Vol. 68, No. 2
JOURNAL OF VIROLOGY, Feb. 1994, p. 776-786
0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Phosphorylation at the Carboxy Terminus of the 55-Kilodalton Adenovirus Type 5 EBB Protein Regulates Transforming Activity JOSE G. TEODORO,1 TODD HALLIDAY,2 STEVE G. WHALEN,1 DENNIS TAKAYESU,' FRANK L. GRAHAM,2'3 AND PHILIP E. BRANTON"* Department of Biochemistry, McGill University, McIntyre Medical Sciences Building, Montreal, Quebec, Canada H3G 1Y6,1 and Department of Biology2 and Molecular Virology and Immunology Program,3 McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Received 16 September 1993/Accepted 15 November 1993
The 55-kDa product of early region lB (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation.
The proteins encoded by early region 1B (E1B) of human adenoviruses play important roles in both virus replication and oncogenic transformation of rodent cells. Adenovirus serotype 5 (Ad5) produces two major E1B proteins from the early 2.2-kb mRNA, including a 19-kDa 176-residue polypeptide (176R) and, from an internal initiation site and alternate reading frame, a 55-kDa 496R product (9, 19, 44). Later during infection, a second major E1B mRNA of 1.0 kb, which encodes both 176R and, from the 496R start site, an 84R protein which contains the same 79 amino-terminal residues as 496R (see Fig. 1A), is produced. Two minor E1B mRNAs of 1.26 and 1.31 kb, which produce, in addition to 176R, 156R or 93R proteins (2, 24, 30, 33, 59, 62), are transcribed later in infection. As with 84R, both 156R and 93R contain the amino-terminal 79 residues of 496R. The remaining 77 residues of 156R are identical to the carboxy terminus of 496R, while 93R is terminated by a unique 14-residue sequence. The roles of these additional E1B polypeptides have not been determined. The 496R E1B protein is synthesized early after infection and is found in both the nucleus and cytoplasm (50, 54, 70). It is essential for viral replication and is necessary for the accumulation of late viral mRNAs, late viral protein synthesis, and the shutoff of host metabolism (3, 4, 6, 8, 11, 25, 29, 31, 40, 46, 51, 67). In the case of serotype 12 (Adl2), this E1B product is required for viral DNA replication (10, 34, 57, 73). In addition, it plays a role in cell transformation. In combination with early region 1A (ElA), the 19- and 55-kDa E1B proteins are each able to transform primary rodent cells independently, but with increased efficiency in combination (6, 8, 40, 63, 67, 72). The 19-kDa protein appears to function to protect cells from the toxic effects of ElA products which induce a phe-
nomenon similar to apoptosis and programmed cell death (14, 48, 64). It is likely that 496R of Ad5 derives some or all of its transforming activity through interactions with the p53 cellular tumor suppressor (27, 53). Such complex formation has been shown to inhibit p53-mediated transcriptional activation (66). The mechanism of this repression is not known. Some years ago, 496R was shown to be phosphorylated (54, 65) at both serine and threonine residues (36). In studies designed to characterize 496R-related E1B products, we found that 156R is also phosphorylated whereas 84R and 93R are either unphosphorylated or phosphorylated only at low levels (59). These results suggested that although phosphorylation sites could be present in the first 79 amino acids of 496R, one or more major phosphorylation sites must exist within the carboxy-terminal 77 residues. In the present experiments, we have used both biochemical and genetic means to show that Ser-490, Ser-491, and probably Thr-495 of 496R are phosphorylated. In addition, a mutant in which these serines had been converted to alanine residues was found to be defective in viral replication and to transform primary rat cells at very reduced efficiency. This mutant 496R protein formed complexes with p53 at normal levels but only partially inhibited p53 transactivation activity. These results suggested that phosphorylation at carboxy terminal sites is required for cell transformation and may regulate the effects of 496R on p53 or some other function. MATERIALS AND METHODS Cells and viruses. Human KB cells were cultured on 150mm-diameter dishes (Corning Glass Works, Corning, N.Y.) in ot-minimal essential medium supplemented with 10% fetal calf serum and were normally infected with mutant or wild-type (wt) AdS at a multiplicity of 35 PFU per cell, as described previously (50). The virus used as wt was d1309 (26). NIH 3T3 cells were cultured under similar conditions in 60-mm-diameter dishes. Plaque assays were performed on human KB cells and AdS-transformed 293 cells (22).
* Corresponding author. Mailing address: Department of Biochemistry, McGill University, McIntyre Medical Sciences Building, 3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6. Phone: (514) 398-7263. Fax: (514) 398-7384. Electronic mail (Internet) address:
[email protected]. 776
VOL. 68, 1994
REGULATION OF Ad5 E1B PROTEIN BY PHOSPHORYLATION
A.
176R E 496R 84R 93R 156R
B.
E1 B mRNA 2.2 kb
I 1
1_79 1_79
T1 T2
T3 25
20
1.0 kb
1.31 kb
1.26 kb
425
430
435
V-N-L-N-G V-F-D-M-T
P-E-SM--K T43
30
40
|450
I iZo I el45
S-G-H-A-S V-E-S-G-C E-T-Q-E-S 35
80f184
-
F1 5
P-SE-RIG V-P-A-G-F
M-E-R{RIN
2.2 kb
80_156 N
0
496
80393
1_79 1
M-Kjl-W-K I V-L-F4Y-D E-T-RIT-R I T44 T45 T46 T47 T48 455
45
P-A-T-V-V F-R-P-P-G D-N-T-D-G
465
4W
C-R-P-C-E C-G-G-KjH l-RjN-Q-P T49
50
60
55
470
G-A-A-A-A A-G-G-S-0 A-A-A-A-G 65
777
70
T50
475
480
V-M-L-D-V T-E-E-L-R P-D-H-L-V
75
e-4i96
A-E-P-M-E P-E-S-R-P G-P-S-G-M
L-A-C-T-R
D-E-D[TD
A-E-F-G- S
T51
T52
79
M-N-V-N --------490 491
wt
Gly Ser Ser Asp GGC TCT AGC GAT
11
C.
I
wt
pm49OA/ GGC GCG GCC GAT 491A Gly Ala Ala Asp
Bgi 11
..ATG ACC ATG AAG ATC TGG AAG GTG CTG..
...M- T
-
435
M- K- I
-
W- K
440
-
V- L...
Ban*il
in 3328 (+ )
BemHI
..ATG ACC ATG AAG ATC CAA TTG TTA TCC GCT CAC AAT TGG GTC AGG ATC TGG AAG GTG CTG..
...M- T
-
M
-
K- I
-
Q-
L- L- S
-
A- H- N- W- V- R- I
-
W- K
-
V- L...
435
BanMI
in 3328 ()
BivI
..ATG ACC ATG AAG ATC CTG ACC CAA JTG TGA GCG GAT AAC AAT TGG ATC TGG AAG GTG CTG.. ...M- T - M - K- I - L- T- Q- L- END 435
FIG. 1. Ad5 E1B gene products, amino acid sequences, and mutants. (A) Protein regions relative to the AdS ElB mRNAs that encode them and the corresponding numbers of residues. =, sequences unique to 176R; _, sequences present in 496R; = sequences unique to 84R; 1, sequences unique to 93R. (B) Amino acid sequences and mutants. Amino acids of 496R present also in 156R have been displayed along with the predicted tryptic peptides (shown below the sequence) and positions of potential phosphorylation sites (underlined). Shown also (lower right) are the changes introduced to yield mutant pm49OA/491A. (C) Production of mutants in3328(+) and in3328( -). The position of the BglII site in wt and those of the inserted sequences in both orientations have been presented. The amino acid sequences are shown below, and those encoded by the inserts are underlined. ,
E1B mutants. Mutants with defects in 496R were prepared by two methods. Two mutants were produced by insertion mutagenesis according to the method of Bautista and Graham (7). These mutants, termed in3328(+) and in3328(-), were generated by the insertion of a synthetic oligonucleotide bounded by sequences recognized by BamHl into the BglII site located following nucleotide 3328 within the coding sequence for 496R (Fig. 1C). Mutant in3328(+) produced a 496R protein containing an 11-amino-acid insert between Ile-438 and Trp-439. Mutant in3328(-), which contained the insert in the opposite orientation, produced a truncated 496R product because of the presence of a stop codon just downstream of Ile-438. Another mutant, termed pm49OA/491A, in which the codons for Ser-490 (TCT) and Ser-491 (AGC) had been altered to those of alanine (GCG and GCC, respectively), was produced by PCR-assisted oligonucleotide-directed mutagenesis (see Fig. 1B). The altered Ad5 DNA fragments were
subcloned into plasmid pXC38, which contains the complete ElA-plus-ElB sequence (bases 22 to 5788), to produce El plasmids pXC-in3328(+), pXC-in3328(-), and pXC-490A/ 491A. These mutations were also introduced into plasmid p1716/2072, a derivative of pXC38 containing mutations which alter the codons of Met-1 and Met-120 of ElB-176R and thus eliminate production of this polypeptide without altering E1B496R (40). Such plasmids have been designated p3328(+)19K-, p3328(-)-19K-, and p490A/491A-19K-. Mutations were rescued into Ad5 virions by the method of McGrory et al. (39), and those in the pm1716/2072 and 19K- backgrounds were noted. The fidelity of all mutants was confirmed by DNA sequencing, restriction enzyme digestion, or Southern blot analysis, as described previously (16, 40, 60). Mutant pm2019/ 2250, which produces 176R but no 496R (40), was also used in studies. Antisera and immunoprecipitation. Antipeptide serum
some
778
TEODORO ET AL.
58-N2 (or a similar 58-NI antibody), which is specific for the amino terminus of 496R and which also recognizes the 496Rrelated EIB products 84R, 93R, and 156R, has been described previously (40, 59), as has anti-peptide serum 58-Cl, which recognizes 496R and 156R (65). Rat monoclonal antibody 9C10 (Oncogene Science) (70), which recognizes an epitope towards the amino terminus of 496R, was also used in some experiments, as was the 496R-specific 2A6 monoclonal antibody (54). Cell extracts were immunoprecipitated exactly as described previously (38) by using 25 pl of antipeptide serum orS .l of 9C10 or 2A6. Radioactive labeling. AdS- or mock-infected cells were normally labeled from 16 to 18 h postinfection (hpi) with 100 p.Ci of [35S]methionine (Amersham Corp., Arlington Heights, Ill.; specific activity, 1,300 Ci/mmol) or 1 mCi of 32p; (New England Nuclear) in 4 ml of methionine- or phosphate-free medium. In the case of samples prepared for further biochemical analysis, 1 mCi of [15S]methionine or 2.5 mCi of 32Pi per plate was employed. For pulse-chase experiments, cells were labeled with [35S]methionine for 1 h at 500 p.Ci per plate and then washed twice with (x-minimal essential medium and incubated for various times in medium containing 10 times the normal amount of unlabeled methionine. SDS-PAGE. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using 10 or 15% polyacrylamide separating gels and a 5% stacking gel, as previously described (50). Preparation of tryptic peptides. Regions of polyacrylamide gels containing labeled 496R or 156R were excised, proteins were eluted into 50 mM ammonium bicarbonate (pH 8.0) containing 0.1% SDS and 0.5% (vol/vol) 2-mercaptoethanol, and the mixture was digested with tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (10 mg/ml), as described previously (61). Peptides were oxidized with fresh performic acid prior to analysis by high-performance liquid chromatog-
raphy (HPLC). Analysis of tryptic peptides by HPLC. [35S]methionine- or 32Pi-labeled E1B proteins purified by immunoprecipitation and SDS-PAGE were extracted and treated with TPCK-trypsin (Worthington), as described previously (61). Tryptic peptides were separated at room temperature on a Waters dual pump HPLC system with a 600E controller using an Ultrasphere octyldecyl silane (C18) reverse phase column (4.6 by 250 mm) which had been preequilibrated with solution A (5% formic acid in water). Peptides were eluted from the column either by using a linear 1 to 63% gradient of solution B (5% formic acid in ethanol) for 95 min (as in the experiments shown in Fig. 3), as described previously (15), or by using a stepwise protocol involving elution for 5 min with solution B, followed by 65 min with a 1 to 30% gradient with solution B, and terminated by 30 min with a 30 to 60% gradient with solution B (as in the experiments shown in Fig. 6). The flow rate was 1 ml/min. Detection of labeled peptides was achieved with an on-line LB507A isotope detector (Berthold). Phosphoamino acid analysis by TLC. 32Pi-labeled proteins were subjected to acid hydrolysis, and after being mixed with nonradioactive phosphoamino acid markers the material was subjected to electrophoresis at pH 3.5 in pyridine-acetic acid-0.5M EDTA-double-distilled H20 (5:50:2:945 by volume) on cellulose thin-layer chromatography (TLC) plates (Polygram CEL300), as described previously (61). The positions of phosphoamino acids were determined by staining with ninhydrin or by autoradiography. p53-binding assay. DNA from expression plasmid P53pSP65 containing the sequence for murine p53 (27) was linearized by digestion with HindlIl, and transcription was performed by
J. VIROL.
pm490AI
wt
491A 35S
32p 35s 32p M
i
92
1'
A
200 92.5
69
U 496R
46
30j
22K 21 K
FIG. 2. SDS-PAGE analysis of EIB proteins of wt and pm49OA/ 491A. KB cells mock infected or infected with wt AdS or pm49OA/ 491A were labeled with either [31 S]methionine or "Pi, immunoprecipitated by using 58-N2 serum, and analyzed by SDS-PAGE. The positions of 496R, 84R, 93R, 156R, and the 21- and 22-kDa proteins have been indicated at the right. Lane M, 14C-labeled molecular weight markers (indicated, in thousands, at the left).
using 2 pLg of DNA and SP6 RNA polymerase (Pharmacia). Capped RNAs were translated by using rabbit reticulocyte lysates (Promega) in the presence of [35 S]methionine (Amersham; translation grade; specific activity, 1,300 Ci/mmol) according to the manufacturer's specifications. The p53-binding assay was performed essentially as described by Kao et al. (27). Extracts were prepared according to Manley et al. (37) from
uninfected KB cells or KB cells infected with wt~or mutant AdS and harvested at 16 hpi. Protein concentration was determined by the Bio-Rad assay using bovine serum albumin as a standard. Increasing amounts of extract were mixed with S RId of translation mixture containing 35S-labeled p53, and complex formation was allowed to occur for 30 min at 40C, after which time 496R complexes were immunoprecipitated by using 20 pAl of 58-N2 serum and separated by SDS-PAGE using 10% polyacrylamide gels. The amount of p53 binding was determined from dried gels by using a FUJIX BAS 2000 Phosphorimager. The amount of 496R was determined from identical immunoprecipitates which were separated by SDS-PAGE, transferred to nitrocellulose, and then treated with 496Rspecific monoclonal antibody 2A6, which was visualized by using 1251-conjugated goat anti-mouse secondary antibody and quantified by using a FUJIX Bas2000 Phosphorimager. Determination of transactivational repression of GAIA-p53 by 496R. Repression by 496R of p53 transcriptional activating activity was performed by a modification of the procedure of Yew and Berk (66). The DNA constructs used in the assay (see Fig. 9A) include G5Elb-CAT, which consists of five GAL4-
VOL. 68, 1994
REGULATION OF Ad5 E1B PROTEIN BY PHOSPHORYLATION
0.6 1 A.
E0
779
B.
0.4 0.2
0 I-
0.2
0
5:
0.8
1.5
0 1. 0 0.4
cr
0.5 20
40
60
80
20
40
60
80
TIME OF ELUTION (min) FIG. 3. HPLC analysis of tryptic peptides from 496R and 156R. 496R and 156R labeled with [35S]methionine or 32pi were isolated by immunoprecipitation and SDS-PAGE, and tryptic peptides were analyzed by HPLC, as described in Materials and Methods. The amount of radioactivity has been shown on the ordinate and the time of column elution (min) on the abscissa. (A and C) 35S- and 32 Pi-labeled 496R, respectively; (B and D) 35S- and 32Pi-labeled 156R, respectively. B to K (A and B) and I to III (C and D), peptides and phosphopeptides, respectively (see the text).
binding sites linked to the E1B TATA sequence followed by the chloramphenicol acetyltransferase (CAT) coding sequence (18), and expression vectors for GAL4 fusions including mGAL4D-p53(1-393) and mGAL4D-VP16, which contain the entire coding sequences for human p53 and the activation domain of herpes simplex virus activator VP16, respectively, linked to the DNA binding region of GAL4 (18). In addition, vectors expressing wt or mutant Ad5 496R were constructed as follows. The coding region of 496R from pXC38 DNA present in the BssHII fragment bounded by nucleotides 1974 and 4106
P-Ser
1
0
P-Thr
P-Tyr 1 wt 496R
2
3
4
5
490A/ 491A
wt 496R
wt
490A/
496R
491A
peak 11
pek
III
peek IV
FIG. 4. Phosphoamino acid analysis. 32Pi-labeled 496R, 156R, and peptides derived from 496R were hydrolyzed, and the resulting phosphoamino acids were analyzed by TLC, as described in Materials and Methods. Samples included 496R derived from wt Ad5 (lane 1) and from mutant pm49OA/491A (lane 2), peptides II (lane 3) and III (lane 4) from wt 496R, and peptide IV from pm49OA/491A isolated by HPLC (lane 5) (see Fig. 6). The positions of phosphoamino acid markers determined by staining with ninhydrin have been indicated.
of the AdS coding sequence was treated with T4 polymerase and introduced in the antisense orientation into the SmaI site of pGEM7f- (Promega) by blunt-end ligation. The EcoRIBamHI fragment was then subcloned into the Moloney retroviral vector pLXSN (1) in the sense orientation to form vector pLXSN-55K. pLXSN-490A/491A was created by removing the KpnI-AfllI fragment (nucleotides 2048 to 3533) of the 496R coding sequence in pLXSN-55K and replacing it with the same fragment from mutant p490A/491A. For the repression assay, NIH 3T3 cells were plated at a density of 2 x 105 cells per 60-mm-diameter plate and were transiently transfected (23) by using 0.5 ,ug of G5Elb-CAT reporter DNA, 0.5 ,ug of mGAL4D-p53(1-393) or 0.1 p.g of mGAL4,,-VP16 DNA, and I jig of DNA from pLXSN, pLXSN-55K, or pLXSN-490A/ 491A. Transfection efficiency was standardized by including 1 p.g of RSV,B-Gal (47) and measuring P-galactosidase activity. Sonicated salmon sperm DNA (Pharmacia) was added to make a total of 10 ,ug of DNA per plate. Cells were glycerol shocked for 3 min at 12 h posttransfection and harvested 36 h later. CAT assays were performed as previously described by Gorman et al. (21) by using cell extracts containing the same amounts of ,B-galactosidase activity. CAT activity was quantified from TLC plates by using a phosphorimager. Transformation of baby rat kidney cells. Cells prepared from kidneys of 5- or 6-day-old Wistar rats were transfected with 5 p.g of plasmid DNA, as described previously (40). DNA concentrations were determined by fluorometry and by quantitative analyses of restriction enzyme-cleaved plasmid preparations on 1 % agarose gels. Parallel experiments indicated that 5 ,ug represented an amount of DNA that was within the linear range of transforming activity. Following 2 or 3 weeks in culture, transformed foci were visualized by Giemsa staining.
780
TEODORO ET AL.
RESULTS
_m
KB wt
J. VIROL.
in(-)
in(+)
58N1 9C10 9C10 58N1 9C10 58N1
Phosphorylation of E1B proteins. Previous studies had indicated that 496R is phosphorylated at both serine and threonine residues (36, 54, 65) and that one or more sites were located in regions common to ElB-156R (59). The latter is composed of the amino-terminal 79 and carboxy-terminal 77 residues of 496R and was found to migrate on SDS-PAGE as two distinct forms (59). Figure 2 shows an analysis by SDSPAGE of extracts from Ad5-infected KB cells which had been labeled with [35S]methionine (lane 2) or 32Pi (lane 1) and then immunoprecipitated by using antipeptide serum 58-N2, which recognizes the amino terminus of 496R. Only the more slowly migrating 156R species was highly phosphorylated. Figure 2 also indicates that no 32p; was evident in the positions of 84R and 93R, which share only 79 amino-terminal residues with 496R. However, it was likely that the phosphoproteins termed 21K and 22K, which migrated slightly more slowly than the major 84R and 93R species, probably represented minor phosphorylated forms of these proteins as they were found to be immunoprecipitated by sera specific for the unique carboxy termini of these products (58). These results suggested that 496R contains major phosphorylation sites within the 77 carboxy-terminal residues shared by 156R and that one or more minor sites might exist within the 79 amino-terminal residues. Figure 1B shows that this carboxy-terminal region contains four serine and six threonine residues. Serines 422 and 424 and threonines 435 and 471 are present within tryptic peptides T43, T44, and T51, which all contain methionine. Serines 490 and 491 as well as threonine 495 are present within the carboxy-terminal peptide T52, which lacks methionine. These serine residues lie adjacent to an acidic region characteristic of sites phosphorylated by casein kinase II (43). The 79-residue amino-terminal region also contains several potential phosphorylation sites, notably Ser-30, which is present within a sequence characteristic of sites phosphorylated by the cdc2 or MAP families of protein kinases, and Ser-23 (and possibly serines 20 and 27) which could represent potential casein kinase II sites. All of these amino-terminal residues are present within the large, methionine-containing tryptic peptide T4. Analysis of tryptic peptides by HPLC. To study the phosphorylation of 496R further, 496R and 156R isolated from [35S]methionine- and 32Pi-labeled cells were digested with trypsin, and the tryptic peptides were analyzed by HPLC. Figure 3 shows the profiles obtained with 496R which, upon complete digestion, should yield 10 methionine-containing peptides. Figure 3A shows approximately this number of 35S-labeled peptides (indicated as B-K). Three 32Pi-labeled phosphopeptides were produced from 496R (Fig. 3C). A minor species (species I) which eluted early in the gradient probably represented free phosphate, which had previously been found to elute from the column in a similar position (61). Two other phosphopeptides (peptides II and III) were clearly evident. Phosphopeptide II eluted in a position close to that of 35Slabeled peptide C (Fig. 3A), but phosphopeptide III clearly did not correspond to any of the methionine-containing peptides. The 156R protein is predicted to yield four methioninecontaining peptides, three of which should correspond to peptides present in 496R, and one (which spans the region following Asn-79 removed by splicing to form the 1.26-kb mRNA) unique species. Figure 3B shows that 156R contained fewer 35S-labeled peptides than 496R, and all except peptide Dl eluted in positions similar to peptides found in 496R (Fig. 3A). Interestingly, the phosphopeptide pattern found with 156R (Fig. 3D) was identical to that observed with 496R. Phosphoamino acid analysis of 32Pi-labeled 496R indicated
1
200
2
3
4
w
5
6
92.5
69
* 496R 46
30
L
FIG. 5. SDS-PAGE analysis of 496R synthesized by in3328 mutants. KB cells infected with wt Ad5, infected with mutants in3328( -) or in3328(+), or mock-infected (lane 1) were labeled with [35S]methionine, immunoprecipitated by using 9C10 or 58-Ni antibodies, and analyzed by SDS-PAGE. The position of 496R has been indicated at the right and those of 14C-labeled molecular weight markers (in
thousands) have been indicated at the left.
that phosphoserine and a small amount of phosphothreonine were present (see Fig. 4, lane 1). Peptide II of 496R isolated by HPLC also contained both phosphoamino acids in about equal amounts (Fig. 4, lane 3), whereas peptide III contained only phosphoserine (Fig. 4, lane 4). These results indicated therefore that the major sites present in 496R must reside at serine and threonine residues in tryptic peptides common to 156R. At least one of these sites must be a serine residue within phosphopeptide III, which lacks methionine. Another (phosphopeptide II) must contain both serine and threonine sites, possibly, but not necessarily, in a methionine-containing peptide. Analysis of carboxy-terminal insertion mutants. Although several potential phosphorylation sites exist within regions of 496R common to 156R, studies were carried out to focus on sites within peptide T52 at the carboxy terminus as this peptide lacks methionine and contains both serine (Ser-490 and Ser491) and threonine (Thr-495) residues which exist in the context of sites recognized by casein kinase II. The first approach was to generate mutants containing an insertion after nucleotide 3328 and following the codon for Ile-438 of 496R (Fig. 1B). Mutant in3328(+) should encode a modified 496R product which contains an 11-amino-acid insert between Ile438 and Trp-439. Mutant in3328(- ) contained the insertion in the reverse orientation, thus producing a translation stop site downstream of Ile-438 forming a truncated 496R product which lacks the carboxy-terminal 48 residues (Fig. 1C). These mutations were rescued into virus and used to infect KB cells. Following incubation with [35S]methionine, 496R was immu-
781
REGULATION OF Ad5 EiB PROTEIN BY PHOSPHORYLATION
VOL. 68, 1994
3.0
1 A.
C. 1.4
0.
1.5 a
0
I' 1.0
#on
1.0
__I __
h
-A.
| D.
B.
I0
4
0
3
1.0
4~~~~ 0.9
2' 20
40
60
:~~~~ 40
20
80
60
80
TIME OF ELUTION (min) FIG. 6. HPLC analysis of phosphopeptides of 496R and 156R synthesized by wt Ad5 or mutant pm49OA/491A. Tryptic peptides from 32Pi-labeled 496R and 156R produced by wt Ad5 or mutantpm49OA/491A were analyzed by HPLC as described in the legend to Fig. 3. 496R from wt AdS (A) and pm490A/491A (B) and 156R from wt Ad5 (C) and pm490A/491A (D) are shown. I to IV, phosphopeptides (see the text).
noprecipitated by using either 58-Ni serum or a 496R-specific monoclonal antibody, 9C10 (70). With mutant in3328( -), no 496R-related protein was evident from use of either 9C10 (Fig. 5, lane 3) or 58-Ni (lane 4) serum. With in3328(+), 496R was detected (Fig. 5, lanes 5 and 6), but clearly at levels far less than with wt AdS (lane 2). It had been observed previously that mutations affecting 496R frequently generate unstable forms of this EiB protein (27, 34, 66, 67, 72, 73), and such appeared to be the case with the in3328 mutants. Analysis of 496R from mutant pm49OA/491A. To determine directly whether or not serines 490 and 491 were sites of phosphorylation, a mutant in which both of these residues were changed to alanines (Fig. 1B) was prepared. These mutations
introduced into plasmid pXC38, which contains the entire ElA and EiB coding sequences, and plasmid DNA was used to produce mutant virus pm49OA/491A. Figure 2 shows the pattern of EBB proteins precipitated from [35S]methionineand 32Pi-labeled cells by using serum 58-N2. Mutant pm49OA/ 491A synthesized levels of 496R (Fig. 5, lane 4) which were comparable to that of wt Ad5 (lane 2), as were those of the 496R-related proteins 84R, 93R, and 156R. Thus, unlike the in3328 mutants, pm49OA/491A seemed to produce stable ElB products. Interestingly, 496R appeared to be phosphorylated
were
at a lower level withpm49OA/491A (Fig. 5, lane 3) than with wt
(lane 1). In addition, the phosphorylated form of 156R was barely detectable. These results suggested that Ser-490 and/or Ser-491 were sites of phosphorylation and that such phosphorylation may have been largely responsible for the shift in gel migration of one of the 156R species. To study this effect further, 32P--labeled 496R and 156R were isolated frompm490A/491A- and wt AdS-infected cells, and tryptic phosphopeptides were analyzed by HPLC. Figure 6A shows that wt 496R again yielded the same two major phosphopeptides, II and III. Withpm49OA/491A, both of these species were absent, but a new species, termed peptide IV, was present (Fig. 6B). An identical set of phosphopeptide patterns were also observed for 156R produced by wt (Fig. 6C) and pm49OA/491A (Fig. 6D). Analysis of the phosphoamino acid content of peptide IV from 496R indicated the presence only of phosphothreonine (Fig. 4, lane 5). While other interpretations were possible, these data suggested that Ser-490, Ser-491, and Thr-495 present in tryptic peptide T52 are phosphorylated and that conversion of the serine residues to alanine in pm49OA/491A yielded a T52 peptide phosphorylated only at Thr-495. It should be pointed out that analysis of tryptic peptides by TLC indicated that with both wt andpm49OA/491A
TABLE 1. Plaque formation by mutant and wt Ad5 Virus
d1309 (wt) in3328(+) in3328( - ) pm49OA/491A
Plaque titer (109 PFU) and ratio for cells of indicated typea Average HeLa/293 Experiment 2 Experiment 1 _______________________________ _______________________________ratio HeLa
293
HeLa/293 ratio
HeLa
293
HeLa/293 ratio
8.1 1.8 0.6 0.4
9.5 11.3 9.5 5.0
0.86 0.10 0.06 0.08
8.0 1.0 0.2 0.2
11.3 11.4 11.3 6.3
0.71 0.08 0.02 0.04
0.79 0.09 0.04 0.06
% of wt ratioaverage
100 11 5 8
a Mutant and wt virus stocks were prepared on 293 cells, and plaque formation on HeLa or 293 cells was determined in duplicate assays with several dilutions of virus.
782
J. VIROL.
TEODORO ET AL.
0 h
5 h
10 h
1 2 3 4 5 6
1 2 3 4 5 6
15 h 1 2 3 4 5
6
20 h
25 h
30 h
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
I
FIG. 7. SDS-PAGE analysis of late viral protein synthesis and host shutoff. Mock-infected KB cells or KB cells infected with a series of E1B mutants were labeled at 5-h intervals for 1 h with [35S]methionine, and equal amounts of cell extract were analyzed by SDS-PAGE. The times of
labeling have been indicated. Lanes: 1, mock-infected cells; 2, wt-dl309; 3, pm2019/2250; 4, pm1716/2072; 491A(19K-).
additional minor labeled species were observed (data not shown), indicating that other minor phosphorylation sites probably exist in 496R. Role of phosphorylation of Ser-490/491 in viral replication. Because 496R is essential for viral replication, two types of experiments to study the importance of phosphorylation at serines 490 and 491 were carried out. The plaquing efficiency of pm49OA/491A was studied by using HeLa cells and 293 cells which express ElA and E1B proteins constitutively, and the results compared with those obtained by using wt AdS and mutants in3328(+) and in3328(-). Table 1 shows that with wt AdS, plaque titers on 293 and HeLa cells were similar. With in3328(+), the efficiency on HeLa cells was only about 10% of that observed on 293 cells, indicating that viral replication was affected by synthesis of low levels of the altered 496R product. With in3328(-), plaquing efficiency on HeLa cells was reduced by more than 10-fold. This effect was not surprising, as this mutant produced a very low level of a truncated 496R product. Interestingly, pm49OA/491A also showed a similar decrease in plaquing efficiency on HeLa relative to that on 293 cells. These results suggested that phosphorylation at serines 490 and 491 may affect one or more of the lytic functions of 496R.
5,
pm49OA/491A; 6, pm49OA/
496R functions in the regulation of late viral gene expression, in the accumulation of late viral mRNAs (4, 8, 11, 25, 29, 46, 51), and in the shutoff of host cell metabolism (3, 4, 25, 67). To examine these functions, KB cells were infected with wt or various ElB mutants including pm49OA/491A; pm2019/2250, which produces ElB-176R but no 496R (29); pm1716/2072, which produces 496R but not 176R (40); and pm49OA/ 491A(19K-), which is the 490A/491A mutant introduced into a pm1716/2072 background. At 5-h intervals, mock-infected and infected cells were labeled for 1 h with [35S]methionine and then whole cell lysates were analyzed by SDS-PAGE. Figure 7 shows that by 10 hpi late viral protein synthesis was detectable in infected cultures and the level of synthesis continued to increase up to 30 hpi. The only exception was pm2019/2250 (Fig. 7, lanes 3), which lacks 496R and which showed lower rates of late viral protein synthesis at all times. Similarly, synthesis of host cell proteins, evident in lanes containing extracts from mock-infected cells (Fig. 7, lanes 1), decreased after 20 hpi in all infected cultures except those infected withpm2019/2250, which showed no apparent shutoff. pm1716/2072 (Fig. 7, lanes 4) also may have been slightly deficient in this function. Nevertheless, both pm490A/491A (Fig. 7, lanes 5) and pm49OA/491A(19K-) (lanes 6) were
TABLE 2. Transformation of BRK cells by Ad5 ElA and wild-type or mutant E1B' Average no. of foci per plate (no. of plates) and % of wt average for indicated expt
Plasmid 1
2
3
0 (8), 0 No DNA 0 (3), 0 44 (8), 100 69 (3), 100 291 (3), 100 42 (4), 100 pXC38 (wt) 42 (3), 60 7 (4), 18 pXC-3328(+) 4 (4), 10 pXC-3328(-) 11 (3), 16 151 (3), 52 pXC490A/491A 15 (8), 34 p2019/2250 8 (4), 18 p1716/2072 13 (8), 30
p490A/491A p3328(+)
p3328(-)
5
4
0 (3), 171 (3), 152 (3), 89 (3), 120 (3), 23 1 1 2
0 100 89 52 70
(3), 14 (3), 1 (3), 1 (3), 1
Average %
6
7
8
ofwtaverage
0 (4), 0 73 (4), 100
0 (16), 0 24 (16), 100
0 (14), 0 24 (36), 100
39 (4), 54
11 (16), 46
10 (36), 41
0 100 56 33 53 34 24 1
25 (4), 34 0 (4), 0
1 (18), 3
1 1
a Primary BRK cells were transfected with 5 ,ug of plasmid DNA as described in Materials and Methods. The average number of foci per dish and the percent transforming efficiency relative to wt have been presented.
VOL. 68, 1994
REGULATION OF Ad5 ElB PROTEIN BY PHOSPHORYLATION
indistinguishable from wt (lanes 2), indicating that these functions did not appear to be affected by removal of the phosphorylation sites at Ser-490 and Ser-491. Role of phosphorylation of Ser-490/491 in 496R-mediated cell transfonnation. Studies to assess the effects of the Ser490/491 mutations on the ability of EBB products to participate with ElA in cell transformation were carried out. Primary baby rat kidney cells were transfected with pXC38 plasmid DNA, which expresses the entire wt Ad5 ElA and ElB regions, or with pXC38 containing various ElB mutations. Table 2 shows the results of a series of experiments in which the transforming efficiencies of mutants have been compared to that of wt. As shown previously (40), p2019/2250 and p1716/2072, which contain defects that block synthesis of 496R and 176R, respectively, yielded transformants at about 25 to 35% the efficiency of wt pXC38. The efficiencies of transformation by pXC3328(+), which contains an insert near the carboxy terminus, and pXC-3328(-), which produces a truncated 496R product, were reduced. Interestingly, pXC49OA/491A also transformed at a reduced efficiency, at a level comparable to that of p2019/2250, which produces no 496R at all. Because 176R and 496R can each independently cooperate with ElA to transform BRK cells (6, 8, 40, 63, 67, 72), it was necessary to introduce mutations into a p1716/2072 background in order to assess the independent transforming ability of 496R mutants. Table 2 shows that both p3328(+) and p3328(-) formed transformants very poorly, suggesting that the carboxy-terminal region of 496R is essential. Furthermore, p49OA/491A was also almost completely defective. To ensure that this reduction in transforming efficiency was not due to diminished stability of the 496R-490A/491A mutant protein, pulse-chase experiments in which KB cells infected with either wt orpm49OA/491A were labeled for 1 h with [35S]methionine and then incubated for increasing numbers of 5-h periods in nonradioactive medium were carried out. The results obtained indicated a half-life of about 8 h for mutant 496R and about 10 h for wt, indicating that no great difference in stability existed (data not shown). The half-lives for 156R and 84R were shown to be about 4 h for both mutant and wt (data not shown). Thus, it appeared likely that phosphorylation at Ser-490/491 is essential for the transforming activity of 496R. Interactions with p53. Transforming activity of 496R appears to derive at least in part from interactions with the p53 tumor suppressor (27, 53). p53 functions as a transcriptional regulator, and complex formation with 496R has been shown to repress transcriptional activation by a GAL4-p53 fusion product (66). To examine the ability of the mutant Ala-490/491 protein to interact with p53, extracts from infected cells were incubated with 35S-labeled p53 synthesized in a coupled in vitro transcription/translation reaction and then 496R was purified by immunoprecipitation and analyzed by SDS-PAGE. Figure 8A shows a typical analysis in which equal amounts of extract were used from mock-infected cells and those infected with wt, pm2019/2250, pm1716/2072, pm49OA/491A, or pm49OA/491A(19K-). Little p53 was detected with mockinfected cells or with cells infected by pm2019/2250, which produces no 496R protein; however, significant levels were detected with all other viruses. Figure 8B shows the results of a quantitative binding assay in which increasing amounts of extract from wt- and pm49OA/491A-infected cells were used. The amount of 35S-labeled p53 was quantified and compared with the levels of 496R detected by Western blotting (immunoblotting). Clearly, the binding curves were very similar, suggesting that phosphorylation at Ser-490/491 did not appear to greatly affect complex formation with p53. To compare the abilities of wt and Ala-490-491 496R to
783
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A.
:~~~~~~~~~~~~~9,
P_t;
B. I--
(n
Z
100'
0
c
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a)
0
75,
a, O
ai-t0 0..
50'
800 1200 400 AMOUNT OF 496R PROTEIN (Phosphorimager units) FIG. 8. Complex formation between 496R and p53. [35S]methionine-labeled p53 synthesized in vitro in a coupled transcription/ translation reaction, as described in Materials and Methods, was incubated with extracts from mock- or AdS-infected cells. 496R complexes were purified by immunoprecipitation with 58-N2 serum and analyzed by SDS-PAGE. p53 binding was quantified from dried gels by Phosphorimager. The amount of 496R was determined by Western blotting analysis using 2A6 antibody and 125I-conjugated goat anti-mouse antibody, which was quantified by Phosphorimager. (A) SDS-PAGE analysis. The lane at the left shows the gel profile of an aliquot of the in vitro translation reaction, and the position of migration of p53 has been noted. Viruses used for infection are as indicated. (B) p53-binding curves. Results from a representative experiment showing the amount of 35S-labeled p53 binding as a function of 496R present in increasing amounts of cell extract have been presented.
repress p53 transactivation activity, a similar study in which the ability of a GAL4-p53 fusion protein to activate expression of a reporter construct containing the CAT gene was assessed was conducted by transient transfection of NIH 3T3 cells. Figure 9A shows the constructs used in these experiments. CAT
J. VIROL.
TEODORO ET AL.
784
A.
E1B
CAT
GAL4 sits TATA
G5E1 b-CAT
13 1e2 1 AS ; 11I1t 1213141b I
(reporter)
= L..
..MMML-
l
I
-0-
PSV40
mGAL4D-p53 (1-393)
0-
PSV40
with the CAT reporter construct alone (Fig. 9B, lanes 1 and 2). Expression of wt 496R (Fig. 9B, lane 4) reduced the level of CAT activity detected in controls (lanes 6 and 3) by almost 90%. With the Ala-490A1491A mutant, such inhibition was only about 50% (Fig. 9B, lane 5). Little effect of 496R on CAT activity regulated by the GAL4-VP16 fusion product (Fig. 9B, lanes 7 to 9) was detected. These results indicated that the Ala-490A/491A mutations rendered 496R partially defective for p53 repression.
iG---71
DISCUSSION Previous studies suggested that although minor phosphorylation sites might exist within the 79 amino-terminal residues MOMLV/MSV of the AdS 496R E1B protein, one or more major serine and 4961 r pLXSN-55K threonine sites must exist near the carboxy terminus (59). 1 ow.Present experiments focused primarily on serine residues 490 B. and 491, which exist within tryptic peptide T52 (Fig. 1). Mutant alapm490A/491A, in which these serines were converted tophos0 87 0 100 13 48 100 129 1I 33 nines, yielded a 496R product which showed reduced (±0) (±0) (±4) (*7) (±6) (±37) (± 39) C.i g 0vF.gj 0f'.~ :", phorylation and lacked both prominent tryptic phosphopepCAX f;X s< 0, m t -tl S''Id^0 tides normally detected with wt 496R but contained a novel phosphopeptide. It was possible that this peptide contained a site not normally phosphorylated in 496R; however, it was more likely that this novel species represented peptide T52, which is phosphorylated only at Thr-495. T52 from wt 496R contains both phosphoserine and phosphothreonine, whereas this novel phosphopeptide contained only the latter. The fact ~ that it eluted later from the HPLC column would be predicted Vifor a peptide which has lost two negatively-charged phosphoamino acids. It was not clear from these studies whether both Ser-490 and Ser-491 were phosphorylated, although it was 1-i likely. Both represent possible casein kinase II substrate sites z (43), and, in a very similar sequence in the E7 protein of -0 z z zv0 i human papillomavirus, both adjacent serine residues were 5 ,§
