimeric aggregates and that this formation is promoted by Rb (Fig. 4). The PuralphabulletRb complex is disrupted by Rb phosphorylation ( Fig. 1and Fig. 6) and by the presence of the single-stranded DNA recognition element (Fig. 4). Rb phosphorylation begins prior to entry of cells into S-phase. The presence of single-stranded DNA at origins of replication could provide a reinforcing mechanism for desequestering Puralpha from Rb since Puralpha has a 10-fold preference for binding to the purine-rich single strand of its recognition element. While it is intriguing to speculate a role for Puralpha in creating such single-stranded regions, there is presently no evidence for this in cells. For most Rb-binding proteins the functional consequences of the interaction are not known. In the present case we have established the nature of Rb binding to Puralpha in vivo and in vitro. Studies of association of Puralpha and Rb in the cell cycle, beyond the scope of the present publication, will help to detail functional aspects of the interaction.

Rb phosphorylation occurs primarily on Ser and Thr residues and is modulated during the cell cycle, Rb phosphorylation beginning in late G1(1, 2) . Each of the three Rb-binding viral proteins described above plays a role in cell transformation, and, in the case of T-antigen, in the initiation of viral DNA replication. E2F is involved in regulation of transcription of several genes, most if not all of which are themselves involved indirectly in regulation of DNA synthesis. For example, the dhfr gene, regulated by E2F(16) , controls a step in the generation of nucleotides necessary for DNA synthesis. Certain D-type cyclins which associate with the Rb protein also associate with the DNA polymerase processivity factor proliferating cell nuclear antigen(6) . Nonetheless, gene knockout experiments call into question a universal role for Rb in the cell cycle control of DNA replication. Homozygous rb fetal mice develop apparently normally until day 12-14 of embryogenesis, at which point they die(49, 50) . At death, defects are found in development of both the hematopoietic system and the hindbrain, two systems which require a cessation of proliferation for terminal differentiation. Since DNA replication clearly occurs in the early embryonic cells of rb mice, Rb cannot be essential for initiation at all origins of replication at that stage. However, it is also clear that an aberrant lack of cessation of proliferation occurs in the blood and brain tissues. It is conceivable that this loss of control occurs at the level of initiation of DNA replication, particularly if certain Rb-binding proteins, possibly including Puralpha, influence replication initiated at only a specific subset of chromosonal loci. It is also conceivable that Rb itself does not play a primary role in cell cycle control of proliferation, but that other Rb-like proteins, such as p107 or p130(51, 52, 53, 54, 55, 56) are more directly involved. The p107 protein in human U937 cells is found in a complex with cyclin A and the cdc2 kinase p33, two proteins thought to be involved in entry of cells into S-phase(57, 58) . The p130 protein associates with CDK2 and cyclins A, D(1), D(2), D(3), and E(53, 54) . It is not presently known whether Puralpha associates with p107 or p130, although both of these proteins possess homology to the Rb T-antigen-binding domain.

It is of interest regarding Puralpha that rb mice are arrested in development with defects in the hematopoietic system. The human puralpha gene has recently been localized to chromosome band 5q31(59) . Partial deletion of 5q is commonly observed in myelodysplasias and acute nonlymphocytic leukemias, causing clinical and hematological symptoms termed the 5q syndrome. Recent observations indicate that 5q31 is the most common deleted locus among 5q individuals(3, 60) . It is conceivable that Puralpha and Rb participate in a common regulatory pathway that can be altered aberrantly in different ways to cause defects in development or cancer.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA55219 (to E. M. J.) and EY05758 (to W.-H. L.) and a grant from the Council for Tobacco Research (to W.-H. L.). 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.

§
To whom correspondence should be addressed. Tel.: 212-241-7510; Fax: 212-534-7491; johnson{at}msvax.mssm.edu.

(^1)
The abbreviations used are: Rb, retinoblastoma protein; aa, amino acid; GST, glutathione S-transferase; CMV, cytomegalovirus.

(^2)
S. Haas, P. Thatikunta, A. Steplewski, E. M. Johnson, K. Khalili, and S. Amini, submitted for publication.

(^3)
C. P. Krachmarov, L. G. Chepenik, S. M. Barr, K. Khalili, and E. M. Johnson, submitted for publication.

(^4)
L. G. Chepenik, C. P. Krachmarov, E. M. Johnson, and K. Khalili, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Andrew Bergemann and Diana H.-J. Lee for experimental aid and advice. We are indebted to Dr. Thomas Moran and the Mount Sinai Hybridoma Core Facility for the preparation of anti-Puralpha monoclonal antibodies and for supplying anti-HA antibody 12CA5. Ernest F. Savransky provided excellent technical assistance.


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