Cell, Vol. 65, 1063-1072,

June

14, 1991, Copyright

0 1991 by Cell Press

The Retinoblastoma Protein Copurifies with E2F-I, an El A-Regulated Inhibitor of the Transcription Factor E2F Srilata Bagchi,” Roberto Weinmann,t and Pradip RaychaudhuriS *Department of Biochemistry (M/C 536) *Center for Research in Periodontal Disease and Oral Molecular Biology College of Dentistry (M/C 860) University of Illinois at Chicago Chicago, Illinois 60680 tThe Wistar Institute of Anatomy and Biology Philadelphia, Pennsylvania 19104-4268

Summary Recently, we identified an inhibitory protein, E2F-I, that blocks the DNA-binding activity of the transcription factor E2F. We also showed that the adenovirus ElA protein reverses this inhibitory activity of E2F-I, thereby restoring the DNA-binding activity of E2F. We have now further purlfied this inhibitory activity and show that the most purified preparation of E2F-I contains a 105 kd ElA-binding protein. This 105 kd ElAbinding protein cross-reacts with two different antibodies against the retinoblastoma (RB) gene product. Moreover, the RB gene product copurifies with E2F-I activity. Taken together, we conclude that the product of the RB gene is a part of E2F-I and is involved in the regulation of E2F activity. Introduction Transcriptional control of eukaryotic mRNA genes involves complex interaction of multiple DNA-binding proteins with regulatory sequences within promoter and enhancer elements (Johnson and McKnight, 1989; Mitchell and Tjian, 1989). In several instances, these DNA-binding proteins are found in inactive forms within cells, which prevents them from binding to their cognate regulatory sequences (see Nevins, 1989 for a review). One of the most well-documented examples of this form of regulation is that of the factor NF-lcB, which regulates transcription of several cellular as well as viral genes (reviewed in Lenardo and Baltimore, 1989). In unstimulated cells, NF-KB is present in the cytosol in an inactive form that is associated with an inhibitory protein, IKB. In stimulated cells, IKB is removed from the complex and active NF-KB migrates to the nucleus where it is able to stimulate transcription from cognate promoters (Baeuerle and Baltimore, 1988). More recently, two other DNA-binding proteins have been shown to be regulated through the interaction of specific inhibitory proteins. The DNA-binding activity of MyoD, a muscle gene transcription regulatory protein, is inhibited by heteromeric association with an inhibitory protein, Id (Benezra et al., 1990). Similarly, the DNA-binding activity of APl can be blocked by the inhibitory protein, IP-1 (Auwerx et al., 1991). Thus, there are now multiple examples

of transcriptional control mediated by inhibitory proteins that blockthe DNA-bindingabilityof keyregulatoryfactors. We have recently identified one such inhibitor, E2F-I, that specifically inhibits DNA binding of the cellular transcription factor E2F (Raychaudhuri et al., 1991). E2F has been shown to be involved in the transcription of several cellular and viral genes (see Nevins, 1989). More interestingly, the activity of E2F has been shown to be the target of various regulatory processes. E2F was initially identified as a cellular factor involved in the El A regulation of the adenovirus early E2 gene (Kovesdi et al., 1986). However, more recent analyses of the regulatory properties of E2F indicated that this factor is in fact regulated by two adenovirus early genes, El A and E4 (Babiss, 1989; Reichel et al., 1989; Raychaudhuri et al., 1990). The product of the E4-6/7 open reading frame, a 19 kd polypeptide, associates with E2F and induces the formation of a stable dimeric complex on the adenovirus E2 promoter (Huang and Hearing, 1989; Hardyet al., 1989; Raychaudhuriet al., 1990). This observation encouraged us to look for cellular proteins that would associate with E2F and control its activity. Recent analysis of E2F activity in the extracts of various cell lines has indicated that it can be found complexed with other cellular proteins and that these E2F complexes cannot interact with the E4 gene product (Bagchi et al., 1990). More importantly, ElA dissociates these cellular complexes and releases free E2F that then forms a functional complex with the E4 gene product (Bagchi et al., 1990). By fractionating mouse L cell extracts, we have recently identified two distinct activities that interact with E2F in an ElA-regulated manner (Raychaudhuri et al., 1991). One activity, E2F-BF, associates with E2F to form a larger complex that has a slower mobility in DNA band shift assays. A second activity, E2F-I, specifically blocks the DNA-binding ability of E2F by forming a complex with E2F. ElA reversed the effects of EPF-BF and E2F-I on E2F. In the case of EPF-BF, ElA dissociated the slow mobility E2F/EPF-BF-DNA complex to release the faster moving EPF-DNA complex; in the case of E2F-I, ElA reversed the inhibition and allowed EPF-DNA complex formation. Using a series of ElA mutants, we also showed that both conserved domains 1 and 2 of ElA are involved in the control of interactions between E2F and E2F regulatory proteins (Raychaudhuri et al., 1991). Furthermore, the ElA mutants that are unable to disrupt the interaction of E2F-BF and E2F-I with E2F are defective in stimulating transcription from the adenovirus E2 promoter (Raychaudhuri et al., 1991). In this communication, we have focused on the E2F-I activity that blocks the DNA-binding activity of E2F, largely because of its immediate implication in the E2F-dependent transcription. We describe characterization of E2F-I and demonstrate that the tumor suppressor gene product, pl05-RB, is a functional component of this E2F DNAbinding inhibitory activity, E2F-I.

Cell 1064

Table 1. Purification

of E2F-I Total

Steps

of Purification

1. Heparin-agarose 2. O%-40% ammonium 3. 4. 5. 6.

DEAE-Sepharose Phosphocellulose Hydroxylapatite Glycerol gradient

sulfate

Total Activity

Specific

(ms)

Protein

W)

Wmg)

500 220

NW NDb

-

10,000 7,000 4,000 1,000

500 1,750 20,000 100,000

20 4 0.2 0.01

sedimentation

a A unit (U) is arbitrarily defined as the amount of E2F-I activity required 6 pg of F9 cell nuclear extract. b Activity of E2F-I is not detectable (ND) at these steps of purification.

Purification of E2F-I E2F-I is assayed by adding column fractions to reaction mixtures containing partially purified E2F from HeLa cells or nuclear extracts of F9 teratocarcinoma cells in the presence of the other components necessary for E2F DNA binding. E2F in HeLa ceils and mouse F9 teratocarcinoma cells is largely present in free form; thus, E2F from these sources serves as a suitable substrate for assaying E2F regulatory proteins (Bagchi et al., 1990; Raychaudhuri et al., 1991). We detected E2F-I activity in the extracts of mouse L cells only after fractionation. Whole cell lysates

-

200 kd

,. -

116kd

-

Figure

1. SDS Gel Electrophoresis

97 kd

-

67 kd

-

45kd

of Glycerol

Gradient-Purified

E2F

Two hundred and fifty microliters of the glycerol gradient peak fraction of E2F-I activity was TCA precipitated and analyzed in an 8% polyacrylamide gel containing 0.1% SDS. Molecular markers (Eio-Rad, high M.W., not shown) were applied in a parallel lane. Protein bands were visualized by Coomassie blue staining.

to inhibit DNA-binding

activity

of a fixed

amount

Activity

of E2F that is present

in

of mouse L cell extracts were fractionated by heparinagarose chromatography as described before (Raychaudhuri et al., 1987, 1989). The majority of the E2F DNA-binding activity eluted as an E2FIE2FBF complex between 0.25 M and 0.6 M KCI from this column (data not shown). Proteins eluting between 0.1 M and 0.25 M KCI contained a small amount of the EPFIEPF-BF complex, a significant amount of free E2F-BF, and E2F-I. However, since both E2F-BF and E2F-I compete for E2F, the E2F-I activity was not readily apparent at this stage of fractionation. The presence of E2F-I in such a preparation became obvious during a chromatography of the heparin-agarose 0.25 M KCI eluate over a DEAE-Sepharose column. This chromatographic step separated away E2F-BF and allowed identification of E2F-I (Raychaudhuri et al., 1991). E2F-I was then further purified through phosphocellulose, hydroxylapatite, and glycerol gradient centrifugation. The purification steps are described in the Experimental Procedures, and the results are summarized in Table 1. The peak active fraction from glycerol gradient was analyzed by SDS-PAGE, followed by Coomassie blue staining. The purified E2F-I contained four major bands of molecular sizes 105 kd, 80 kd, 58 kd, and 56 kd (Figure 1). Purified E2F-I Preparation Contains a 105 kd ElA-Binding Protein E2F-I inhibits the DNA-binding activity of E2F; recently, we have shown that the adenovirus ElA gene products can restore the DNA-binding ability of E2F-l-inhibited E2F (Raychaudhuri et al., 1991). Moreover, analysisof aseries of ElA mutant gene products made in reticulocyte lysates revealed that both conserved regions (CR) 1 and 2 are involved in the restoration of E2F DNA-binding activity. These two regions of the ElA protein are also involved in binding cellular proteins (Whyte et al., 1989). This suggested that the El A-mediated reversal of E2F DNAbinding activity may involve a direct interaction of the ElA protein with the E2F/E2F-I complex. Since a stable interaction between E2F and ElA was not detected, we suspected that E2F-I must contain the ElA-binding component(s). To investigate this possibility, we employed purified E2F-I and purified bacterially produced ElA protein. As shown in Figure 2, ElA protein made in Escherichia coli can restore E2F DNA-binding activity after inhibition by the E2F-I protein. Three different amounts of ElA protein

The Retinoblastoma 1065

Protein

Copurifies

with E2F-I

EIA

E2F-I

-

f

CRI+CRZ --

CR3

__-

-PBg 8”-

I

2

::

Z:_&

.

l

l

+

+

2 “0

g

l

+

Competitor

-116Kd

-

/_:, ‘:,

I

2

3

4

Figure 2. Reversal of E2F-I Activity Synthetic ElA Peptides

5

6

by Bacterially

7

8 Produced

-

97Kd

-

67Kd

43 Kd

9 EIA and

Purified E2F-I was first incubated for 20 min with E2F under the conditions of the E2F-I assay, and then indicated amounts of purified El A, produced in E. coli, were added. Incubations were continued for another 20 min, followed by gel analysis. For experiments in lanes 6 and 7, a synthetic peptide (49-mer), CR3 corresponding to residues 140189 of the ElA protein, was used in place of EIA; in lanes 6 and 9 a synthetic peptide (37-mer), CR1 + CR2, corresponding to residues 3754 and 121-139 of the EIA protein was used in place of EIA.

were assayed, 20 ng, 50 ng, and 100 ng (lanes 3,4, and 5) after a 20 min incubation of E2F with E2F-I in reaction mixtures that contained the other components necessary for E2F-specific DNA binding (Yee et al., 1989). We also used a synthetic peptide (37-mer) corresponding to the amino acid sequence present in CR1 (residues 37 to 54) and 2 (residues 121 to 139) of ElA protein in these assays. As shown in Figure 2 (lanes 8 and 9) this peptide could also restore the DNA-binding activity of E2F after E2F-I inhibition, while a control peptide (49-mer, corresponding to CR3, residues 140 to 189 of the El A protein) had no restoration activity. These results confirmed our previous finding that CR1 and CR2 of the ElA protein are sufficient for the reversal of the E2F-I effect. To see a direct interaction of ElA and E2F-I, we labeled the E2F-I preparation with ?. The 1Z51-labeled E2F-I was first-incubated with purified ElA and then immunoprecipitated with an ElA monoclonal antibody (Oncogene Science). As shown in Figure 3, we saw specific coprecipitation of a %labeled polypeptide with an apparent molecular size of 105 kd. To address the issue of the specificity

Figure 3. Coimmunoprecipitations in Purified E2F-I

of EIA-Binding

Proteins

Present

1251-labeled E2F-I was first mixed with no peptide or 10 pg of the CR1 + CR2 peptide (see legend to Figure 2) or IO pg of the CR3 peptide (see legend to Figure 2) and then incubated for 1 hr with 1 rrg of the purified EIA protein. After incubation the reaction mixtures were immunoprecipitated with 3 ug of ElA antibody as described in the Experimental Procedures. The immunoprecipitates were analyzed by 6% SDS-PAGE followed by autoradiography.

of these interactions, we used ElA peptides as competitors in these immunoprecipitation assays. Since a synthetic peptide corresponding to CR1 and CR2 was able to replace ElA in functional assays (see Figure 2, lanes 8 and 9), we decided to use that peptide as a competitor in these immunoprecipitation assays (the ElA monoclonal antibody used in these experiments recognizes an epitope that liesoutside CR1 , CR2, and CR3 of El A protein; Whyte et al., 1989). The experiment in Figure 3 (lane 3) shows that this peptide specifically competed coprecipitation of the 105 kd polypeptide, while the control peptide, apeptide corresponding to the CR3 region of ElA, competed very poorly. Moreover, a control antibody, a monoclonal against ~53, did not precipitate the 105 kd polypeptide (data not shown). Thus, these experiments provided strong evidence for the presence of a 105 kd El A-binding protein in the most purified preparation of E2F-I. In addition, specific competition by a synthetic peptide corresponding to CR1 and CR2 of ElA suggests that binding of 105 kd protein to ElA involves CR1 and CR2 of ElA.

Cdl 1066

-

200Kd

-

ll6Krt

-

97 Kd

-

67Kd

-

45 Kd

Figure 4. lmmunoprecipitation of the 105 kd EIA-Binding Antibodies against the RB Protein

Protein

by

lmmunoprecipitations were carried out essentially as described in the Experimental Procedures except for the experiments in lanes 2,3, and 4 where the antibody (Rb-AbP, 1.5 ug) was preincubated overnight at 4OC without (lane 2) or with 7.5 ug (lane 3) or 20 tug (lane 4) of Rb peptide 1 (Oncogene Science). In lane 5 a different antibody (Rb-Abl, Oncogene Science) against the RB protein was used. The immunoprecipitates were analyzed by 8% SDS-PAGE followed by autoradiography.

Identification of the 105 kd ElA-Binding Protein as the Product of the Retinoblastoma Susceptibility Gene Several cellular proteins have been identified that bind to ElA (Harlow et al., 1986). One of these proteins is the product of the retinoblastoma (RB) gene, which is recognized by CR1 and 2 of the El A protein and has a molecular mass of 105 kd (Whyte et al., 1989). The similarities between the RB gene product and the 105 kd ElA-binding polypeptide in the E2F-I preparation prompted us to investigate whether these were the same proteins. We took two approaches to determine this. First, a polyclonal human RB peptide antibody (Rb-Ab2, Oncogene Science), raised against a C-terminal peptide, was used to immunoprecipitate the 105 kd polypeptide present in the 1251-labeled E2F-I preparation (Figure 4). As a control, antibodies previously blocked with two different amounts of the peptide, Rb (peptide 1) (Oncogene Science), against which the antisera

was raised, were used (lanes 4 and 5). Clearly, the 105 kd polypeptide present in purified E2F-I preparation was specifically recognized by the antibody against the RB peptide. Moreover, as shown in lane 6, another antibody against the RB protein (Rb-Abl, Oncogene Science) cross-reacted with the 105 kd protein. Thus, the 105 kd El A-binding polypeptide in the E2F-I preparation crossreacts with two different antibodies against ~10.5RB. In these assays, we have seen specific precipitation of two other polypeptides with apparent molecular sizes of 58 kd and 56 kd, and at this point we do not clearly understand the significance of these bands. Our second approach was to see whether the RB protein copurifies with E2F-I activity during various chromatographic procedures. E2F-I was purified from a heparinagarose 0.25 M eluate through DEAE-Sepharose, phosphocellulose, hydroxylapatite, and finally through glycerol gradient centrifugation. The RB gene product in the column fractions was assayed by Western blots using a polyclonal peptide antibody(Rb-Ab2). Ascan be seen in Figure 5, we could detect a 105 kd polypeptide that cross-reacted with the RB protein-specific antibody in the fractions where E2F-I activity was detected (data for DEAE-Sepharose column not shown). Moreover, the peak of the 105 kd polypeptide coincided with the peak of E2F-I activity. Thus, there is a clear correlation between the E2F-I activity and the RB gene product, which reinforces the notion that the RB protein is a component of the E2F-I activity. Finally, we wanted to assess the contribution of the 105 kd ElA-binding protein in E2F-I activity. To do this we have used the polymerase chain reaction (PCR) and a prokaryotic expression vector pGEX-2T (Smith and Johnson, 1988) to generate a chimeric glutathione S-transferase (GST)-ElA fusion protein. The chimeric protein was then purified following a previously described procedure (Smith and Johnson, 1988) in the form of glutathioneSepharose conjugates. Glutathione-Sepharose beads containing the El A fusion protein or just glutathione transferase were used to deplete the 105 kd ElA-binding protein from a purified preparation of E2F-I. As shown in Figure 6A, incubation of E2F-I with ElA fusion proteincontaining beads, GST-ElA-Sepharose, resulted in the depletion of E2F-I activity, whereas beads conjugated to GST alone, GST-Sepharose, did not deplete E2F-I activity. We extracted the proteins from the GST-ElA-Sepharose beads and assayed for RB protein by Western blots; the result is shown in Figure 6B. Antibody against the RB protein recognized a 105 kd band, whereas RB antibody blocked with a peptide, against which the antibody was raised, did not recognize the 105 kd band, suggesting that the ElA fusion protein depletes E2F-I activity by removing pl05-RB from the preparation. From this experiment and others described above we conclude that pl05-RB is involved in the inhibition of E2F DNA-binding activity. Discussion Work presented in this communication is of significance in three ways. First, we have characterized a regulatory protein, E2F-I, that inhibits DNA-binding activity of E2F.

The Retinoblastoma 1067

Protein

with E2F-I

Phospho-cellulose

A. E2F-I

Copurifies

Hydroxyl-apatite

6.

Assay EZF-I Assay

EBF-DNA Complex

Fr. No.

121416I8202224262830323436

p I05-RB

-

Assay

plO5-RB

202224262830323436

C. Glycerol

Fr. No.

Gradient

IO 12 I4 16 18 20 22 24 26 28 30

Fr.No.

Fr. No. plO5-RB

Sedimentation

Figure 5. Copurification E2F-I Activity

Assay

EZF-DNA Complex

-

2

3 4

5

6 7 8

9 10 II I2 I3 I4 I5 16 17

Fr.No.

p 105.RB Assay

~10%RB

IO 12 14 16 18 20 22 24 26 28 30

p 105.RB Assay

12141618

EZF-I

EZF-DNA Complex

of the RB Protein with

Chromatographies were carried out as described in the Experimental Procedures. The column fractions were assayed for E2F-I activity in gel retardation assays and for the RB protein in Western blot assays as described in the Experimental Procedures. For the E2F-I activity assay, 2 ul samples of the indicated fractions were analyzed, whereas for the p105-RB assay 250 ul samples of the same fractions were analyzed. (A) Phosphocellulose column. (B) Hydroxylapatite column. (C)Glycerol gradient sedimentation.

-

2 3 4 5 6 7 8 9 IO II

I2

I3 I4

I5

I6

I7

Second, we have shown the product of the RB gene, pl05RB, is a functional component of the E2F-I activity. Third, taken together, these experiments elucidate a biochemical function for the tumor suppressor gene product ~105RB. The Role of E2F-I in Controlling the DNA-Binding Activity of E2F E2F is a cellular transcription factor whose DNA-binding activity has been shown to be regulated by the growth status of cells. For example, proliferating mouse F9 teratocarcinoma cells contain high levels of E2F in an apparently active form. Upon CAMP- and retinoic acid-induced differentiation, these cells become relatively quiescent and contain very little active E2F (Reichel et al., 1987). In addition, extracts of 3T3 cells rendered quiescent by serum starvation’ have low levels of E2F in the active form; however, when extracts were prepared from serum-stimulated 3T3 cells, a significant increase in E2F activity was observed (Mudryj et al., 1990). A more dramatic change in E2F activ-

Fr.No.

ity is observed during infection with adenovirus (Kovesdi et al., 1986). The activity of this factor is critical for the transcription of the adenovirus early E2 gene. Two other adenovirus early genes, ElA and E4, act in concert to ensure the availabifity of active E2F (Bagchi et al., 1990; Raychaudhuri et al., 1990). One of the E4 gene products, a 19 kd protein, associates with E2F to induce formation of a stable dimeric complex at the two adjacent E2F-binding sites in the E2 early gene promoter (Huang and Hearing, 1989; Raychaudhuri et al., 1990). This observation suggested that E2F activity could be regulated by proteinprotein interactions. Recently, we have identified several cellular proteins that interact with E2F (Bagchi et al., 1990; Raychaudhuri et al., 1991). Of interest here is an inhibitory protein, E2F-I, that blocks the DNA-binding activity of E2F. E2F-I inhibits DNA binding of E2F most likely by forming an E2F/E2F-I complex in a manner very similar to other well-studied inhibitors of sequence-specific DNA-binding proteins, such as NF-~6. The discovery of E2F-I itself is significant

Cell 1068

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