mapping of a phosphorylation site in the 176r (19 kda) early region 1b protein of human adenovirus...

VlROLOGY168, 119-127(1989)

Mapping of a Phosphorylation Site in the 176R (19 kDa) Early Region 1 B Protein of Human Adenovirus Type 5

C. JANE McGLADE, MICHEL L. TREMBLAY,’ AND PHILIP E. BRANTON”

Molecular Virology and Immunology Program, Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 325

Received August 3, 1988; accepted September 27, 1988

The 176-residue (176R) early region 1 I3 (El 6) protein of human adenovirus type 5 (Ad5) was shown to be phosphory-

lated at serine in lytically infected KB cells at a level estimated to be about one phosphate group per 28 176R molecules. Through the analysis of peptides generated by cleavage with cyanogen bromide and Sraphylococcus aureus V-8 prote-

ase the phosphorylation site was mapped to Ser-164. Using site-directed mutagenesis, a mutant was produced in

which the codon for Ser-164 was changed to that of asparagine while leaving the coding sequence for the overlapping 496R protein unchanged. This virus, which replicated well on human KB cells, produced normal levels of 176R, but in

an unphosphorylated form. The mutant transformed baby rat kidney cells in cooperation with El A at an efficiency about one-half that obtained with wt El 6. These data therefore gave little indication that phosphorylation is essential for the

function of 176R. 0 1999 Academic Press. Inc.

INTRODUCTION

The products of both early region 1 B (El B) and 1 A (El A) of human adenoviruses are required for complete oncogenic transformation (cf. Branton et a/., 1985). El B of adenovirus type 2 (Ad2) produces two major early mRNAs which yield products of 175, 495, and 82 residues (175R, 495R, and 82R), and minor mRNAs that encode proteins of 92R and 155R which, like 82R, share amino-terminal sequences with 495R (Perri- caudet eta/., 1979; Virtanen and Pettersson, 1983; An- derson et a/., 1984; Lewis and Anderson, 1987). The corresponding El B products of the adenovirus type 5 (Ad5) serotype (176R, 496R, 84R, 92R, and 156R) are predicted to be almost identical to those of Ad2 (Perri- caudet et a/., 1979; Bos et a/., 198 1). It is now apparent that both 176R and the amino-terminal portion of 496R are important for oncogenesis (Chinnadurai, 1983; Rowe et al., 1984b; Babiss et al., 1984; Subramanian et al., 1984b; Takemori et al., 1984; Barker and Berk, 1987). Mutants of Ad5 or adenovirus type 12 which produce abnormal 176R proteins are typically transformation defective and produce a rapidly cytocidal (cyt) infection characterized by the rapid degradation (deg) of viral and cellular DNA (Mak and Mak, 1983; Subra- manian et a/., 1984a; White et al., 1984b). The 176R protein functions in the regulation of early viral gene expression (White et a/., 1986; Jochemsen et a/., 1987; Yoshida et a/., 1987) and it is possible that effects of

’ Present address: National Institutes of Health, Room 338, Build- ing 6, Bethesda, MD 20892.

’ To whom requests for reprints should be addressed.

176R on ElA transcription are responsible, either directly or indirectly, for the cytldeg phenotype (White and Stillman, 1987).

The 176R protein is found largely in association with membranes (Persson et al., 1982; Rowe et al., 1983), primarily in the nuclear envelope and endoplasmic reticulum (White et al., 1984a; McGlade ef al., 1987), although small amounts may also be present in the plasma membrane (FBhring et al., 1983; White et al., 1984a). It is likely that membrane association is achieved, at least in part, by the presence of fatty acids covalently linked to the 176R molecule (Grand et al., 1985; McGlade et al., 1987). Analysis of 176R on 15% polyacrylamide gels revealed two closely migrating 176R species, termed 19K and 18.5K, which yielded identical V-8 protease digestion patterns (Rowe et al., 1983; McGlade et al., 1987). Both of these species appeared to be full length, acylated 176R molecules (Mc- Glade et a/., 1987), and thus some other form of post- translational modification must be responsible for the generation of these two electrophoretically separable forms.

In the present studies we report that 176R isolated from lytically infected human KB cells is phosphorylated at low levels at Ser-164. Both 19K and 18.5K were found to be phosphorylated at relatively equal levels, suggesting that phosphorylation is not the basis for the multiple 176R species.

MATERIALS AND METHODS

Cells and viruses

Human KB cells were cultured on loo-mm dishes (Corning) using a-minimum essential medium supple-

119 0042-6822/89 $3.00 CopyrIght 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

120 MCGLADE, TREMBLAY. AND BRANTON

mented with 10% fetal calf serum and were infected with wild-type Ad5 (or the mutant pm2204, see below) at a multiplicity of infection of 35 plaque-forming units per cell, as described previously (Rowe et a/., 1983).

Antisera

The 176R protein was immunoprecipitated from extracts of infected cells using a rabbit anti-peptide serum, 19-Cl, which is specific for the carboxy terminus of 176R (McGlade et a/., 1987). E 1 A proteins were immunoprecipitated using M73 monoclonal antibody (Harlow et a/., 1985). El B-496R was precipitated using a rat monoclonal antibody (anti-Ad5 55 kDa Onco- gene Science). In some experiments the hamster anti- tumor serum 148 (Rowe et al., 198413) was used to pre- cipitate E 1 A and E 1 B products.

Radioactive labeling

Ad5- or mock-infected cells were incubated from 8 to 12 hr postinfection (p.i.) with 100 &i of [35S]methionine (Amersham Corp.; sp act 1300 Ci/mmol) in 2.5 ml of methionine-free medium. Labeling with [32P]orthophosphate (New England Nuclear; carrier free) was during the same time period at 2 to 5 mCi per plate in 2.5 ml of phosphate-free medium. Leucine labeling was carried out under similar conditions using 100 &i of [3H]leucine (New England Nuclear; sp act 140 Ci/ mmol) in leucine-free medium.

Cell extracts and immunoprecipitation

Cell extracts were prepared in RIPA buffer [0.05 n/r Tris, pH 7.2, containing 150 mfl/l NaCI, 0.1% SDS, 0.1% (v/v) sodium deoxycholate, O.lo!o (v/v) Triton X- 100, and 100 KIU of aprotinin/ml] and immunoprecipitation was carried out using 25 ~1 of 19-Cl serum, in either the presence or absence of excess 19-C peptide, using 10~1 of M73 serum, or using 25 ~1 of 14B serum, as previously described (Yee et al., 1983; Mc- Glade et al., 1987).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Samples were analyzed on 15% polyacrylamide gels and labeled proteins were detected in dried, fluoro- graphed gels by autoradiography, as described previously (Rowe et a/., 1983; Branton and Rowe, 1985).

Phosphoamino acid determination

32P-Labeled 176R was eluted from polyacrylamide gels by shaking the appropriate gel fragment overnight at room temperature in 0.05 Mammonium bicarbonate containing 0.1% SDS and 0.5% (v/v) 2-mercaptoetha-

nol. The solution was combined with 50 pg of human globin as carrier and the mixture made 20% trichlor- acetic acid, left on ice for 3 hr, and then centrifuged at 12,000 g for 30 min. The pellet containing the precipitated protein was redissolved in 6 Iv HCI and heated for 2 hr at 110” in sealed tubes. The HCI was removed in vacua, and hydrolysates were analyzed along with purified phosphoamino acid markers by two-dimensional electrophoresis on cellulose thin-layer plates (20 cm X 20 cm X 0.1 mm; Polygram CEL 300) as described previously (Branton et a/., 198 1).

Cleavage of 176R with cyanogen bromide

Areas of polyacrylamide gels containing labeled 176R were excised and the gel pieces were treated for 4 hr at 23” with cyanogen bromide at 30 mg/ml in 0.1 N HCI containing 5% formic acid and 0.4% (v/v) 2-mercaptoethanol, as described by Pepinsky (1983). To re- move excess CNBr and to neutralize the samples, the gel pieces were incubated twice for 5 min with water, twice with 0.25 M Tris (pH 6.8) and then they were equilibrated in gel sample buffer [lo0 mMTris, pH 6.8, containing 2% SDS, 2% 2-mercaptoethanol (v/v), and 10% glycerol (v/v)]. The gel slices were loaded onto a 15% polyacrylamide gel for electrophoresis, as described above.

Staphylococcus aureus V-8 protease digestion

Labeled 176R protein was recovered from polyacry- amide gels as described above and then resuspended in 0.1 M ammonium bicarbonate to a final concentra- tion of 10 mg of 176R and carrier protein per milliliter. Staphylococcus aureus V-8 protease (1 yg) was added and the mixture was allowed to digest for 2 hr at 37”. The reaction was stopped by boiling in the presence of 2% SDS and the mixture was combined with either an equal volume of double-strength sample buffer for electrophoresis or diluted in RIPA buffer for immunoprecipitation with 1 g-Cl serum.

Oligonucleotide-directed mutagenesis

The mutantpm2204 was constructed by oligonucleotide mutagenesis using the method of Zoller and Smith (1984) as modified by Kunkel (1985). As shown in Fig. 1 A, the template used for mutagenesis was the M 13 phage mp18 which contained the Ad5 sequence from plasmid pXC38 between the Kpnl and HindIll sites (bases 2048 to 2804). The phosphorylated synthetic oligonucleotide containing the point mutation (see Fig. 1 B) was used as a primer for the synthesis of the com- plementary strand to yield double-stranded replicative form DNA. The mutation changed base 2204 from G to A, thus altering the codon for residue 164 in 176R from

PHOSPHORYIATION SITE OF ElB PROTEIN 121

Ban II

2191 2204 2220

wt . I

. . . . CGGCAGGAGCA-ATGGAACCCGAGA...... 176R ArgGlnGluGln~ProTrpAsnProArg 496R AlaGlyAlaGluProMetGluProGlu

em2204 . . . . CGGCAGGAGCAGAACCCATGGAACCCGAGA...... 176R ArgGlnGluGln~ProTrpAsnProArg 496R AlaGlyAlaGluProMetGluProGlu

C AMINO ACID SEQUENCE OF AD5 ElB-176R

1 5 10 15 MET:GLU-ALA-TRP-GLU CYS-LEU-GLU-ASP-PHE SER-ALA-VAL-ARG-ASN

t 20 25

LEU-LEU-GLU-GLN-SER SER-ASN-SER-THR-SER TRP-PHE-TRP-ARG-PHE

35 40 45 LEU-TRP-GLY-SER-SER GLN-ALA-LYS-LEU-VAL CYS-ARG-ILE-LYS-GLU

t 50 55 60

ASP-TYR-LYS-TRP-GLU PHE-GLU-GLU-LEU-LEU LYS-SER-CYS-GLY-GLU t t t t

65 JO 75 LEU-PHE-ASP-SER-LEU ASN-LEU-GLY-HIS-GLN ALA-LEU-PHE-GLN-GLU

t 80 85 90

LYS-VAL-ILE-LYS-THR LEU-ASP-PHE-SER-THR PRO-GLY-ARG-ALA-ALA

95 100 105 ALA-ALA-VAL-ALA-PHE LEU-SER-PHE-ILE-LYS ASP-LYS-TRP-SER-GLU

t 110 115 120

GLU-THR-HIS-LEU-SER GLY-GLY-TYR-LEU-LEU ASP-PHE-LEU-ALA-MET t

125 130 135 4 HIS-LEU-TRP-ARG-ALA VAL-VAL-ARG-HIS-LYS ASN-ARG-LEU-LEU-LEU

140 145 150 LEU-SER-SER-VAL-ARG PRO-ALA-ILE-ILE-PRO THR-GLU-GLU;GLN-GLN

t 155 160 165

GLN-GLN-GLN-GLU-GLU ALA-ARG-ARG-ARG-ARG GLN-GLU-GLN-ER-PRO t t t P

170 175 176 TRP-ASN-PRO-ARG-ALA GLY-LEU-ASP-PRO-ARG GLU

FIG. 1. Generation of mutant pm2204 and sequence of 176R. (A) Plasmids pXC38 and pXC2204 and phage Ml 3 mp18 have been illustrated,

including relevent nucleotides and restriction enzyme sites (K, Kpnl; B, Banll; H, HindIll). (B) DNA sequence of El B region containing nucleotide 2204 for wt Ad5 and the mutant pm2204. The amino acid sequences of the 176R and 496R proteins have been illustrated as has the position of the Banll restriction site in the wr DNA sequence. (C) Amino acid sequence of the El B-l 76R protein. The amino acid sequence of 176R (Perricaudet et al., 1979; Bos et a/., 1981) has been presented, including the positions of CNBr (t) and S. aureus V-8 protease (t) cleavage sites

and the phosphorylation site at Ser-164, as determined from the data presented in Figs. 2-4.

122 MCGLADE. TREMBLAY. AND BRANTON

serine to asparagine and removing a Banll restriction enzyme site. As shown in Fig. 1 B, this change was the only possibility so as not to alter the amino acid sequence of the 496R protein which is produced from a different reading frame in an overlapping sequence. A mutant phage was identified by sequencing using the dideoxynucleotide method with suitable oligonucleo- tides as primers, and the Kpnl to HindIll fragment was inserted into pXC38 to yield the plasmid pXC2204 (see Fig. 1A). The presence of the mutant sequence was confirmed by digesting pXC2204 with Banll. The mutation was rescued into Ad5 virions by cotransfection of 293 cells (Graham et al., 1977) with pXC2204 and pJM 17 DNA according to the method of McGrory et al, (1988) to yield the mutant pm2204. The presence of the mutation in virion DNA was confirmed by Banll digestion and Southern blot analysis of fragments using M 13 mpl8 as a probe. Digestion of pm2204 DNA with a number of restriction enzymes indicated that no other abnormalities were evident.

Transformation of baby rat kidney cells

Cells prepared from kidneys removed from 6-day-old Wistar rats were transfected with 5 pg of plasmid DNA using a modified calcium phosphate-DNA coprecipita- tion technique (Graham and van der Eb, 1973; Weeks and Jones, 1983). DNA concentrations were determined by absorbance at 260 nm, by calorimetric assays using diphenylamine, and by quantitative analysis of restriction enzyme cleaved plasmid preparations on polyacrylamide gels. Following 2 to 3 weeks in culture, transformed foci were visualized by staining with Giemsa.

RESULTS

Detection of phosphorylated forms of the El B-i 76R protein

Previous studies had indicated that the Ad5 ElA products and the 496R El B protein, but not the 176R ElB polypeptide, are phosphorylated (Malette et al., 1983; Yee et a/., 1983; Rowe et a/., 1984b). To investi- gate phosphorylation more thoroughly, Ad5- or mock- infected cells were labeled with [35S]methionine or with a high level of [32P]orthophosphate, and 176R was immunoprecipitated using an anti-peptide antiserum, 19- Cl, which is specific for the carboxy terminus of 176R (McGlade el a/., 1987). 32P-Labeled E 1 A products and 496R were precipitated using a mixture of monoclonal antibodies specific for these proteins. Precipitates were examined by SDS-PAGE and incorporation of 32P into 176R was assessed using periods of time for autoradiography that were considerably longer than those

WT pm2204 ________-_------ _-m-_-e e--w-

=s 32 P 35s

32 P

X C D E F G I J K

FIG. 2. Phosphorylation of 176R in cells infected by wt Ad5 and mutant pm2204. Cells infected with either wt Ad5 or the mutant

pm2204, and mock-infected cells, were labeled with [%]methionine or [32P]orthophosphate and cell extracts were immunoprecipitated

with 19-Cl, in some cases in the presence of the 19-C peptide, or

with a combination of M73 anti-ElA and anti-496R monoclonal antibodies. Lanes A-F, wtAd5-infected cells; lanes A-B, 35S-labeled

samples with 1 g-Cl serum in the absence (A) and presence (B) of 19-C peptide; lanes C-F, 3ZP-labeled samples with 1 g-Cl serum in

the absence (C) and presence (D) of 19-C peptide, mock-infected cells with 1 g-Cl serum (E), and infected cells with monoclonal anti-

bodies to EIA products and 496R. Lanes G-K, pm2204-infected

cells; lanes G-H, %-labeled samples with 19-Cl serum in the absence (G) and presence (H) of 19-C peptide; lanes I-K, 32P-labeled

samples with 19.Cl serum in the absence (I) or presence (J) of 19-C serum and with antibodies to ElA products and 496R. The positions of ‘%-labeled molecular weight markers are shown at the left of the

figure.

used for our studies on El A products and 496R. Figure 2 shows that 19-Cl serum precipitated 35S-labeled 19 and 18.5K 176R species (lane A). Both forms were also detected in 32P-labeled extracts from infected (lane C) but not mock-infected cells (lane E), and precipitation was blocked by addition of 19-C peptide (lanes D and B). Although it is not apparent from the exposures used for Fig. 2, the level of 32P incorporation into 176R was much lower than that obtained with ElA products or 496R (lane F). These data indicated that both 176R species were phosphorylated at low levels.

Identification of the phosphoamino acid in 176R

The phosphoamino acid present in 176R was identified by acid hydrolysis of gel-purified 32P-labeled 176R

PHOSPHORYLATION SITE OF ElB PROTEIN 123

TABLE 1

%Labeled 496R (cpm) 3256

%-Labeled 176R (cpm) 1089

Number of Met residues in 496R 15

Number of Met residues in 176R 2

Molar ratio 176R/496R 2.5

Ratio 3ZP-labeled 176R/3ZP-labeled 496R 0.0335

Average number of phosphatesI496R molecule 2.7

Estimated number of phosphates/l 76R molecule 0.036

B AdSinfected cells were labeled with either [3ZP]orthophosphate

or [%]methionine and labeled 176R and 496R proteins were precipitated using 148 hamster anti-tumor serum. Regions in the gel con-

taining labeled 176R and 496R were excised and assayed for radioactivity as described previously (Branton and Rowe, 1985). The level

of phosphorylation of 176R was estimated from the ratios of 32P/35S, knowing the number of methionine residues in each molecule (Perri-

caudet et a/., 1979; Bos et al., 1981) and the average number of

phosphates per 496R molecule, estimated previously to be 2.7 (Ma- lette et al., 1983).

followed by two-dimensional electrophoresis on thin- layer plates. Labeled material was found in three spots, one of which comigrated with phosphoserine marker, a second with free phosphate, and a third in the position of incompletely hydrolyzed peptides (data not shown). Thus 176R is phosphorylated at one or more serine residues.

Estimation of the level of 176R phosphorylation

It was possible to estimate the level of phosphorylation of 176R because, in a previous study using known amounts of [35S]methionine and [32P]orthophosphate, we had estimated that each 496R molecule contains on average about 2.7 phosphate groups (Malette et a/., 1983). Ad5infected cells were labeled with either radioactive methionine or or-thophosphate and the El B products were immunoprecipitated with 14B hamster anti-tumor serum which recognizes both 176R and 496R. Both of these products have long half-lives (Branton and Rowe, 1985) and labeling was carried out at a time when the rate of synthesis was maximal (Rowe et al., 1984a). The precipitates were subjected to SDS-PAGE and regions in the gel containing 176R or 496R were excised and the gel fragments were assessed for radioactivity. As shown in Table 1, knowing the number of methionine residues in each of these molecules, the fact that 496R contains on average 2.7 phosphate residues per molecule, and the ratios of 35S/ 32P for each of these El B species, it was possible to estimate that 176R proteins are phosphorylated at a level of 3.6%, or one phosphate residue for about 28 176R molecules. Thus it appeared that only a subfrac-

tion of 176R molecules is phosphorylated, in all likeli- hood at a single site.

Mapping of the phosphorylation site

To begin to map the site of phosphorylation, 176R labeled with [32P]orthophosphate, [35S]methionine, or [3H]leucine was gel purified and then reanalyzed by SDS-PAGE following treatment with CNBr which cleaves polypeptides at methionine residues. As shown in Fig. lC, 176R contains two methionine residues, at positions 1 and 120, and thus CNBr treatment will yield peptides of a single amino acid (the amino- terminal methionine residue), one of 1 19 amino acids from residues 2 to 120, and one of 56 amino acids (56R) from residues 121 to 176. Because CNBr re- moves the sulfur atom during cleavage, no radioactivity will be detectable in any of the peptides generated from 35S-labeled samples. However, as cleavage with CNBr is not completely efficient, one might expect to see 35S- labeled peptide comprising residues 1 to 120 (120R), in addition to undigested full-length molecules. Figure 3 (lane B) shows that, as predicted, CNBr treatment of the 35S-labeled material yielded labeled 120R peptide in addition to undigested 176R molecules. With the

CNBr

Met 3* P Leu --

A B C D

176 R

56 R

FIG. 3. Cleavage pattern of 176R by CNBr. Ad5-infected cells were labeled with [35S]methionine, [32P]orthophosphate, or [3H]leucine

from 8 to 12 hr p.i., cell extracts were immunoprecipitated with 19- Cl serum, and the precipitates were analyzed by SDS-PAGE. Gel pieces containing labeled 176R were excised and either treated with CNBr or left untreated prior to reanalysis on SDS gels, as described

under Materials and Methods. (A) Untreated 35S-labeled 176R. (B) CNBr-treated 35S-labeled 176R. (C) CNBr-treated 3*P-labeled 176R.

(D) CNBr-treated 3H-labeled 176R.

124 MCGLADE, TREMBLAY, AND BRANTON

[3H]leucine-labeled material (lane D), both 120R and 56R were detected, in addition to undigested 176R. With 32P-labeled material (lane C), label was found only in the undigested 176R and in the 56R peptide, suggesting that phosphorylation occurs exclusively in the carboxy-terminal 56 residues. Only three serine residues are present in this region, at positions 137, 138, and 164 (see Fig. 1 C).

Ser-164 is present in the carboxy-terminal S. aureus V-8 peptide which lacks methionine but contains leu- tine and comprises amino acids 163 to 176 (see Fig. 1 C) and which should be precipitable by the 19-Cl anti- peptide serum. Infected cells were labeled with either [35S]methionine or [32P]or-thophosphate and 176R was gel purified and digested with S. aureus V-8 protease. Some of this material was analyzed directly by SDS- PAGE and the remainder was immunoprecipitated with 19-Cl serum in the presence or absence of 15 pg of 19-C peptide. Figure 4 (lane B) shows that with 176R examined directly, 35S-labeled samples yielded several peptides or partial proteolytic products. With the 32P- labeled sample (lane C) only a single peptide was observed which did not comigrate with any of the 35S-labeled species. The 1 g-C1 serum precipitated this 32P- labeled peptide (lane F) but no 35S-labeled species (lane D), and this peptide was not precipitated when 19-C peptide was added to the immunoprecipitation mixture (lane G). In other studies a species labeled with [3H]leu- tine was found to be precipitable with 19Cl serum and to comigrate with the 32P-labeled peptide (data not shown). These data indicated that the phosphorylation site is present in the carboxy-terminal V-8 peptide, and thus at Ser-164. They also suggested that this must be the only site of phosphotylation in 176R.

Confirmation of Ser-164 phosphorylation site by site-directed mutagenesis

Using site-directed mutagenesis, a mutant pm2204 was prepared that contained a single-point mutation at base 2204, thus changing residue 164 in 176R from serine to asparagine without affecting the coding sequence for the El B 496R product (see Fig. 1 and Mate- rials and Methods). To confirm that Ser-164 was the single site of phosphorylation on 176R, cells infected with either pm2204 or WT Ad5 were labeled with [35S]methionine or [32P]orthophosphate and 176R was immunoprecipitated using 19-Cl serum. To determine if ElA proteins and the El B-496R product were produced in normal amounts by pm2204, extracts were reprecipitated with a mixture of monoclonal antibodies against these products. Figure 2 shows that while both wt and pm2204 produced 35S-labeled 19K and 18.5K 176R proteins (lanes A and G), the 32P-labeled 176R

0 15 3zp

D E F-

FIG. 4. Cleavage pattern of 176R with S. aureus V-8 protease. The

176R protein labeled with [3ZP]orthophosphate or [35S]methionine was isolated from SDS-polyacrylamide gels and treated with S.

aureus V-8 protease, as described under Materials and Methods. The resulting peptides either were analyzed directly on a 15% poly- actylamide gel (lanes B and C) or they were first immunoprecipitated

with 1 g-Cl serum (lanes D-G) in the absence (lanes D and F) or pres-

ence (lanes E and G) of 15 pg of 19-C peptide, as described under Materials and Methods. Lane A “C-labeled molecular weight mark-

ers (New England Nuclear).

species were detected only in w&infected cells (lanes C and I). Mutant pm2204 produced levels of El A products and 496R that were comparable to those of wt (lanes F and K). These data showed that pm2204 produces 176R, but in an unphosphorylated form, and thus that Ser-164 must be the single phosphorylation site on this molecule. In addition, the presence of the 19K and 18.5K forms of 176R with pm2204 indicated that phosphorylation plays no role in the generation of these two species.

Role of phosphotylation in 176R

Failure to phosphorylate Ser-164 of 176R was found to have little effect on viral replication as mutant


TABLE 2

TRANSFORMATION OF PRIMARY BABY RAT KIDNEY CELLS’

Number of foci per dish (total number of foci)

Total number of foci

Plasmid Experiment 1 Experiment 2 (total dishes)

pXC38 36,40, 38,40 29, 32, 25, 29,

(154) 40,35, 24, 27 395 (12)

(241) pXC2204 18,22. 17, 13 14, 17, 16, 15,

(70) 16, 18, 18, 14 198 (12)

(128) Noneb 0, 0 (0) 0, 0 (0) 0 (4)

a Cells were transfected with equal concentrations of plasmid DNA

as described under Materials and Methods. The results of two separate experiments using independent plasmid DNA preparations have

been presented and include the number of transformed cell foci per dish and the total number of foci observed in all plates.

b Cells were transfected with 5 pg of carrier herring sperm DNA

only.

pm2204 replicated in KB cells about as efficiently as wt Ad5 and the plaques that formed had no unusual morphology (data not shown). To study the effect on transformation, baby rat kidney ceils were transfected with the plasmids pXC38 (wt) or pXC2204 (Asn-164) which contain both the ElA and El B region. Table 2 shows that pXC2204 transformed with only about one- half the efficiency of WT. This type of assay is prone to some variability, and thus while these results suggested that phosphorylation at Ser-164 may have some effect on the transforming function of 176R, it does not appear to be essential.

DISCUSSION

We have now shown that the El B-l 76R protein is post-translationally modified in two ways. It is acylated via palmitate and myristate residues (McGlade et a/., 1987) and it is likely that this modification plays some role in its association with cellular membranes. We report here a second modification, that of phosphorylation at Ser-164. Neither acylation nor phosphorylation appears to explain the generation of the 19K and 18.5K 176R species seen in 15% polyacrylamide gels. Both species are immunoprecipitated by anti-peptide sera generated against the amino and carboxy termini (Mc- Glade et al., 1987) and thus, unless a previously unde- tected El B mRNA exists that contains a short in-frame splice in the interior of the 176R coding sequence, they are presumably both full-length 176R molecules.

Phosphorylation at Ser-164 was estimated to occur only at a level of about one per 28 176R molecules.

These results indicated that only a subfraction of molecules, perhaps those present in a specific intracellular location, are phosphorylated. Large quantities of 176R are found in the nuclear membrane in association with nuclear lamina, and in the endoplasmic reticulum (White et al., 1987; McGlade et al., 1987). In addition, small quantities have been detected in the plasma membrane at the external cell surface (FBhring et a/., 1983; White eta/., 1984a). It is possible that phosphorylation helps to localize 176R molecules to one of these cellular compartments, or that the protein kinase that recognizes the Ser-164 site is itself compartmental- ized. A second possibility is that 176R is actually phosphorylated at much higher levels but that phosphate is rapidly removed by phosphoprotein phosphatases. This latter idea seems less likely as the addition of phosphatase inhibitors to infected cells in viva and to solutions used to prepare cell extracts had little effect on the level of phosphate detected in 176R (C. J. Mc- Glade and P. E. Branton, unpublished results).

The Ser-164 phosphorylation site (Arg-Arg-Arg- Arg-Gln-Glu-Gln-Ser-Pro) is preceded by a series of basic residues and thus it is possible that it may be phosphorylated at very low efficiency by cyclic AMP- dependent protein kinase which recognizes the sequences Arg-Arg-X-Ser and Lys-Arg-X-X-Ser (cf. Edelman et al., 1987). It should also be noted that the Ser-164 site shares a common feature with phosphorylation sites found in other nuclear oncogene products including Ser-89 (Tremblay et al., 1988) and Ser-219 (Tsukamoto el al., 1986; Tremblay et al., 1988) of Ad5 El A proteins, Thr-124 and Thr-701 of SV40 large T antigen (Scheidtmann eta/., 1982), Ser-81, Ser-187 (Has- sauer et a/., 1986), and Ser-278 (Bockus and Schaff- hausen, 1987) of polyoma large T antigen, and Ser-37 and Ser-312 of p53 (Samad et al., 1986; Meek and Eck- hart, 1988). All are located in regions that are rich in proline and the phosphorylated serine or threonine residue is immediately followed by proline. It is possible that the presence of proline affects protein conforma- tion such that these sites are more accessible to protein kinases. However, these amino acid sequences do not correspond to any of the recognition sites of known serine/threonine protein kinases (Edelman eta/., 1987). Thus, as also suggested by Walter and colleagues (Hassauer eta/., 1986; Grasser eta/., 1988) all of these sites may be phosphorylated by the same previously unidentified cellular protein kinase that utilizes proline to recognize the phosphate acceptor. Studies are in progress to examine this possibility.

The importance of Ser-164 phosphorylation will be clarified only through further studies on the biological properties of the mutant ~17~2204. The fact that this mutant replicated as well as wtAd5 was not surprising

126 MCGLADE, TREMBLAY, AND BRANTON

as it has been shown that 176R is not necessary for lytic growth (Barker and Berk, 1987). Plasmids containing the pm2204 mutation transformed with only about one-half the efficiency of wt, but due to the inherent variability of this assay it is difficult to know if this effect is significant. It is also unclear that any greater effect was possible under the conditions employed as re- duced levels of transformation similar to those obtained in the present study were observed using con- structs in which 496R was the only El B protein ex- pressed (Bernards et al., 1986). These data suggest that in assays using El plasmids, either 496R or 176R alone is able to cooperate with El A to produce some transformed foci. We are currently introducing the pm2204 mutation into plasmids that do not express 496R (cf. Barker and Berk, 1987) to examine the role of phosphorylation more carefully.

ACKNOWLEDGMENTS

Our thanks to Silvia Cers, Monica Graham, and Julie Gordon for their excellent technical assistance, to Ed Harlow for the gift of serum

M73, and to Gernot Walter and Frank Graham for useful discussions.

This work was supported through grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.

C.J.M. and M.L.T. are Research Students, and P.E.B. is a Terry Fox Career Scientist of the National Cancer Institute of Canada.

REFERENCES

ANDERSON, C. W., SCHMITT. R. C., SMART, J. E.. and LEWIS, J. B. (1984). Early region 1 b of adenovirus 2 encodes two coterminal proteins

of 495 and 155 amino acid residues. f. Viral. 50, 387-396. BAEIISS, L. E., FISHER, P. B., and GINSBERG, H. S. (1984). Effect on

transformation of mutations in the early region-l b-encoded 2 1

and 55-kilodalton proteins of adenovirus 5. /. Viral. 52, 389-395. BARKER, D. D., and BERK. A. 1. (1987). Adenovirus proteins from both

El B reading frames are required for transformation of rodent cells

by viral infection and DNA transfection. Virology 156, 107-l 2 1. BERNARDS, R., DE LEEUW, M. G. W., HOUWELING, A., and VAN DER Ee,

A. J. (1986). Role of the adenovirus early region 1 B tumor antigen

in transformation and lflic infection. Virology 150, 126-l 39.

BOCKUS, B. J., and SCHAFFHAUSEN, B. (1987). Localization of the phos- phorylations of polyomavirus large 1 antigen. J. Viral. 61, 1 155-

1163. Bos, J. L., POLDER, L. J., BERNARDS, R., SCHRIER. P. I., VAN DEN ELSEN,

P. J., VAN DER Es, A. J., and VAN ORMONDT, H. (1981). The 2.2 kb

El b mRNA of human Ad1 2 and Ad5 codes for two tumor antigens starting at different AUG triplets. Ce//27, 121-l 31.

BRANTON, P. E., BAYLEY, S. T., and GRAHAM, F. L. (1985). Transforma-

tion by human adenoviruses. Biochim. Biophys. Acta 780,67-94. BRANTON, P. E., LASSAM, N. J., DOWNEY, J. F., YEE, S.-P., GRAHAM,

F. L., MAK, S., and BAYLEY, S. T. (1981). Protein kinase activity im-

munoprecipitated from adenovirus-infected cells by sera from tumor-bearing hamsters. J. Viral. 37, 60 l-608.

BRANTON, P. E., and ROWE, D. T. (1985). Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells. 1. Viral. 56, 633-638.

CHINNADURAI, G. (1983). Adenovirus 2 Ip+ locus codes for a 19 kd

tumor antigen that plays an essential role in cell transformation. Celi33, 759-766.

EDELMAN, A. M., BLUMENTHAL, D. K., and KREBS, E. G. (1987). Protein serineithreonine kinases. Annu. Rev. Biochem. 56, 567-613.

F~HRING, F., GALLIMORE, P. H.. MELLOW, G. H., and RASKA, K., JR.

(1983). Adenovirus 12 specific cell surface antigen in transformed cells is a product of the El B early region. Virology 131, 463-472.

GRAHAM, F. L., SMILEY, J., RUSSELL, W. C., and NAIRN. R. (1977). Char- acterization of a human cell transformed by DNAfrom human ade-

novirus type 5. J. Gen. Viral. 39, 59-77.

GRAHAM, F. L., and VAN DER Ee, A. J. (1973). A new technique for the assay of adenovirus 5 DNA. Virology 52,456-467.

GRAND, R. J. A., ROBERTS, C., and GALLIMORE, P. H. (1985). Acylation of adenovirus type 12 early region 1 b 18-kDa protein. FfBS Lett.

181,229-235.

GRASSER, F. A., SCHEIDTMANN, K. H., TUAZON, P. T., TRAUGH, J. A.,

and WALTER, G. (1988). In vitro phosphorylation of SV40 large T antigen. Virology 165, 13-22.

HARLOW, E., FRANZA, B. R., JR., and SCHLEY, C. (1985). Monoclonal antibodies specific for adenovirus early region 1A proteins: Exten-

sive heterogeneity in early region 1A products. J. Viral. 55, 533-

546.

HASSAUER, M., SCHEIDTMANN, K. H., and WALTER, G. (1986). Mapping

of phosphorylation sites in polyomavirus large T antigen. /. Viral. 58,805-8 16.

JOCHEMSEN, A. G., PELTENBURG, L. T. C., TE PAS, M. F. W., DE WIT,

C. M., Bos, J. L., and VAN DER EB, A. J. (1987). Activation of adenovirus 5 ElA transcription by region El B in transformed primary rat

cells. EMBOJ. 6, 3399-3405.

KUNKEL, T. A. (1985). Rapid and specific site-specific mutagenesis

without phenotype selection. Proc. Nat/. Acad. Sci. USA 82, 488- 492.

LEWIS, J. B., and ANDERSON, C. W. (1987). Identification of adenovirus

type 2 early region 1 B proteins that share the same amino termi-

nus as do the 495R and 155R proteins. /. Viral. 61, 3879-3888.

MAK, I., and MAK, S. (1983). Transformation of rat cells by cyt mu-

tants of adenovirus type 12 and mutants of adenovirus type 5. J. VifOl. 45, 1 107-l 1 17.

MALE~E, P., YEE, S.-P., and BRANTON, P. E. (1983). Studies on the

phosphorylation of the 58,000 dalton early region 1B protein of

human adenovirus type 5.1. Gen. Viral. 64, 1069-l 078.

MCGLADE, C. J., TREMBLAY, M. L., YEE, S-P., Ross, R., and BRANTON,

P. E. (1987). Acylation of the 176R (19-kilodalton) early region 1 B protein of human adenovirus type 5. J. Viral. 61,3227-3234.

MCGRORY, W. J.. BAUTISTA, D. S., and GRAHAM, F. L. (1988). A simple

technique for the rescue of early region 1 into infectious human adenovirus type 5. Virology 163,614-617.

MEEK, D. W., and ECKHART, W. (1988). Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells. Mol. Cell. Biol.

8,461-465.

PEPINSKY, R. B. (1983). Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a

novel peptide-mapping technique. J. Biol. Chem. 258, 11,229-

1 1,235.

PERRICAUDET, M., AKUSJARVI, G., VIRTANEN. A., and PETTERSSON, U.

(1979). Structure of two spliced mRNAs from the transforming re-

gion of human subgroup C adenoviruses. Nature (London) 281, 694-696.

PERSSON, H., KATZE, M. G., and PHILIPSON, L. (1982). Purification of a

native membrane-associated adenovirus tumor antigen. J. Viral.

42,905-917. ROWE, D. T., BRANTON, P. E., and GRAHAM, F. L. (1984a). The kinetics

of synthesis of early viral proteins in KB cells infected with wild- type and transformation-defective host-range mutants of human

adenovirus type 5.1. Gen. Viral. 65, 585.


ROWE, D. T., BRANTON. P. E., YEE, S.-P., BACCHETTI, S., and GRAHAM,

F. L. (1984b). Establishment and characterization of hamster cell

lines transformed by restriction endonucleasefragments of adeno-

virus type 5. /. Viral. 49, 162-l 70.

ROWE, D. T., GRAHAM, F. L., and BRANTON, P. E. (1983). Intracellular

localization of adenovirus type 5 tumor antigens in productively

infected cells. Vkology 129,456-468.

SAMAD, A., ANDERSON, C. W., and CARROLL, R. B. (1986). Mapping of phosphomonoester and apparent phosphodiester bonds of the

oncogene product p53 from simian virus 40-transformed 3T3

cells. Proc. Nat/. Acad. SC;. USA 83, 897-901.

SCHEIDTMANN, K. H., ECHLE, B., and WALTER, G. (1982). Simian virus

40 large T antigen is phosphorylated at multiple sites clustered in

two separate regions. /. viral. 44, 1 16-l 33.

SUBRAMANIAN, T., KUPPUSWAMY, M., GYSEJERGS, J., MAK, S., and CHIN-

NADURAI, G. (1984a). A 19.kDa tumor antigen coded by early region

El b of adenovirus 2 is required for efficient synthesis and for pro- tection of viral DNA. /. Biol. Chem. 259, 1 1,777-l 1,783.

SUBRAMANIAN, T., KUPPUSWAMY, M., MAK, S., and CHINNADURAI, G.

(1984b). Adenoviruscyt+ locus, which controlscell transformation and tumorigenicity, is an allele of /p+ locus, which codes for a 19.

kllodalton tumor antigen. J. Viral. 52, 336-343. TAKEMORI, N., CLADARAS, C., BHAT, B., CONLEY, A. J., and WOLD,

W. S. M. (1984). cyt gene of adenovirus 2 and 5 is an oncogene for transforming function in early region 1 and encodes the El B

19,000-molecular-weight polypeptide. /. Viral. 52, 793-805.

TREMBLAY, M. L., MCGLADE, C. J., GERBER, G. E., and BRANTON, P. E. (1988). Identification of the phosphotylation sites in early region 1A

proteins of adenovirus type 5 by amino acid sequencing of peptide fragments. J. Biol. Chem. 263, 6375-6383.

TSUKAMOTO. A. S., PONTICELLI, A., BERK, A. J., and GAYNOR, R. B.

(1986). Genetic mapping of a major site of phosphorylation in ade-

novirus 2 ElA proteins. J. Viral. 59, 14-22. VIRTANEN, A., and PETTERSSON, U. (1983). Organization of early region

1 B of human adenovirus type 2: Identification of four differentially

spliced mRNAs. 1. Viral. 54, 383-391. WEEKS, D. L., and JONES, N. C. (1983). E 1 A control of gene expression

is mediated by sequences 5’ to the transcriptional starts of the

early viral genes. Mol. Cell. Biol. 3, 1222-I 234. WHITE, E.. BLOSE, B., and STILLMAN, 8. (1984a). Nuclear envelope

localization of adenovirus tumor antigen maintains the integrity of cellular DNA. Mol. Cell. Biol. 4, 2865-2875.

WHITE. E., FAHA, B., and STILLMAN, B. (1986). Regulation of adenovirus gene expression in human WI38 cells by an El B-encoded tumor antigen. Mol. Cell. Biol. 6, 3763-3773.

WHITE, E., GRODZICKER, T., and STILLMAN, B. W. (1984b). Mutations

in the gene coding for the adenovirus early region 1 B 19,000-molecular weight tumor antigen cause degradation of chromosomal DNA. 1. Viral. 52,41 O-41 9.

WHITE. E., and STILLMAN, B. (1987). Expression of adenovirus El B

mutant phenotypes is dependent on the host cell and on synthesis of El A proteins. /. Viral. 61, 426-435.

YEE, S-P., ROWE, D. T., TREMBLAY, M. L., MCDERMOTT, M., and BRAN- TON, P. E. (1983). Identification of human adenovirus early region

products using antisera against synthetic peptides corresponding to the predicted carboxy termini. J. Viral. 46, 1003.

YOSHIDA, K.. VENKATESH, L., KUPPUSWAMY, M., and CHINNADURAI, G. (1987). Adenovirus transforming 19.kD T antigen has an en-

hancer-dependent trans-activation function and relieves enhancer repression mediated by viral and cellular genes. Genes Dev. 1, 645-658.

ZOLLER, M. J.. and SMITH, M. (1984). Oligonucleotide-directed mutagenesis: A simple method using two oligonucleotide primers and single-stranded DNA template. DNA 3,479-488.

mapping of a phosphorylation site in the 176r (19 kda) early region 1b protein of human adenovirus...

Documents