Download - Cell 1988 DeCaprio

Cell, Vol. 54, 275-283, July 15, 1998, Copyright 0 1989 by Cell Press

SV40 Large Tumor Antigen Forms a Specific Complex with the Product of the Retinoblastoma Susceptibility Gene James A. DeCaprio: John W. Ludlow: James Figge, Jin-Yuh Shew,t Chun-Ming Huang,* Wen-Hwa Lee,t Erika Marsilio, Eva Paucha, and David M. Livingston* l Division of Neoplastic Disease Mechanisms Dana-Farber Cancer Institute

and Harvard Medical School Boston, Massachusetts 02115 t Department of Pathology and Center for

Molecular Genetics School of Medicine University of California at San Diego La Jolla, California 92093 * PharMingen 11555 Sorrento Valley Road San Diego, California 92121

Summary

Monkey cells synthesizing SV40 large T antigen were lysed and the extracts immunoprecipitated with either monoclonal anti-T antibody or monoclonal antibody to ~110-114, the product of the retinoblastoma susceptibility gene (Rb). T and ~110-114 coprecipitated in each case, implying that the proteins are complexed with each other. Substitution and internal deletion mutants of T that contain structural alterations in a ten residue, transformation-controlling domain failed to complex with ~110-114. In contrast, T mutants bearing structural changes outside of this domain bound to ~110-114. These results are consistent with a model for transformation by SV40 which, at least in part, involves T/p110 -114 complex formation and the perturbation of Rb protein and/or T function.

The transforming region of the DNA tumor virus, SV40, encodes two proteins, the large (T) and small (t) tumor antigens. While both are active in the induction of a neoplastic phenotype in cultured cells, T, alone, is an obligatory par- ticipant in this process (Sleigh et al., 1978; Lewis and Mar- tin, 1979; Kriegler et al., 1984; Brown et al., 1986; Jat et al., 1986; Bike1 et al., 1987). Indeed, it is clear that, when present in high enough concentration, T can perform all of the functions needed for either the transformation of established cells in culture or tumor formation in suitable ro- dents.

T, a largely nuclear phosphoprotein of 708 amino acids, has multiple biochemical activities. Included are an ability to initiate rounds of SV40 DNA replication, partly depen- dent on specific binding to the SV40 replication origin; ~53, AP2 (Mitchell et al., 1987) and DNA polymerase-a binding; adenine nucleotide binding; nonspecific DNA binding; ATPase and helicase activities; stimulation of host cell DNA and ribosomal RNA synthesis; and transac- tivation of selected RNA polymerase II promoters, includ-

ing the SV40 late promoter (for review, see Livingston and Bradley, 1987). Some of these activities are controlled by discrete functional domains. Thus far, a number, although not yet all, of these intrinsic biochemical functions have been tested and found to be unlinked to its transforming function (Stringer, 1982; Sompayrac et al., 1983; Manos and Gluzman, 1984; Sompayrac and Danna, 1984; Pipas et al., 1984; Peden and Pipas, 1985; Manos and Gluzman, 1985; Sompayrac and Danna, 1985; Fischer-Fantuzzi and Vesco, 1985; Paucha et al., 1986; Rutila et al., 1986; Mohr et al., 1987; Sompayrac and Danna, 1988). Indeed, the biochemical actions of T that are essential to its oncogenic behavior are not yet defined.

On the other hand, genetic analysis of the relationship between T primary structure and its transforming activity has revealed useful insights. It is now clear that the amino- terminal fifth of T has significant transforming activity (Colby and Shenk, 1982; Clayton et al., 1982; Sompayrac and Danna, 1984; Pipas et al., 1984; Asselin and Bastin, 1985; Sompayrac and Danna, 1985; Pan et al., 1985; Som- payrac and Danna, 1988). In recent analyses of the structure-function relationships underlying transformation by T, Kalderon and Smith (1984) Cherington et al. (1988), and Chen and Paucha (unpublished data) have noted depen- dence of this activity on the intact nature of a colinear segment extending from residues 105 to 114. Specifically, a number of mutants in this region were defective in induc- ing focus formation and soft agar growth by established lines of rat and mouse cells (Kalderon and Smith, 1984; Cherington et al., 1988; Chen and Paucha, unpublished data). This region contains several T phosphorylation sites, is not required for origin-specific DNA binding (Paucha et al., 1986; Arthur et al., 1988) and is not essential for the nuclear location ofT (Kalderon et al., 1984a; Kalderon et al., 1984b; Lanford and Butel, 1984; Smith et al., 1985). Importantly, this small segment bears predicted structural homology to a portion of one of the two discrete transformation-controlling domains of the adenovirus 2 and adenovirus 5 ElA gene products, i.e., conserved transforming domain 2 = residues 121 to 139 (Stabel et al., 1985; Figge et al., 1988). Domain 2 has been shown to be essential to ElA cell growth perturbing activity (Lillie et al., 1986; Moran et al., 1986a; Moran et al., 1986b; Zerler et al., 1986; Lillie et al., 1987; Moran and Mathews, 1987; Kuppuswamy and Chinnadurai, 1987; Murphy et al., 1987; Schneider et al., 1987; Zerler et al., 1987; Whyte et al., 1988; Moran and Zerler, 1988; Velcich and Ziff, 1988). Moreover, Moran and co-workers have shown that this region of T will substitute functionally for ElA domain 2 in a relevant chimeric protein (E. Moran, unpublished data). Given the small size and homology of these two transformation control domains, Figge et al. (1988) predicted that this region could act as a binding site for a cellular protein active in the T and ElA transforming mechanisms.

Recently, the Rb susceptibility gene, which when deleted or otherwise inactivated predisposes to the development of malignant retinoblastoma, has been identified

Cdl 276

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kd

205

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Null cd?b 349 245 419

I I I I I I TM TM TM TM TM

B

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419 c&b 245 Null

I 1 23 45 67 6

Figure 1. lmmunoprecipitation of Various Transfected CVlP Extracts with Anti-T and Anti-Fib Monospecific Antibodies

(A) CVlP ceils were transfected with pPVU-O(T) or pBR328(M) and labeled with radioactive methionine, as detailed in Experimental Procedures. Each culture was then extracted and the lysate immunoprecipitated with the antibodies noted at the top of the figure. Null is the nonreactive, IgGs. monoclonal antibody described in the text. Anti-Rb IgG (a-Rb) is a monospecific rabbit antibody to a tpE-Rb fusion protein, and 349 and 245 are abbreviated designations of the monoclonal anti-Rb antibodies noted in the text. 419 refers to PAb419, an anti-T monoclonal antibody. The upper arrow (left border) points to pllO-114. The lower arrow points to T (8) COS-1 (lanes 1, 3, 5, 7) an SV40 transformed derivative of CV1 cells (Gluzman, 1981) and CV-1P (lanes 2, 4, 6, 8) cells were labeled, in parallel, with radioactive methionine, extracted, and immunoprecipitated as denoted in Experimental Procedures. The following antibodies were used to generate the relevant immunoprecipitates: PAb419 (lanes 1 and 2); rabbit anti-Rb IgG (lanes 3 and 4); Rb-PMG3-245 (lanes 5 and 6); and null monoclonal antibody (lanes 7 and 8):Thelowest arrow points to ~53.

(Friend et al., 1986; Lee et al., 1987a; Fung et al., 1987). The Rb gene encodes a nuclear phosphoprotein (ppllOeb) associated with DNA-binding activity (Lee et al., 1987b). Inactivation of both alleles of the Rb gene has been found in all retinoblastomas as well as in some osteosarcomas and soft tissue sarcomas, even without prior history of retinoblastoma (Friend et al., 1987; Mendoza et al., 1988; J. Shew et al., unpublished data). The recent observation that Rb inactivation can occur in human breast cancer (Lee et al., 1988) indicates that this gene may have a broader role in oncogenesis than might otherwise have been anticipated.

127 of ElA are necessary for specific binding of this protein to ElA (Harlow et al., unpublished data). Following this ex- ample, we report here the finding that T also forms a specific complex with the Rb product and that stable complex formation, at least in part, depends upon the intact nature of the 105-114 T transforming region.

Results

Detection of a 110-114 kd T-Associated Protein CV-1P (monkey) cells were transfected with either a plas-

mid containing the entire SV40 early region linked to an intact early promoter element (pPVU-0; Kalderon and Smith, 1984) or with the backbone plasmid (pBR328) into which the aforementioned SV40 sequences had been cloned. The culture was labeled with %-methionine for

Interestingly, Harlow and co-workers (White et al., submitted) have shown that ppllOnb is one of several known ElA-associated proteins (Yee and Branton, 1985; Harlow et al., 1986). They have also found that residues 121 to

SV40 Large T Binds to Rb Protein 277

C

hd hd

116 - 116 -

64 - 64 -

56 - 58 -

46 - 48 -

autorad blot 245

T n

hd

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a4 -

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autorad

D

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blot 419

Figure 2. Western Blotting Analysis of Various lmmunoprecipitates

CV-1P cells transfected with either pPVU-O(T) or pBR328(M) were labeled with radioactive methionine, extracted, and the various lysates immunoprecipitated with either rabbit anti-Rb IgG (a-Ffb) or PAb419. Af- ter electrophoresis, the separated proteins were transferred to nitrocellulose, and the imprinted membranes were reacted with either monoclonal anti-Rb antibody (245) (B) or PAb419 (419) (D). After incubation with enzyme-linked second antibody and exposure to the chromogenic substrate, a photograph was taken of each blot (B) and (D). Autoradio- grams of the same blots are shown in (A) and (C), respectively. The ~53 kd band noted in both right-hand panels is murine IgG heavy chain.

3 hr, one day after removing the DNA. Crude cell lysates were then prepared, and aliquots were subsequently immunoprecipitated with various antibodies. PAb419 is an lgGp monoclonal antibody that reacts with an N-terminal epitope present in SV40 T and t (Harlow et al., 1981). An lgG2 mouse monoclonal antibody (null), not found to recognize any cellular or viral proteins in immunoprecipitation experiments, served as a negative control for PAb419. It was the generous gift of Dr. Deborah Morrison. Monospecific anti-Rb antibodies were used to precipitate other aliquots of these cell extracts. Among them were purified IgG from a rabbit serum raised against an SDS gel band-purified frpE-human Rb fusion protein (anti-Rb IgG;

Lee et al., 1987b), and two IgG, mouse monoclonal anti- Rb antibodies, Rb-PMG3-245 and ffb-PMG3-349 (C.-M. Huang et al., unpublished data). These anti-Rb antibodies were known to precipitate specifically an 110-114 kd protein (pllO-114) from various human cell lines, except those derived from retinoblastoma cells (Lee et al., 1987b). The Rb protein of monkey and human shares an identical molecular massof 110-114 kd (J. Shew et al., unpublished data). As shown in Figure 1 (top arrow), ~110-114 can be seen in the pBR328-transfected cell immunoprecipitates generated with each of the three anti-Rb antibodies. In each of the lysates from pPVU-0 transfected cells, a 94 kd protein (p94; lower arrow), which comigrated with T immunoprecipitated by PAb419, was also detected with each anti-Rb antibody. The ~110-114 was also present in each of these p94-containing lanes. In the same experiment, PAb419 immunoprecipitates were found to contain 94 kd T and a protein which comigrated with the anti-Rb reactive ~110-114. In most experiments of this type, a clear 1 lo-114 kd doublet band was seen (see Figure 5B). Furthermore, in most anti-T immunoprecipitates of T-containing cells, a triplet structure in the 110-114 kd region was noted. An- other anti-T monoclonal antibody (PAb423, Harlow et al., 1981), which reacts with a C-terminal epitope, also precipitated T and a 110-114 kd band that comigrated with the Rb product (Figure 5A). In contrast, neither p94 nor ~110-114 were detected in PAb419 precipitates of backbone plasmid-transfected cells (Figure 1A).

Analogous results were obtained with extracts of COS-1 cells, an SV40 transformed derivative of CV-1 (Gluzman, 1981; Figure 1B). Specifically, PAb419, rabbit anti-Rb IgG, and a monoclonal anti-Rb antibody all precipitated p94 and ~110-114.

In another experiment, similarly labeled extracts were immunoprecipitated, in parallel, with anti-Rb IgG or PAb419. The precipitated products were electrophoresed and transferred to nitrocellulose. The imprinted membranes were incubated with either PAb419 or an anti-Rb monoclonal antibody. Complexes were detected with an appropriate second antibody coupled to alkaline phosphatase (Figures 28 and 2D). These developed blots were also subjected to autoradiography (Figures 2A and 2C). The results qhow that ~110-114, coprecipitated with T by PAb419, reacted directly with an anti-Rb monoclonal antibody, while the 94 kd band coprecipitated with ~110-114 by anti-Rfl IgG reacted directly with PAb419. The data in Figure 2 also show that ~110-114 was only present in the PAb419 immunoprecipitate of T-containing cell extracts. It was not detected in an identical precipitate of a pBR328- transfected cell extract.

Comparative Partial Pmteolytic Analysis of ~110-114 Precipitqted by Anti-Rb Antibody and Coprecipitated with T by Antl-T Antibody The lower two members of the ~110-114 triplet coprecipitated with T by PAb419 and the comigrating, ~110-114 doublet directly precipitated with anti-Rb were separately excised from an SDS-polyacrylamide gel and exposed to limiting quantities of Staphylococcal V8 protease. As shown in Figure 3A, partial digests of these two proteins,

Cell 278

A kd 123456 kd 1 2 3 4

29

generated with similar quantities of enzyme, revealed a number of common polypeptide fragments. Analogous results were obtained with 1251-labeled pl lo-114 isolated by antiT and anti-/% immunoprecipitation, respectively (data not shown). Thus far, the identity of the uppermost member of the aforementioned T-coprecipitating, ~110-114 triplet is unclear. In addition, the 35S-labeled 94 kd bands directly precipitated by PAb419 and coprecipitated with ~110-114 by anti-r% antibody also share multiple V8-generated fragments (Figure 38).

Genetic Analysis of TlpllO-114 Complex Formation A series of plasmids encoding various mutant species of T were transfected, in parallel, onto CV-1P cells. pPVU-0 served as a positive control in this experiment. The identity and location of the various T mutations used here are illustrated in Figure 4. Included are full-sized proteins con-

Transforming Region of SV40 Large T Antigen

wr

Kl

K7

C105D114

PVU- 1

T50-Ll l

105 114 CYS SER GLU GLU MET PRO SER SER ASP ASP

LYS

LYS ASN

GLY LYS

,....._....__ nR . . . ..___....._..........

,........._.._.......... ~~~-..~ . . . . . . ..__...............-............... ~. 92

FOCUS Formation

+

Figure 4. Primary Structure of 105-114 Re- gion T Antigen Mutants

soft Agar23 tzir&&

The sequences of the various mutants used in the experiments described in Figures 5 and 6 are noted. All mutants have been described previously, except C105D114, which was re-

+ cently constructed and sequenced. The results of focus-forming and agar growth assays of rodent cells transfected by plasmids encoding these mutant T species were recorded by Kalderon and Smith (1964), Cherington et al., (1988)s, and Chen and Paucha (unpublished

nd nd

I - 124

Figure 3. Partial Digestion of ~110-114 and p94 with Staphylococcal V8 Protease

(A) (lanes 1 and 2) shows the products of digestion of a5S-methionine labeled, gel-purified pliO-114 (the lower two members of the triplet family) coprecipitated from pPVU-0 transfected CV-1P cells by PAb419. Five hundred nano- grams (lane 1) and 50 ng (lane 2) of V8 protease were incubated with the target protein. Lanes 3 and 4 are digests of ~110-114, precipitated, in parallel, by rabbit anti-Rb IgG from pWU-0 transfected cells, and lanes 5 and 6 contain digests of this protein precipitated by this antibody from pBR328-transfected cells. Again, 50 ng (lanes 3 and 6) and 500 ng (lanes 4 and 5) of V8 protease were used. Digestion and analy sis of products were performed as described in Experimental Procedures. (8) Cleavage patterns of TsS-methionine labeled p94 digested with 50 ng (@es 1 and 4) and 500 ng (lanes 2 and 3) of V8 protease. Lanes 1 and 2: digests of p94 from pPVU-0 transfected cells precipitated with PAb419. Lanes 3 and 4: digests of p94 coprecipitated, in parallel, from the same cells with rabbit anti- Rb IgG.

taining single or do.uble amino acid substitutions and two in-frame, internal deletion mutant species of T A summary of the known activities of these plasmids in promoting focus formation (Kaldeion and Smith, 1984; Chen and Paucha, unpublished data) or soft agar growth by rodent cells (Cherington et al., 1988) is also noted in Figure 4. The T species encoded by mutants Kl, K7, C105Dl14, PVU-1, and T50-Lll all failed to coprecipitate efficiently with ~110-114 in PAb419 and anti-/% IgG immunoprecipitates, unlike wild-type T encoded by pPVU-0 (Figures 5 and 6). The defect in complex formation in the case of K7 may not be as marked as in the cases of the other mutants, given the finding of a small amount of T in the relevant anti-M IgG immunoprecipitate. In contrast, dl0 (lys 128- thr), a mutant which encodes full-sized T bearing a single amino acid substitution leading to defective nuclear localization (Kalderon et al., 1984a), clearly bound pilO-114, as

data)3. (-) means that the efficiency of focus or agar colony formation was less than 10% of that of wild-type T-producing cells tested in parallel. T50-Lll will be further discussed in Chen and Paucha.

:%;O Large T Binds to Rb Protein

A

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123 4 5 6 7 6 91011

- +

-

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Figure 5. lmmunoprecipitation of Wild-Type and Mutant T-Containing Cell Extracts

(A) Extracts of methionine-labeled CV-1P cells, transfected with either wild-type or mutant T-encoding plasmids, were immunoprecipitated with either PAb419, rabbit anti-Rb IgG, or PAb423, as described in Experimental Procedures. The plasmids tested were: pBR326 (lanes 1 and 10); pPVU-0 (lanes 2 and 11); Kl (lane 3); K7 (lane 4); WU-1 (lane 5); T50-Lll (lane 6); dl0 (lane 7); X46 (lane 6); and U19 (lane 9). The antibodies used were: PAb419 (lanes l-9); anti-Rb IgG (lane 10); PAb423 (lane 11). The upper arrow points to pIlO-114. The lower one points to ~53. (6). The same procedures employed in the experiment described in (A) were employed here. The plasmids used were: U24 (lanes 1 and 2); pPVU-0 (lanes 4, 6, and 6); and pBR326 (lanes 3, 5, and 7). The antibodies were: PAb419 (lanes 1, 3, 4); rabbit anti-Rb IgG (lanes 2, 5, and 6); and null (lanes 7 and 6)

did X46 (lys 127-thr) a mutant characterized by a wild-type phenotype (Kalderon and Smith, 1984). It was previously shown that d10 and X46 both generated foci on Rat-l cells normally (Kalderon and Smith, 1984). In addition, the origin-specific DNA-binding-defective mutants, U19 (ser 152-asn; arg 154-1~s; Figure 5A) and U24 (ala 149~val; Fig- ure 55) (Kalderon and Smith, 1984; Paucha et al., 1986) also bound ~110-114 like wild-type T Taken together, these results suggest that the T sequence between residues 105 and 114 functions in both the T transforming pathway and in stable T/pllO-114 complex formation. In keeping with these conclusions, E. Moran (unpublished data) has found that an ElASV40 T chimera in which the 101-118 region of T was substituted for ElA domain 2 has both transforming and /%-binding activity. Thus far, we have no evidence for or against the involvement of other amino acid sequences of T in complex formation. However, since mutations affecting nuclear localization and origin binding did not inhibit pliO-114 binding, the domains controlling these functions are probably not involved.

Discussion

The data presented here show that, in addition to binding to the host cell proteins, ~53, DNA polymerase-a, and AP2

(Mitchell et al., 1987), SV40 T can bind specifically to pllO-114, the product of the retinoblastoma susceptibility gene (Lee et al., 1987b). The evidence for this suggested identity of pliO-114 bound to T is both immunologic and structural. Specifically, the protein comigrated with the Rb product in SDS-polyacrylamide gel electropherograms and cross-reacted with it immunologically in experiments performed with three different monospecific Rb antibodies. Moreover, their V8-produced fragment maps were similar.

Support for the claim that T forms a stable complex with ~110-114 comes from the repeated finding that both proteins can be coprecipitated with either monoclonal anti-T or monoclonal anti-Rb antibodies. Coprecipitation is not a sign that these two proteins share common epitopes, since the presence of T was required to observe ~110-114 in the autoradiogram of immunoprecipitates generated with anti-T monoclonal antibodies. Second, two different anti-T monoclonal antibodies, PAb419 and PAb423 (Har- low et al., 1981) directed against disparate epitopes, both coprecipitated ~110-114. Third, monoclonal anti-T antibody failed to recognize pllO-114 and monoclonal anti-Fib antibody failed to recognize T in Western blotting. Given the first two results, it is unlikely that a hypothetical common epitope, not detected in the Western blotting experi-

Cell 280

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Figure 6. Further Analysis of Various 1 Mutants

The same procedures employed in the experiments described in Fig- ure 5 were employed here. CWP cells were transfected with the following plasmids: Kl (lanes 1 and 6); K7 (lanes 2 and 7); C105D114 (lanes 3 and 8); pPVU-0 (lanes 4,9, and 11); pBR326 (lanes 5 and IO). Extracts were immunoprecipitated with either PAb419 (lanes l-4); rabbit anti- Rb IgG (lanes 5-9); or null monoclonal antibody (lanes 10 and 1 I), The upper arrow points to ~110-114, the middle one points to T, and the lower one points to ~53.

ments, was missed because it is denaturation-sensitive. Thus, coprecipitation indicates a physical association between T and the Rb product.

The stoichiometry of T/pllO-114 complex formation is unknown. This is, at least in part, because all of the experiments relevant to this point involved immune isolation and the inherent potential therein for incomplete precipitation and coprecipitation of the relevant proteins. However, results such as those shown in Figure 1 imply that a significant fraction of the T molecules detectable with PAb419 in at least one cell line can be coprecipitated with anti-Rb monoclonal antibody. Since PAb419 is known to bind rela- tively efficiently to T and since the experiments reported here were all performed in antibody excess, this suggests that a significant fraction of the T antigen in the relevant transfected cells is associated with ~110-114. Further- more, the COS-1 results imply that a significant fraction of the immunoreactive Rb protein can be coprecipitated with T

Other evidence (Figures 2 and 6) shows that while pllO-114 does not appear to bind to ~53 in the absence of T, ~53 did appear in an anti-Rb immunoprecipitate when wild-type T was present (e.g., Figure 6, lower arrow). The simplest explanation for these findings is that at least some of the T associated with ~110-114 is also complexed to ~53. This remains to be proven as a general phenome- non. If it can be so demonstrated, one wonders whether DNA polymerase-a will also be identified in complex with T and pllO-114, given the known competition of ~53 and DNA polymerase-a for binding to T (Braithwaite et al., 1987; Gannon and Lane, 1987). The fact that multiple mutant T species, unable to bind pllO-114, bound ~53 eliminates the possibility that pliO-114 binding to T is really the result of it binding exclusively to ~53, which is, in turn, complexed to T. Similarly, all of the mutapt species tested in the experiments described here en&e intact t. Therefore, we cannot eliminate the possibility that t contributes to ~110-114 binding by T.

The genetics of the T-Rb association indicate that complex formation is a specific process. A small, colinear sequence (residues 105-114) that, at least in part, governs the transforming function of T is essential to ~110-114 binding. Indeed, the close correlation between transforming ability and ~110-114 binding suggests that complex formation is a step in the T-mediated transforming mecha- nism. On the other hand, even if this is true, the results do not say that it is the only biochemical act which T must perform in this regard. .$I of this notwithstanding, if one assumes that T/pllO-114 complex formation contributes to transformation maintenance, certain hypothetical ex- planations of how this might be achieved deserve con- sideration. The somatic elimination of function of the remaining copy of the Rb gene underlies hereditary retinoblastoma development (Knudson, 1971; Cavenee et al., 1983; Friend et al., 1986; Lee et al., 1987a; Fung et al., 1987; Lee et al., 1987b). Thus, pliO-114 can be viewed as a suppressor of neoplastic behavior, although how it might do this is unknown. Since it is a nuclear phosphoprotein with associated DNA-binding activity (Lee et al., 1987b), one wonders whether it can recognize specific sequences and, if so, whether it serves as a transcriptional regulator or as an inhibitor of passage through an important choke point in the cell cycle. Numerous other possibilities exist. Nevertheless, the knowledge that it normally suppresses growth raises the possibility that a dominant nuclear oncogene product like T or ElA (White et al., submitted) modifiesor neutralizes its function, thereby contributing to the development of transformed behavior. A formal prediction of this model is that synthesis of an Rb mutant, non- defective in T binding but fully able to suppress neoplastic growth, would, in turn, neutralize T transforming function. Alternatively, synthesis of more ~110-114 than could be bound by T might lead to the same effect.

In keeping with the aforementioned discussion, it is also formally possible that Rb protein inhibits one or more T transforming functions. In that case, one might argue that the fraction of T not bound to ~110-114, if it exists, is the active species in disturbing growth control. Against this and in favor of the hypothesis that T dominates Rb is the

El,40 Large T Binds to Rb Protein

finding that T mutants in the 105 to 114 region, defective in transformation, failed to bind ~110-114. Similarly, it seems reasonable to ask whether the T/pllO-114 complex acquires new functions that lead to the perturbation of cell growth and is not a property of either species alone. Fi- nally, since SV40 is a lytic virus in monkey cells and a number of T functions are essential to its normal life cycle, one wonders what role the T/pllO-114 complex might play in the propagation of the virus in its natural host.

Experimental Procedures

Cell Culture and DNA Transfection CVlP and COS-1 cells were grown at 3pc in a humidified, 10% COT containing atmosphere in Dulbeccos modified Eaglds medium (DMEM; Gibco) supplemented with 10% newborn bovine serum (Flow Laboratories) on plastic surfaces. PlOO plates of ~80% confluent CV- 1P cells were transfected by the calcium phosphate method (Graham and van der Eb, 1973). Twenty micrograms of plasmid DNA was utilized for each PlOO plate. Glycerol shock was not employed. The DNA calcium phosphate precipitate was left in the culture medium for 1518 hr. The medium was then drained, and the cells washed twice and refed.

Antibodies The preparations of PAb419 and PAb423 (Harlow et al., 1981) used here were pooled tissue culture supernatants from actively growing hy- bridoma cultures. They were not purified further. Rabbit anti-Rb IgG raised against a rrp E-fib fusion protein was prepared as described (Lee et al., 1987b). The monoclonal anti-Rb antibodies were also used as purified IgG fractions. Both are IgG, species and required the addition of rabbit anti-mouse IgG prior to immune complex isolation on protein ASepharose (Huang et al., unpublished data).

Immunoptecipitation and SDS-Polyacrylamide Gel Electmphoresls Transfected cells and COS-I in 100 mM dishes were labeled with 3 ml of 35S-methionine (1083 Cilmol; 200 t&i/ml; NEN) in methioninefree DMEM for 3 hr at 3pc. Cells were then lysed with 1 ml of ice-cold EBC buffer (50 mM Tris-HCI [pH 8.01, 120 mM NaCI, 0.5% Nonidet P-40, and 0.1 TIU of aprotinin [Sigma]) for 20 min at 4C. The lysates were then cleared of nuclei and debris by centrifugation in a microfuge (Fisher) for 15 min. The supernatants were aliquoted, mixed with 0.5 ml of NET- N (20 mM Tris-HCI [pli 8.01, 100 mM NaCI, 1 mM EDTA, and 05% Nonidet P-40) and the appropriate antibody (10-80 ul). The mixture was rocked for 1 hr at 4%. Then, 20 ul of a I:1 mixture of freshly washed and suspended protein ASepharose (Pharmacia) in TBS-BSA (25 mM Tris-HCI [pH 8.01 and 120 mM NaCl containing 10% bovine se rum albumin, Pentex-crystalline [Miles Laboratories]) was added. Incu- bation proceeded for an additional 20 min at 4OC. The protein A-Sepha- rose beads (Pharmacia) were then washed five times with 1 ml of NET-N at 4OC. SDS-polyacrylamide gel electrophoresis was followed by fluorography (Chamberlain, 1979).

lmmunoblotting lmmunoprecipitates of transfected cells were prepared as described above. After electrophoresis, the proteins were transferred to nitrocellulose in transfer buffer (25 mM Tris-HCI, 192 mM glycine, 20% [v/v] methanol and 0.01% SDS [pH 8.51) for 4 hr (liibin et al., 1979). The nitrocellulose was then blocked by incubation in TBS containing 4% BSA (Promega) for 2-14 hr and then incubated for 2-14 hr in 15 ml of TBST (10 mM Tris-HCI [pH 8.01, 150 mM NaCI, 0.05% Tween 20 [Bi- orad], and 4% BSA [Promega]) and 150 ul of PAb419 or 75 ul of Rb- PMG3245. The blots were then probed with alkaline phosphatase- conjugated rabbit anti-mouse IgG (Promega) and developed for color according to the manufacturers specifications.

Partial Pfoteolytic Mapplng Peptide mapping of pllO-114 and T was performed as described by Cleveland et al. (1977). 35S-labeled proteins of interest, separated in 7.5% SDS-polyacrylamide gels, were localized by autoradiography of

unfixed, dried slabs. Following excision of the radiolabeled protein- containing strips, the gel pieces were swelled and equilibrated for 15 min at room temperature in buffer A (125 mM Tris-HCI [pH 8.81, 0.1% SDS, 1 mM EDNA, and 1 mM 8-mercaptoethanol). The equilibrated gel pieces were then inserted at the bottom of sample wells atop a 15% SDS-polyacrylamide gel prepared with stacking and running gel solu- tions rendered 15 mM in ED%A and overlaid with buffer A containing 20% glycerol and 0.0001% bromophenol blue. Finally, 10 pi of buffer B (buffer A containing 10% glycerol) and a given amount of Staphylo- coccus aureus V-8 protease (Sigma) were applied as a layer over the buffer in each slot. Electrophoresis (at room temperature) was performed at 18 mA (constant current) until the blue dye neared the bottom of the stacking gel, at which time the current was turned off for 30 min. Electrophoresis was then resumed at 30 mA(constant current) until the blue dye had just run off the gel. Visualization of protein standards and radiolabeled peptides was accomplished by Coomassie blue staining and fluorography, respectively.

Acknowledgments

We are grateful to Drs. E. Harlow, J. Horowitz, E. Moran, and R. Wein- berg for sharing with us the results of their work prior to its publication. We also wish to thank Christopher Wright and Dr. Alan Smith for help ful suggestions and Ann Desai for her expert help in constructing this manuscript. The work was supported by grants from the National Cancer Institute (to D. M. L.) and the National Eye Institute (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 be hereby marked b&ertisemenf in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 10, 1988; revised June 21, 1988.

Arthur, A. K., H&s, A., and Fanning, E. (1988). Expression of simian virus 40 T antigen in Escherichie co/i: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J. Virol. 62, 1999-2608

Asselin, C., and Bastin, M. (1985). Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts. J. Virol. 56, 958-968.

Bikel, I., Montano, X., Agha, M. E., Brown, M., McCormack, M., Bol,tax, J.. and Livingston, D. M. (1987). SV40 small t antigen enhances the transformation activity of limiting concentrations of SV40 large T antigen. Cell 48, 321-330.

Braithwaite, A. W., Sturzbecher, H.-W., Addison, C., Palmer, C., Rudge, K., and Jenkins, J. R. (1987). Mouse ~53 inhibits SV40 origin-depen- dent DNA replication. Nature 329, 458-460.

Brown, M., McCormack, M.. Zinn, K. G., Farrell, M. P. Bike& I., and Livingston, D. M. (1988). A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage- independent growth. J. Virol. 60, 290-293.

Cavenee, W. K., Dryja, T. t?, Phillips, R. A., Benedict, W. F., Godbout, R., Gallie, B. L., Murphree, A. L., Strong, L. C., and White, R. L. (1983). Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779-784.

Chamberlain, J. P (1979). Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Anal. Biochem. 98, 132-135.

Cherington, V., Brown, M., Paucha, E., St. Louis, J., Spiegelman, B. M., and Roberts, T M. (1988). Separation of simian virus 40 Iarge-T- antigen-transforming and origin-binding functions from the ability to block differentiation. Mol. Cell. Biol. 8, 1380-1384.

Clayton, C. E., Murphy, D., Lovett, M., and Rigby, F! W. J. (1982). A fragment of the SV40 T-antigen gene transforms. Nature 299, 59-61.

Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, Lt. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sul- fate and analysis by gel electrophoresis. J. Biol. Chem. 3, 1102-1108.

Colby, W. W., and Shenk, T (1982). Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells. Proc. Natl. Acad. Sci. USA 79, 5189-5193.

Cell 282

Figge, J., Webster, T., Smith, T F., and Paucha. E. (1988). Prediction of similar transforming regions in simian virus 40 large T, adenovirus ElA, and myc oncoproteins. J. Virol. 62, 1814-1818.

Fischer-Fantuzzi, L., and Vesco. C. (1985). Deletion of 43 amino acids in the NH,-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells. Proc. Natl. Acad. Sci. USA 82, 1891-1895.

Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T f? (1988). A human DNA segment with properties of the gene that predisposes to retinoblastoma and os- teosarcoma. Nature 323, 843-848.

Friend, S. H., Horowitz, J. M., Gerber, M. R., Wang, X.-F, Bogenmann, E., Li, F. R. and Weinberg, R. A. (1987) Deletions of a DNA sequence in retinoblastomas and mesenchymal tumors: Organization of the sequence and its encoded protein. Proc. Natl. Acad. Sci. USA 84, 9059-9083.

Fung, Y.-K. T., Murphree, A. L., TAng, A., Qian, J., Hinrichs, S. H., and Benedict, W. F. (1987). Structural evidence for the authenticity of the human retinoblastoma gene. Science 236, 1857-1881.

Gannon, J. V.. and Lane, D. l? (1987). p53 and DNA polymerase a-compete for binding to SV40 T antigen. Nature 329, 458-458.

Gluzman, Y. (1981). SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-182.

Graham, F. L., and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,458467.

Harlow, E., Crawford, L. V., Pim, D. C., and Williamson, N. M. (1981). Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol. 39, 881-869.

Harlow, E., Whyte, P, Franza, B. R., Jr., and Schley, C. (1988). Associa- tion of adenovirus early-region 1A proteins with cellular polypeptides. Mol. Cell. Biol. 6, 1579-1589.

Jat, P S.. Cepko, C. L., Mulligan, R. C., and Sharp, P A. (1986). Recom- binant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens. Mol. Cell. Biol. 6, 1204-1217.

Kalderon, D., and Smith, A. E. (1984). In vitro mutagenesis of a putative DNA binding domain of SV40 large% Virology 739, 109-137.

Kalderon, D., Richardson, W. D., Markham, A. F, and Smith, A, E. (1984a). Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 377, 33-38.

Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984b). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509.

Knudson, A. G., Jr. (1971). Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. USA 68, 820-823.

Kriegler, M., Perez, C. F., Hardy, C., and Botchan, M. (1984). Transfor- mation mediated by the SV40 T antigens: separation of the overlapping SV40 early genes with a retroviral vector. Cell 38, 483-491.

Kuppuswamy, M. N., and Chinnadurai, G. (1987). Relationship between the transforming and transcriptional regulatory functions of adenovirus 2 Ela oncogene. Virology 759, 31-38.

Lanford, R. E., and Butel, J. S. (1984). Construction and characteriza- tion of an SV40 mutant defective in nuclear transport of T antigen. Cell 37, 801-813.

Lee, W.-H., Bookstein, R., Hong, F., Young, L.-J., Shew, J.-Y., and Lee, E. Y.-H. P (1987a). Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235, 1394-1399.

Lee, W.-H., Shew, J.-Y., Hong, F. D., Sery, T W., Donoso, L. A., Young, L.-J., Bookstein, R., and Lee, E. Y.-H. F? (1987b). The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature 329, 642-645.

Lee, E. Y.-H. l?, To, H., Shew, J.-Y., Bookstein, R., Scully, P, and Lee, W.-H. (1988). Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science, in press.

Lewis, A. M., Jr., and Martin, R. G. (1979). Oncogenicityof simian virus 40 deletion mutants that induce altered 17kilodalton t-proteins. Proc. Natl. Acad. Sci. USA 76, 4299-4302.

Lillie, J. W., Green, M., and Green, M. R. (1986). An adenovirus Ela protein region required for transformation and transcriptional repres- sion Cell 46, 1043-1051.

Lillie, J. W., Loewenstein. l? M., Green, M. R., and Green, M. (1987). Functional domains of adenovirus type 5 Ela proteins. Cell 50, 1091-I 100.

Livingston, D. M., and Bradley, M. K. (1987). Review: the simian virus 40 large T antigen-a lot packed into a little. Mol. Biol. Med. 4,83-80.

Manos, M. M., and Gluzman, Y. (1984). Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation. Mol. Cell. Biol. 4, 1125-1133.

Manos, M. M., and Gluzman, Y (1985). Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. J. Virol. 53, 120-127.

Mendoza, A. E., Shew, J.-Y., Lee, E. Y.-H. i?, Bookstein, R., and Lee, W.-H. (1988). A case of synovial sarcoma with abnormal expression of the human retinoblastoma susceptibility gene. Hum. Path. 79, 487-489.

Mitchell, R J., Wang, C., and Tjian, R. (1987). Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is in- hibited by SV40 T antigen. Cell 50, 847-861.

Mohr, I. J., Stillman, B., and Gluzman, Y. (1987). Regula&n of SV40 DNA replication by phosphorylation of T antigen. EMBO J. 6.153-160.

Moran, E., and Mathews, M. B. (1987). Multiple functional domains in the adenovirus ElA gene. Cell 48, 177-178.

Moran, B., and Zerler, 8. (1988). Interactions between cell growth- regulating domains in the products of the adenovirus ElA oncogene. Mol. Cell. Biol. 8, 1756-1784.

Moran, E., Grodzicker, T., Roberts, R. J., Mathews, M. B., and Zerler, B. (1986a). Lytic and transforming functions of individual products of the adenovirus ElA gene. J. Virol. 57 785-775. Moran, E., Zerler, B., Harrison, T M., and Mathews, M. B. (1986b). Iden- tification of separate domains in the adenovirus ElA gene for immortal- ization activity and the activation of virus early genes. Mol. Cell. Biol. 6, 3470-3480.

Murphy, M., Opalka, B., Saja&ykowski, R., and Schulte-Holthausen, H. (1987). Definition of a region required for transformation in Ela of adenovirus 12. Virology 759, 49-56.

Pan, S., Sompayrac, L. M., Knowles, B. B.. and Danna, K. J. (1985). A fragment of the simian virus 40 early genome can induce tumors in nude mice. J. Virol. 53, 988-989.

Faucha, E., Kalderon, D., Harvey, R. W., and Smith, A. E. (1986). Sim- ian virus 40 origin DNA-binding domain on large T antigen. J. Virol. 57 50-84.

Peden, K. W. C., and Pipas, J. M. (1985). Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation. J. Virol. 55, l-9.

Pipas, J. M., Chiang, L. C., and Barnes, D. W. (1984). Influence of genetic, cellular, and hormonal factors on simian virus 40-induced transformation. In Cancer Cells 2/0ncogenes and Viral Genes, G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 355-382.

Rutila, J. E., Imperiale, M. J., and Brockman, W. M. (1986). Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants. J. Virol. 58, 528-535.

Schneider, J. F, Fisher, F., Goding, C. R., and Jones, N. C. (1987). Mutational analysis of the adenovirus Ela gene: the role of transcriptional regulation in transformation. EMBO J. 6, 2053-2080.

Sleigh, M. J., Topp, W. C., Hanich. R., and Sambrook, J. F (1978). Mu- tants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell 74, 79-88.

Smith, A. E., Kalderon, D., Roberts, 8. L., Colledge, W. H., Edge, M., Gillett, f?, Markham, A., Paucha, E., and Richardson, W. D. (1985). The nuclear location signal. Proc. R. Sot. Lond. B. 226, 43-58.

Sompayrac, L. M., and Danna, K. J. (1984). Less than 40% of the simian virus 40 large T antigen-coding sequence is required for transformation Mol. Cell. Biol. 4, 1661-1663.

Sompayrac, L., and Danna, K. J. (1985). The simian virus 40 sequences between 0.189 and 0.423 map units are not essential to im- mortalize early-passage rat embryo cells. Mol. Cell. Biol. 5, 11911194.

.SS;O Large T Binds to Rb Protein

Sompayrac, L.. and Danna, K. J. (1988). A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells. Virology 163, 391-396.

Sompayrac, L. M., Gurney, E.G., and Danna, K. J. (1983). Stabilization of the 53,000 dalton nonviral tumor antigen is not required for transformation by simian virus 40. Mol. Cell. Biol. 3. 290-296.

Stabel, S., Argo& P., and Philipson, L. (1985). The release of growth arrest by microinjection of adenovirus ElA DNA. EMBO J. 4, 2329- 2336.

Stringer, J. R. (1982). Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells. J. Virol. 42. 854-864.

Towbin. l-i., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: proce- dure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350- 4354.

Velcich, A., and Ziff, E. (1988). Adenovirus Ela ras cooperation activity is separate from its positive and negative transcription regulatory function. Mol. Cell. Biol. 8, 2177-2183.

Whyte, P.. Ruley, Ii. E., and Harlow, E. (1988). Two regions of the adenovirus early region ElA proteins are required for transformation. J. Virol. 62, 257-265.

Yee, S.-P., and Branton. P E. (1985). Detection of cellular proteins associated with human adenovirus type 5 early region ElA polypeptides. Virology 747. 142-153.

Zerler, B., Moran, B., Maruyama, K., Moomaw, J.. Grodzicker, T., and Ruley, H. E. (1986). Adenovirus ElA coding sequences that enableras and pmtoncogenes to transform cultured primary cells. Mol. Cell. Biol. 6, 887-899.

Zerler, B., Roberts, R. J.. Mathews, M. B., and Moran, E. (1987). Differ- ent functional domains of the adenovirus ElA gene are involved in regulation of host cell cycle products. Mol. Cell. Biol. 7, 821-829.

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