Cellular targets for transformation by the adenovirus E1A proteins

Cell, Vol 56, 67-75.

January

13, 1989. Copyright

0 1989 by Cell Press

Cellular Targets for Transformation Adenovirus EIA Proteins Peter Whyte; Nicola M. Williamson, Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

and Ed Harlow

Summary Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as plOM?B, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus ElA proteins. To help determine the functional basis of these interactions, the regions of ElA that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, pl05-RB, required the presence of sequences from two noncontiguous regions of the ElA polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of ElA that are required for these interactions coincided precisely with the regions of ElA that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of ElA. Introduction Human adenoviruses have been widely used as models for studying both cellular transformation and the regulation of gene expression. The adenovirus ElA region plays a central role in each of these processes. This region of the viral genome encodes a series of related proteins with multifunctional capabilities. Among the described activities of ElA is the ability to regulate transcription from a wide variety of promoters. Most commonly, this results in activation of transcription from the target promoter, which can be of either viral or cellular origin (for a review see Berk, 1986). In other cases, especially those involving promoters linked to transcriptional enhancers, transcrlption from the targeted promoter is repressed by the action of ElA (Borrelli et al., 1984; Velcich and Ziff, 1985; Hen et al., 1985). The ElA protein also contributes to the transforming capabilities of adenovirus. The ElA and ElB regions together comprise the transforming region of adenovirus (Gallimore et al., 1974; Graham et al., 1974). While expression of ElA alone is sufficient to immortalize primary cells, complete transformation requires the additional expression of the El6 region (Houweling et al., 1980; van den Elsen et al., 1983). Several other oncogenes, including an activated H-ras oncogene and the poiyoma middle T antigen, can also complement ElA by substituting for ElB in these transformation assays (Ruley, * Present address: Fred Hutchlnson Cancer Research Columbia Street, Seattle, WashIngton 98104.

Center. 1124

by the

1983). Similarly, ElA can be replaced by the polyoma large T antigen gene, members of the myc gene family, or the p53 oncogene (Land et al., 1983; Ruley, 1983; Eliyahu et al., 1984; Jenkins et al., 1984; Parada et al., 1984; Schwab et al., 1985; Yancopoulos et al., 1985). Whether or not these functional similarities reflect similar blochemical mechanisms is as yet unknown. At early times during adenovirus infection and in transformed cells expressing ElA, two mRNA species are transcribed from the ElA region (Berk and Sharp, 1978). These two mRNA species, referred to by their sedimentation values of 13s and 12S, each contain a single intron and arise through differential splicing of a common precursor RNA (Perricaudet et al., 1979). Related polypeptides of 289 and 243 amino acids (289R and 243R) are synthesized from the 13s and 125 mRNA species, respectively. The 243R species differs from the 289R species only by the absence of 46 internally located amino acids. Each of these primary translation products undergoes extensive posttranslational modification resulting in a series of highly related proteins that are located in the nucleus of the cell (Harlow et al., 1985). Mutational analysis of the ElA protein coding region has made it apparent that some activities of ElA are distinct from others and are carried out independently by different regions of the protein, indicating that ElA is multifunctional at the biochemical level (for a review, see Moran and Mathews, 1987). In particular, the regions of the protein involved in transactivation can be clearly separated from those Involved in transformation and immortalization. Detailed mapping studies suggest that the transactivation function is carried out by the region that is specific to the 289R polypeptide (Carlock and Jones, 1981; Monte11 et al., 1982; Moran et al., 1986; Lillie et al., 1986, 1987). Two regions common to both the 289R and 243R polypeptides are required for transformation but not for transactivation (Haley et al., 1984; Moran et al., 1986; Zerler et al., 1986; Schneider et al., 1987; Subramanian et al., 1988; Whyte et al., 1988b). Other activities have been attributed to ElA; however, their relationships to the transactivation and transformation functions have not yet been made clear. The same regions of ElA that are involved in transformation also appear to be involved in immortalization and stimulation of DNA synthesis (Braithwaite et al., 1983; Stabel et al., 1985; Kaczmarek et al., 1986; Zerler et al., 1986; Lillie et al., 1987; Schneider et al., 1987). Early studies suggested that transcriptional repression was also linked to immortalization and transformation, but recent reports have described mutants that affect only the transforming ability but do not alter transcriptional repression while other mutants affected only transcriptional repression (Subramanian et al., 1988; Velcich and Ziff, 1988). Another property of ElA is its ability to induce the production of an epithelial cell growth factor (Quinlan et al., 1987). Induction of the epithelial cell growth factor apparently requires both the sequences involved in transformation as well as other regions of the protein (Qulnlan et al., 1988;

Cell

6s

wi-

I

I I I

-~-

560

974

1112A1229

I

2

1542

Deletron

+

Substrtution

CTdll009p

0.0. 151-289

+

leu

CTd1976

O.O.140-289

-?,

leu

aa. 135-289

+

leu

a.0 128-289

+

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0.0. 126-289

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a.a. 125-289

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leu

0.a 86- 120

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e 934

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a.a 2-85

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aa. 2-150

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dl637N

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dl739N

Frgure 1 Structures

of ElA Mutants

Schematrc representatron of each of the mutants of ElA are shown below a representation of the ElA and rts 135 mRNA product Symbols: open boxes. translated regions of the mRNA; strarght black Imes, untranslated regions of the mRNA; hatched boxes, 135 unique region in mutants that retain the alternative splrcrng pattern of the 12s and 13s mRNA. Numbers located above the schematic representatrons refer to the nucleotides at the boundarres of the deletrons. To the right of each mutant is the predicted change of the protein product of that mutant.

Subramanian et al., 1988). Although these studies involving mutagenic analysis of ElA activities have in some cases suggested a relation among the different phenotypes induced by ElA, they have provided little information about the biochemical nature of ElA’s functions. lmmunochemical studies on the ElA proteins have identified a number of cellular proteins that form physical complexes with the ElA proteins and coimmunoprecipitate with ElA (Yee and Branton, 1985; Harlow et al., 1986). The most prominent among these are proteins migrating with relative molecular weights of 300,000 (300K), 107,000

(107K), and 105,000 daltons. The latter two of these correspond to the previously described doublet of approximately 110,000 daltons (Harlow et al., 1986). Recently, we have identified the 105,000 dalton protein as plOdFIB, the product of the retinoblastoma susceptibility gene (Whyte et al., 1988a). Although the functional significance of these interactions has not previously been established, these cellular proteins represent potential targets for the various functions of ElA. In this study, the regions of ElA involved in binding to these cellular proteins are examined in an effort to determine the functional basis for these in-

Cellular Targets for Transformation 69

300K

by ElA

-

107K 105K-

“.--

“.

Figure 2. Analysis of ElAiHost

9

1.

--_-

^

Protein Complexes

.

from Cells Infected with Viruses Expressing

Carboxy-Termlnal

Mutants of ElA

lmmunopreclpltatlons from 35S-methlonlne-labeled HeLa cells Infected with viruses expressing the mutant ElA regions were run on (A) an 8% polyacrylamlde gel or (6) a 15% polyacylamide gel The posItIons of ElA and the copreclpltatlng cellular proteins are shown on the left. The EiA proteins were lmmunoprecipitated with the M58 monoclonal antlbody (or M73 for mutant NTdllOlO); 416 (PAb416: Harlow et al 1981) was used as a negative control monoclonal antlbody

teractions. The regions of EIA involved in binding to these three cellular proteins were found to be the same regions that were previously identified as essential for transformation. The results presented here suggest that ElA exerts its transforming function through these interactions.

Results Mapping of the Regions of ElA Involved in Binding to the 300K, 107K, and pl05-FIB Proteins As an initial step in determining the functional role of the interactions between ElA and the 300K, 107K, and ~105 RB proteins, a genetic analysis was undertaken of the regions of ElA that participate in these interactions. Several sets of deletion mutants (see Figure 1 for structures) were used to delineate these regions of ElA by analyzing the ability of their mutant protein products to form complexes with the cellular proteins. These ElA mutants were introduced into HeLa cells using recombinant adenoviruses. Complexes formed between the mutant ElA proteins and cellular proteins were immunoprecipitated using anti-ElA monoclonal antibodies and analyzed by SDSpolyacrylamide gel electrophoresis. A series of mutations resulting in the production of truncated ElA polypeptides was used to determine the carboxy-terminal boundary of the sequences required for interaction with the 300K, 107K, and pl05-RB proteins (Figure 2). The mutant CTd1976 directs the synthesis of a polypeptide encoded by the sequences common to the first exon of the 12s and 13s mRNAs. The 300K, 107K,

and ~105RB proteins each coprecipitated with the protein product of CTd1976 demonstrating that the sites of interaction with ElA for all three of these proteins are present within the sequences encoded by the first exon of ElA. Mutants containing further deletions of the sequences from the carboxyl terminus demonstrated that a polypeptide consisting of the amino-terminal 127 amino acids of ElA (mutant CTd1940) was capable of interacting with all three cellular proteins. The mutant CTd/961 bound only low levels of ~105RB in the experiment shown (Figure 2). In longer exposures, the pl05-RB protein could be clearly distinguished, and in other experiments, variable amounts of pl05-RB were found coprecipitating with the product of mutant CTd1961. In contrast to these mutants, neither the 107K protein nor pl05-RB were complexed to a polypeptide of 125 amino acids (mutant CTd1934). These results suggested that the amino-terminal 127 amino acids of ElA contain the binding locations for the 300K, 107K, and pl05-RB proteins. Amino acid 127 appears to represent the carboxy-terminal boundary of the sequences necessary for the stable interaction between ElA and the 107K and pl05-RB proteins. The fact that the 300K protein binding site IS retained in the absence of binding to the 107K and pl05-RB proteins demonstrates that the 300K protein interacts with a different site on the ElA protein. Separation of the 107K and pl05-RB proteins required a low percentage acrylamide gel that did not retain the truncated ElA proteins. To ensure that these viruses were synthesizing the mutant ElA proteins, samples from the same immunoprecipitations were run on a 15% polyacrylamide

Cell 70

107K 105K 107K 105K -

-

925K

46~ EIA

-

30K

Frgure 3 Complex Formation between Cellular Proteins and ElA Mutants wrth Internal Deletrons lmmunoprecrprtatrons from 35S-methronrne-labeled HeLa cells infected with viruses expressing Internal deletion mutants of ElA were run on an 8% polyacrylamide gel. The mutant proteins were rmmunopreciprtated wtth the monoclonal antibody M73; 416 (PAb416; Harlow et al.. 1981) was used as a negahve control

gel (Figure 2b). Each mutant synthesized approximately the same amount of an ElA product that migrated in a manner consistent with the size of the deletion. The mutant NCdl contains a deletion of the sequences encoding amino acids 86 to 120 (Moran et al., 1986). The absence of these sequences did not affect complex formation with the 300K, 107K, or pl05-RB proteins (Figure 3). This was somewhat surprising because truncated proteins consisting of less than the first 127 amino acids failed to form complexes with either the 107K or pl05-RB proteins. These results suggested that amino acids 121 to 127 play an essential role in the interactions between ElA and the 107K and pl05-RB proteins. A mutation with a deletion in this region further demonstrated the importance of these amino acids in the 107K and pl05RB interactions The mutant d/922/946 is missing the sequences encoding amino acids 122 to 129. This deletion eliminated the interactions between ElA and both the 107K and ~105 RB proteins but had no effect on the interaction with the 300K protein. Long exposures of the gel shown in Figure 3 revealed what appeared to be a low level of the 107K protein. Although we have been unable to determine whether or not this is in fact the 107K ElA binding protein, it is possible that in the absence of amino acids 122 to 129, low levels of the 107K protein can associate with ElA through weak interactions with amino acids outside of this region. Nonetheless, amino acids 121 to 127 appear to be essen-

Figure 4 lmmunoprecipitatrons of the ElAlHost Protein Complex from Cells Infected with Vrruses Expressing Amino-Terminal Mutants of ElA lmmunoprecrpitations from %-labeled HeLa cells Infected with vrruses expressrng the ammo-terminal mutants of ElA. Positions of ElA and coprecrpitatmg cellular proteins are on the left. The ElA proterns were immunoprecipitated with the anti-ElA monoclonal antrbody M73; 416 (PAb416) was used as a control monoclonal antibody Unequal alrquots were loaded to compensate for differences rn the quantity of ElA protern produced by the drfferent mutants

tial for a strong interaction between EIA and the 107K and pl05-RB proteins. To determine the amino-terminal boundary of the sequences necessary for binding to the cellular proteins, a series of mutants with deletions in the sequences encoding the amino terminus of ElA was examined (Figure 4). Results obtained using these mutants showed that no sequences could be removed from the amino terminus without eliminating binding to the 300K protein. Even a mutation that predicts only a single amino acid change, at position two (mutant pm563; Figure 4) was sufficient to eliminate coprecipitation of the 300K protein. In contrast, mutations at the extreme amino terminus did not affect binding to either the 107K or the plO5-RB protein, confirming that these proteins bound independently of the interaction with the 300K protein. The first 29 amino acids were not required to form the interaction between either the 107K or the pl05-RB proteins (mutant NTdI646; Figure 4); however, extension of the deletion to include the first 85 amino acids abolished the interaction with the p105-RB protein (mutant NTd1814; Figure 4). This result suggests that two noncontiguous regions of the ElA polypeptide are needed for binding to the pl05-RB protein, one region within amino acids 30 to 85 and the other region consisting of amino acids 121 to 127, as described above. It is possible that both do not directly interact with the pl05-RB protein but that one region influences binding through an indirect mechanism. The data presented here do not distinguish between these possibilities. Unlike the pl05-RB

Cellular Targets for Transformation 71

by ElA

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protein, binding to the 107K protein was not affected by the absence of amino acids 2 to 85 (NTd1814) nor was it abolished by deletion of the sequences encoding amino acids 2 to 120 (mutant NTdlS19). The lower level of 107K coprecipitating with the protein product of NTdl919 may be a consequence of the low amount of protein produced by this mutant. As expected, the product of mutant NTd/lOlO was incapable of interacting with the 107K protein. This mutant deletes the sequences encoding amino acids 2 to 150 and therefore is missing all the sequences shown above to be important for binding to the three cellular proteins. The protein product from mutant NTd/lOlO migrated off the gel shown in Figure 4 but is shown on the 15% gel in Figure 2b. A previous study using many of the mutants described here demonstrated that deletion of the region encoding amino acids 30 to 85 results in exclusron of the protein from the nucleus (Quinlan et al., 1988). It was possible that the failure of the ~105RB protein to bind to the protein products of mutants NTd/814 and NTdI919 was due to inaccessibility of the mutant ElA proteins to the pl05-FIB protein. To address this possibility, in vitro binding reactions were performed ustng radiolabeled uninfected HeLa cell lysates and unlabeled infected lysates as previously described (Harlow et al., 1986). In vitro binding reactions provided essentially the same results observed in vivo with immunoprecipitates from infected cells. Mutants NTd/814 and NTd1919 failed to bind detectable levels of pl05-RB but were capable of binding to 107K. Wild-type ElA bound to slightly greater amounts of plO5-RB than 107K, indicating that the failure of the mutants to bind to pl05RB was probably not due to competition and/or greater affinity for the 107K protein (data not shown). The region composed of amino acids 1 to 8.5 contains

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Figure 5. lmmunopreclpltatlons of ElAiHost Protein Complexes from Cells Infected with Internal Deletion Mutants of ElA Shown are immunopreclpltatlons from 3%-methlonlne-labeled HeLa cells infected with VIruses expressing Internal deletion mutants of ElA. The posItIons of the EiA proteins and the cellular proteins copreclpltating with ElA are shown on the left. Numbers on the right represent the molecular weights of standard size markers. The ElA proteins were immunopreclpltated with the M73 monoclonal antibody. 416 was used as a control antlbody.

both the binding site for the 300K protein and one of apparently two regions of ElA necessary for binding to the ~105RB protein; however, the boundary at amino acid 85 was determined only by the extent of the deletion of NCdl. To more rigidly define the boundary of the sequences involved in interacting with these two proteins, a serves of mutants containing deletions of sequences encoding amino acid 85 and amino acids toward the aminoterminus of the protein were analyzed. Examination of the complexes formed between these mutant ElA proteins and the cellular proteins (Figure 5) revealed that deletion of the sequences encoding amino acids 77 to 85 (mutant di787N; Figure 5) did not affect binding to either the 300K protein or the plO5-RB protein. A larger deletion removing the sequences encoding amino acids 69 to 85 (mutant dR63N; Figure 5) resulted in the loss of binding to the 300K protein but did not affect binding to the pi05RB protein. This result indtcates that the 300K protein binds to an epitope or epitopes contained within amino acids 1 to 76. The interaction with the pl05-RB protein did not requtre amino acids 61-85 (mutant d/742N; Figure 5) but was lost when sequences encoding amino acids 30 to 85 were deleted (mutant d1646N; Figure 5). These results indicate that amino acids 30 to 60 contain sequences that are required in addition to amino acids 121 to 127 for stable binding to the pl05-RB protein. Regions of ElA That Are Involved in Transformation Form Binding Sites for Cellular Proteins In a previous publication, many of these mutants were used to identify the minimal regions of ElA that are critical for the transforming function(s) (Whyte et al., 1988b). The transforming ability of one set of mutants, those constructed by deletron from the Nael site at nucleotrde 813

Cell 72

Table 1 Cooper&on

between Adenowrus

ElA Deletion Mutants and the T24 H-ras Oncogene

to Transform

Primary BRK Cells

PlasmId Cotransfected with T24 H-ras

Region of ElA Affected”

Total Number of Foclb

Transformation Efflclency Relative to Wild-type (Percentage)c

none (carrier DNA only) wld-type ElA d/81 1 N

-

0 100 110 164 110 48

d1793N

aa a a. a.a a a.

85 83-85 81-85 79-85

0 115 127 189 127 71

d1787N

aa.

77-85

55

d1805N d/799N

d1763N

a.a. 69-85

dl742N

a.a.

d1739N

a.a. 61-85 a a 30-85 a.a 27-85

d1646N d/637N

62

(21)”

VW

62-85

(00.9,” 0 0

(h 0 0

B Amino acids of ElA deleted by the mutation. In each mutant, the Inserted linker encoded the amino acids pro-arg-gly ii Total number of foci observed m three Independent experiments Ten plates were cotransfected In each experiment. L Percentage of foci observed relatwe to the number of foci observed using wld-type ElA. d Number In brackets represents abortwe foci See text for further details.

toward the Yportion of the gene, have not been previously reported. Table 1 summarizes the results of several focus formation experiments in which plasmids expressing the mutant ElA proteins were cotransfected with a plasmid expressing the T24 H-ras oncogene into primary baby rat kidney ceils. Deletion of the sequences encoding amino acids 77 to 85 only slightly affected the levels of transformed foci observed. In contrast, extension of the deletion to include sequences encoding amino acids 69 to 85 resulted in a significant decrease in the number of foci. Furthermore, the foci formed by these mutants were unstable in that they had a limited proliferative potential and consistently failed to give rise to stably transformed cell lines. As expected, larger deletions also had an impaired transforming ability. A comparison of the regions involved in binding to the 300K, 107K, and ~105RB cellular proteins with the regions identified as being important in cooperating with a second oncogene to transform primary cells revealed a striking correlation between the two (Figure 6). In fact, the data obtained using this set of mutants (Table 1 and Whyte

Figure 6 Comparison

of Regions of ElA involved In Transformation

et al., 1988b) detected no differences between transforming ability and binding to cellular proteins. All mutants defective for binding to any of the 300K, 107K, or ~105RB proteins were also defective for transformation. This correlation suggests that these proteins are cellular targets for the transforming functions of ElA and that interactions with more than one cellular protein are essential for ElA to exert its transforming function. Discussion The results presented in this study strongly imply that ElA exerts its transforming potential through interactions with the 300K, 107K, and pl05-FIB proteins. In no case was stable transformation obtained using a mutant whose protein product failed to bind to each of these three cellular proteins Furthermore, regions of ElA that were not essential for these protein-protein interactions were also nonessential for the transforming functions of ElA. Interaction with at least two and possibly all three of the cellular proteins appears to be necessary for transformation. The correla-

and Regions Involved in Binding to Cellular Proteins

SchematIc representahons fo the ElA region h!ghllghtlng the sequences required for transformation and the regions required for blnding to each of the 300K. 107K, and ~105.RB cellular proteins The boxed regions represent protein coding regions. The hatched region represents the protein coding sequences that are umque to the 13s mRNA The solld regions represent sequences required for the respective functions stated on the right Numbers refer to nucleotlde posItIons The corresponding amino acid sequences for each region shown In black are at the right.

Cellular Targets for Transformatlon by ElA 73

tion between the regions needed for transformation and those needed for interacting with the cellular proteins supports an essential role for the interaction with the 300K protein in transformation. Mutations that led to the disruption of the interaction with the 300K protein completely abolished transformation regardless of whether or not the interactions with the 107K and pl05-RB proteins were left intact. However, the interaction with the 300K protein was not sufficient for transformation. Mutations resulting in the disruption of the interactions with the 107K and ~105RB proteins also destroyed transforming activity, indicating that interaction with at least one or possibly both of the 107K or ~105RB proteins is required in addition to the interaction with the 300K protein. The individual roles of the interactions between ElA and the 107K and ~105RB proteins are difficult to distinguish. Mutations that deleted the binding site for the 107K protein also abolished the interaction with the pl05-RB protein, indicating that the binding sites for 107K and ~105RB have sequences in common. As a consequence, the mutants used in this study do not differentiate between the potential transforming roles of the interactions involving the 107K and ~105RB proteins. Interaction with at least one and possibly both appears to be essential. Further experimentation will be necessary to fully resolve the roles of these two proteins in ElA-mediated transformation. Recent experiments by Moran and Zerler (1988) suggest that the interaction with the 107K protein is important for transformation. In these experiments, mutants containing deletions in the sequences encoding either conserved region one (amino acids 40 to 70) or conserved region two (amino acids 121 to 139) could together complement an activated ras gene. Apparently, the mutant protein products were capable of functioning in trans. Although neither of the proteins produced by these constructs would be expected to interact with the plO5-RB protein, low levels of transformed foci were obtained. These observations would support an essential role for the interaction with the 107K protein, at least in the absence of the interaction with pl05-RB. These experiments might also suggest that the interaction with pl05-RB is not essential for transformation; however, several pieces of information argue against this conclusion. First, the low level of transformed foci observed in these assays raise the possibility of additional events contributing to the transformed state. It is possible that these transformed cells contain either a mutation disrupting the function of the RB-1 gene or another functionally comparable mutation. Second, in a variation of this experiment, we have found that much greater levels of transformed foci can be obtained by including a mutant whose product can bind to both the 107K and plO&RB protein in place of a mutant whose product can bind only to the 107K protein (our unpublished data). Finally, the recent identification of pl05-RB as a product of a gene thought to be involved in tumorigenesis supports a role for this interaction in transformation (Whyte et al., 1988a). A comparison of the ElA sequences from different adenovirus serotypes identified three strongly conserved regions (Kimmelman et al., 1985). The regions have been referred to as conserved regions one, two, and three and

span amino acid residues 40 to 70, 121 to 139, and 140 to 185, respectively. Conserved regions one and two comprise much of the ElA sequences involved in binding to the 300K, 107K, and ~105RB proteins. One likely possibility is that the binding sites for these cellular proteins have been conserved throughout the evolution of the different serotypes. Binding to the 300K protein requires sequences extending to the amino terminus of the protein, indicating that the functionally important regions of ElA are not entirely within the conserved regions. The location of the binding sites for the 300K protein and for pl05-RB also indicate that region one is much more complex than previously thought. Although both the 300K and pl05-RB proteins seem to interact with sequences in this region, the two proteins do not compete for this region of ElA. Both the 300K protein and pl05-RB can interact with the same ElA species (our unpublished data). It is possible that the Interaction with the 300K protein actually requires two noncontiguous regions separated by all or part of amino acids 30 to 60. If this is true, it may be possible to make mutations in this region that disrupt binding to ~105 RB but leave the interaction with the 300K protein intact. The interaction with pl05-RB requires the presence of sequences from each of these two regions. It is possible that only one of these regions actually physically interacts with pl05-RB and that the other region indirectly influences binding. While we cannot eliminate this possibility, our data do indicate that both regions must be present for the interaction to occur. It has been previously suggested that each of the conserved domains forms a functional domain of the protein. Our data present a more complex picture of the roles of conserved regions one and two. A region of ElA (conserved region two) with similarity to a region conserved in papovaviral T antigens has been previously identified (Stabel et al., 1985). This region of ElA contains the binding site for the 107K protein and part of the binding site for the pl05-RB protein. A similar region has also been found in the E7 protein of human papillomaviruses (Phelps et al., 1988). Each of these proteins has been associated with the transforming potential of their respective viruses. Mutants in the analogous region of SV40 T antigen suggest that this region is important for transformation by SV40 large T antigen (Kalderon and Smith, 1984). Furthermore, DeCaprio and colleagues have recently demonstrated the existence of an interaction between the SV40 large T antigen and the pl05-RB protein (DeCaprio et al., 1988). It is possible that interactions with the 107K and/or the pl05-RB proteins is a common transforming mechanism among viral transforming proteins. The identification of multiple interactions as being important for ElA-mediated transformation raises the question of whether they represent three independent pathways or whether the proteins associate to achieve one biochemical event. The results of Moran and Zerler (1988) suggest that the interactions with the 300K and the 107K proteins represent independent events. Furthermore, the overlapping binding sites for the 107K and pl05-RB suggest that these molecules do not both associate with the same ElA molecule. Physical studies on the ElA protein

Cell 74

complex also indicate this to be the case (our unpublished data). It therefore seems likely that each of the three interactions between ElA and the cellular proteins represents an independent event. It is possible that ElA achieves its transforming potential by simply binding to the cellular proteins. As previously discussed (Whyte et al., 1988a), ElA may block the normal function the pl05RB protein by binding to it, thus achieving the same effect as the loss of the RB-1 gene function seen in retinoblastomas and various other tumors (Benedict, 1987; Friend et al., 1987; Harbour et al., 1988; Lee et al., 1988). A mechanism such as this may explain the potent actions of the small regions of ElA that interact with these cellular proteins. Experimental

Procedures

ConstrucGon of Mutants and Viruses The ElA mutants used in this study have been previously described (Whyte et al , 1988b) with several exceptions. The wrus NCdl has been previously described and was provided by E Moran (Moran et al., 1986). Mutants NTd1919 and NTdllOlO were constructed In a manner slmtlar to other mutants with truncations of the amino terminus. An Ncol linker containmg a translatlonal start codon was Introduced into the gene encoding ElA upstream of nucleotides 919 and 1010 at the Clal and Smal sites, respectively. The upstream sequences of ElA were then ligated to the Ncol sites of these mutants as previously described (Whyte et al.. 1988b). A series of mutations extending upstream from the Nael site at nucleotide 813 were constructed by exonuclease Ill plus nuclease Sl digestion from the Nael site followed by Insertion of an Xhol linker. Sequences upstream of the Nael site from constructs containing appropriate deletions were ligated to the Xhol site of a plasmid containing a Xhol linker inserted into the Nael site to provide wild-type sequences downstream of the Nael site. Recombmant viruses containing mutated ElA regions in place of the wild-type ElA region have been previously described for some of these mutants (Quinlan et al., 1988). Viruses containing the remaining mutants were reconstructed using a similar protocol. Infections HeLa cells close to confluency were Infected with 100-200 PFU/cell of adenowrus serotype 5 or recombinant adenoviruses carrymg mutatlons In the ElA region. Infection was carned out for 1 hr in Dulbecco’s modlfled Eagle’s medium (DMEM) without serum, after which the innocula were removed and replaced with media containing 5% fetal bovine serum. All infectlons were performed in HeLa cells obtained from the cell culture faclllty at the Cold Spring Harbor Laboratory. Analysis of Cellular Proteins Complexed to ElA Cultures of Infected HeLa cells were labeled with 0.5 mC1/60 m m dlameter tissue culture plate of an 35S-methionme and 35S-cystelne mixture (Trans %-label, ICN Radiochemicals, Irvme, Califorma). LabelIng took place in 1 ml of DMEM, prepared wlthout methionine, for 4 hr beginning at 7 hr post-Infection. After the labeling period, the cells were rinsed with PBS and then lysed in a 1 ml solution of 50 m M HEPES (pH 7.0), 250 m M NaCI, and 0.1% NP40, as previously described (Harlow et al., 1986). ElA was lmmunopreclpitated using the antl-ElA monclonal antIbodIes Ml, M52, M58, and M73 The monoclonal antlbody PAb416, which recognizes the SV40 large T antigen, was used as a negative control antibody (Harlow et al., 1981). Immunopreopltates were subjected lo SDS-polyacrylamlde gel electrophorests on 8% polyacrylamlde gels except where otherwise stated (Laemml!, 1970). The gels were fluorographed (Bonner and Laskey, 1974), dried, and exposed at -70% for appropriate time Intervals. Transformation Assays The ability of ElA mutants to cooperate with the T24 H-ras oncogene In the transformation of primary baby rat kidney cells was assayed by cotransfectlng plasmlds expressing the ElA mutants with a plasmld expressing the H-ras oncogene as previously described (Whyte et al 1988b)

Acknowledgments We thank our colleagues at Cold Spring Harbor for advice and many helpful discusstons and J. Duffy, D. Green, and M. Ockler for photography and art work. This work was supported by Public Health Service grant CA 13106. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” In accordance with 18 U.S.C. Section 1734 solely to Indicate this fact. Received September

13, 1988, revised November

15, 1988

References Benedict, W. F. (1987) Recessive human cancer susceptibility (Retlnoblastoma and Wllm’s loct) Ad”. Viral. Oncol. 7. 19-34 Berk, A J. (1986). Adenovlrus Annu. Rev Genet. 20, 45-79

promoters

genes

and ElA transactlvation.

Berk, A. J., and Sharp, P A. (1978). Structure of the adenovlrus mRNAs Cell 14, 695-711.

2 early

Bonner, W. M , and Laskey, R. A. (1974). A film detection method for trltium-labelled proteins and nucleic acids in polyacrylamlde gels. Eur. J. Biochem 46, 83-88. Borrelli, E., Hen, R., and Chambon, P. (1984). Adenovirus 2 ElA products repress enhancer-induced stimulation of transcriptlon. Nature 312. 608-612. Braithwalte, A. W., Cheetham, B. F., LI, P., Parish, C. R., WaldronStevens, L K., and Bellett, A. J. D. (1983). Adenovirus-Induced alteratlon of the cell growth cycle: a requirement for expression of ElA but not of ElB. J. Vlrol. 45, 192-199. Carlock, L. R., and Jones, N. C (1981). Transformation-defective mutant of adenovlrus type 5 contaimng a smgle altered ElA mRNA specles. J. Vlrol. 40, 657-664. DeCapno, J. A., Ludlow, J. W., Flgge, J Shew, J.-Y., Huang, C.-M., Lee, W.-H., Marsilio, E., Paucha, E.. and Livingston, D. M. (1988). SV40 large T antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54, 275-283 Ellyahu, D., Raz, A., Gruss, P. Givol, D , and Oren, M. (1984). Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature 372, 646-649. Friend, S. H., Horowitz, J. M.. Gerber, M. R.. Wang, X., Bogenmann, E., Li, F. I’, and Weinberg, R. A. (1987). Deletion of a DNA sequence in retinoblastomas and mesenchymal tumors orgamzation of the sequence and its encoded protein. Proc. Natl. Acad. Sci. USA 84, 9059-9063. Gallimore, P H., Sharp, I? A., and Sambrook, J. (1974). Viral DNA transformed cells. II. A study of the sequences of adenovirus 2 DNA II- nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. 6101. 89, 49-72. Graham, F. L., van der Eb, A. J., and Heijneker, H. L. (1974). Size and location of the transforming region In human adenovirus type 5 DNA. Nature 257, 687-691. Haley, K. P., Overhauser, J., Bablss. L. E , Ginsberg, H. S., and Jones, N. C. (1984). Transformation properties of type 5 adenovlrus mutants that differentially express the ElA gene products. Proc. NatI Acad. Sci. USA 81, 5734-5738 Hansen, M. F., Koufos, A., GallIe, B L., Phillips, R. A., Fodstad, O., Brogger, A., Gedde-Dahl. T., and Cavenee, W. K. (1985). Osteosarcoma and retinoblastoma: a shared chromosomal mechanism revealing recessive predisposition. Proc. Natl. Acad. Sci. USA 82. 62166220 Harbour, J. W., Lal, S., Whang-Peng, J.. Gazdar, A. F, Minna. J. D. and Kaye, F J. (1988). Abnormalltles m structure and expression of the human retinoblastoma+gene In SCLC. Sctence 247, 353-357. Harlow. E.. Crawford, L. V., Plm. D C, and WIlliamson. N. M. (1981) Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Viral. 39, 861-869. Harlow, E., Franza, B., and Schley, C. (1985). Monoclonal

antlbodtes

Cellular Targets for Transformation 75

by ElA

speclflc for adenovlrus early region 1A proteins’ extensive heterogeneity in early region IA products. J Vlrol 55. 533-546. Harlow. E., Whyte, P. Franza. Jr, B R and Schley, C (1986). Assoclation of adenovlrus early-region 1A proteins with cellular polypeptldes Mol Cell BIoI. 6, 1579-1589. Hen. R Borrelli. E and Chambon. P (1985) Represston of the ~mmunoglobultn heavy chain enhancer by the adenovlrus-2 ElA products Science 230. 1391-1394 Houwellng, A, van den Elsen, I? J., and van der Eb. A J. (1980). Parteal transformation of primary rat cells by the leftmost 45% fragment of adenovlrus 5 DNA Vtrology 705, 537-550 Jenkins, J R Rudge. K and Currle, G A (1984). Cellular Immortalizatlon by a cDNA clone encodlng the transformation-associated phosphoproteln p53 Nature 312. 651-654. Kaczmarek. L., Ferguson. B.. Rosenberg. M and Baserga. R (1986). lnductlon of cellular DNA synthesis by purified adenovlrus EIA protelns Virology 152, l-10 Kalderon, D., and Smith, A. E (1984) In vltto mutagenesis of a putative DNA blnding domaln of SV40 large-T Virology 739, 109-137

Qulnlan, M P, Whyte, P, and Grodzlcker. T (1988) Growth factor I”ductlon by the adenovirus type 5 ElA 125 protein is required for immortallzatlon of primary epithellal cells. Mol. Cell. BIoI. 8, 3191-3203. Ruley, H. E (1983). Adenovlrus early region 1A enables viral and cellular transforming genes to transform primary cells in culture Nature 304, 602-606. Schneider. J. F.. Fisher. F, Goding, C. R. and Jones, N. C. (1987) Mutatlonal analysis of the adenovlrus ElA gene: the role of transcrlptlonal regulation in transformation EMBO J. 6, 2053-2060 Schwab, M Varmus, H E.. and Bishop. J M (1985). Human N-myc gene contributes to neoplastlc transformatlon of mammaltan cell in culture Nature 376. 160-162 Squire. J Goddard, A. D.. Canton, M Becker, A.. Phllllps. R A and GallIe, B. L. (1986). Tumour induction by the retlnoblastoma mutation IS Independent of N-myc expressIon. Nature 322, 555-557. Stabel, S Argos. P, and Philipson, L. (1985). The release of growth arrest by mlcrolnjectlon of adenovlrus ElA DNA EM60 J. 4, 23292336.

Klmchl. A , Wang, X-F. Weinberg. R A Chelfetz, S , and Massague. J (1988). Absence of TGF-b receptors and growth lnhibltory responses in retinoblastoma ceils Science 240. 196-199

Subramanian, R.. Kappuswamy, M., Nasr. R. J., and Chlnnadural. G. (1988). An N-terminal region of adenovlrus ElA essential for cell transformatlon and InductIon of an epithelial growth factor. Oncogene 2, 105-112

Klmmelman, D, Miller. J S., Porter, D and Roberts, B. E (1985). ElA regions of the human adenovlruses and the highly oncogenlc slmfan adenovirus 7 are closely related. J. Vlrol 53, 399-409.

van den Elsen, P, Houweling, A., and van der Eb. A. J. (1983) Expression of region ElB of human adenovlrus in the absence of regron ElA IS not sufficient for complete transformation Virology 728. 377-390.

Laemmll. U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227. 680-685

Velclch, A.. and Zlff, E. (1985). Adenovlrus ElA proteins repress transcrIptIon from the SV40 early promoter Cell 40, 705-716.

Land. H.. Parada, L F., and Weinberg, R. A. (1983). Tumorlgenic conversion of embryo flbroblasts requires at least two cooperating oncogenes Nature 304. 596-602.

Velclch, A., and Ziff, E. (1988) Adenovirus ElA ras cooperation activity IS separate from Its posctlve and negative transcrIptIon regulatory functlons. Mol. Cell. BIoI. 8, 2177-2183.

Lee, E. Y. P., To, H., Shew, J., BooksteIn, R , Scully. P, and Lee, W. (1988). InactIvation of the retlnoblastoma susceptiblllty gene in human breast cancers.,Sclence 247, 218-221.

Whyte, P, Buchkovlch, J J., Horowitz, J M.. Friend. S. H Raybuck. M., Weinberg, R. A., and Harlow. E. (1988a). Assoctation between an oncogene and an anti-oncogene: the adenovlrus ElA proteins bind to the retinoblastoma gene product Nature 334, 124-129.

Lee, W.-H., Murphree, A L and Benedict, W F (1984). Expression and ampllflcation of the N-myc gene in prnary retlnoblastoma Nature 309, 458-460 Llllle, J. W, Green, M., and Green, M. R (1986). An adenovlrus Ela protein region required for transformation and transcrlpbonal repress10n. Cell 46. 1043-1051. Llllle, J W, Loewensteln, P M Green, M. R.. and Green, M. (1987) FunctIonal domains of adenovlrus type 5 ElA protetns. Cell 50, 1091-1100. Montell, C, Ffsher, E. F. Caruthers, M. H , and Berk. A. J. (1982) Resolving the functions of overlapplng viral genes by site-specific mutagenesis at an mRNA splice site. Nature 295, 380-384. Moran, B., and Zerler, 6. (1988) InteractIons between cell growth-regulatlng domains in the products of the adenovlrus ElA oncogene. Mel Cell. BIoI. 8, 1756-1764. Moran, E., and Mathews, M B. (1987). Multiple functlonal the adenovlrus ElA gene. Cell 48, 177-178

domalns in

Moran, E , Zerler, B., HarrIson, T. M., and Mathews, M. 8. (1986). Identtflcatlon of separate domalns in the adenovlrus ElA gene for immortaltzatlon activity and the actlvatlon of virus early genes. Mol Cell. BIoI. 6, 3470-3480 Parada, L F., Land, H , Weinberg. R. A., Wolf, D.. and Rotter, V. (1984) Cooperation between gene encodlng p53 tumour antigen and ras m cellular transformation. Nature 372, 649-651. Perricaudet, M., Akuslarvi. G., Virtanen. A., and Pettersson, U (1979). Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses. Nature 278. 694-696. Phelps. W C., Yee, C. L., Mtinger, K., and Hawley, P M. (1988) The human paplllovirus type 16 E7 gene encodes transactivatlon and transformation functions similar to those of adenovirus ElA. Cell 53, 539-547. Quinlan, M. P., Sullivan. N.. and Grodzlcker, T. (1987). Growth factor(s) produced during infection with adenovlrus variant stimulates prollferation of nonestablished eplthelial cells. Proc Natl. Acad. SCI. USA 84. 3283-3287.

Whyte, P, Ruley, H. E., and Harlow, E (1988b). Two regions of the adenovirus early region 1A proteins are required for transformation. J. Vlrol 62, 257-265. Yancopoulos, G. D., Nlsen, P D., Tesfaye, A., Kohl, N. E., Goldfarb, M. P., and Alt, F. W. (1985) N-myc can cooperate with ras to transform normal cells in culture. Proc. Natl. Acad. SCI USA 82. 5455-5459. Yee, S., and Branton, P E. (1985). DetectIon of cellular protems assoclated with human adenovlrus type 5 early region 1A polypeptldes. Virology 747, 142-153. Zerler, B., Moran, B Maruyama, K , Moomaw, J , Grodrlcker, T., and Ruley, H. E. (1986). Adenovlrus ElA coding sequences that enable ras andpmtoncogenes to transform cultured primary cells. Mol. Cell. BIOI 6, 887-899.