Identification of the Phosphorylation Sites in Early … of the Phosphorylation Sites in Early...

THE JOURNAL 0 1988 by The American Soeiety for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol. 263, No. 13, Issue of May 5, pp. 6375-6383, 1988 Printed in U.S.A.

Identification of the Phosphorylation Sites in Early Region 1A Proteins of Adenovirus Type 5 by Amino Acid Sequencing of Peptide Fragments*

(Received for publication, October 1,1987)

Michel L. TremblaySQlI, C. Jane McGlade$§II, Gerhard E. Gerber**, and Philip E. Branton$§$$ From the $Departments of Pathology and **Biochemistry, §Molecular Virology and Immunology Program, McMaster University, Hamilton, Ontario, Canada L8N 325

We have mapped the positions of three of the phosphorylation sites on the 289 and 243 residue (289R and 2433) early region 1A (ElA) proteins of human adenovirus type 5 (Ad5). These proteins, which play roles in both transcriptional control and oncogenic transformation, have identical sequences except for the presence in 289R of 46 additional internal amino acids. Phosphorylation was detected exclusively at serine residues. E1A proteins purified from [36S]methionine- or [S2P]orthophosphate-labeled Ad5-infected cells were digested with trypsin, and two phosphopeptides were isolated by reverse-phase chromatography and subjected to automated Edman degradation. The major species was shown to contain a single phosphorylation site at Ser-219. The second phosphopeptide was shown to contain at least one phosphorylation site at Ser-231. A third phosphorylated tryptic peptide could not be eluted from the column but was isolated using an E1A-specific rat monoclonal antibody. Fol- lowing subcleavage by Staphylococcus aureus V-8 protease, this peptide was shown to contain at least one phosphorylation site at Ser-89. The present data indicate that both the 289R and 243R E1A proteins are phosphorylated at the same sites, at least one in the amino terminal half of the molecule, and at least two toward the carboxyl terminus.

Human adenoviruses induce oncogenic transformation through the action of proteins encoded by two early transcription units, termed ElA’ and E1B (cf. 1). As illustrated in Fig. l.4, E1A transcripts are spliced to produce two major early mRNAs of 13 S and 12 S, and three late mRNAs of 9 S, 10 S , and 11 S (2-9). The proteins encoded by all of these mRNAs, except for the product of the 9 S message, share common amino- and carboxyl-terminal sequences. Of major importance are the products of the 13 S and 12 S mRNAs

* This work was funded through grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Research student. 11 Research student. $$Terry Fox Cancer Research Scientist of the National Cancer

Institute of Canada. To whom correspondence should be addressed. The abbreviations used are: ElA, early region 1A transcription

unit; Ad5, human adenovirus type 5; HPLC, high performance liquid chromatography, SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin layer chromatography; wt, wild-type; TPCK, ~-l-tosylamido-2-phenylethyl chloromethyl ketone.

which encode proteins of 289 and 243 residues (289R and 243R), respectively. These proteins are identical save for the presence of 46 internal residues in the larger molecule (4). E1A products activate transcription of early viral genes (10- 13) as well as some cellular genes (14-16), and the 289R protein appears to be largely responsible for this function (17-20). They also repress the activity of some enhancers, and this function is primarily detected with the 243R protein (17, 19-23). In addition, E1A products induce cell division and immortalization (cf. 1 and references therein) and stim- ulate production of an epithelial growth factor (24).

The 13 S and 12 S E1A mRNAs each produce two acidic nuclear proteins which migrate in polyacrylamide gels with apparent sizes of 52 and 48.5, and 50 and 45 kilodaltons (kDa), respectively (25-29), all of which are greater than the 32 and 27 kDa predicted from the sequence of 289R and 243R. Each of these four E1A species is comprised of at least five or six closely migrating subspecies (30), and it is likely that all of this heterogeneity is generated by differences in post- translational modifications. None of the E1A proteins appear to be glycosylated or acylated or to contain polyADP ribose (31).* However, all E1A products are phosphorylated (25, 27- 29).

Because of the potential significance of phosphorylation in the regulation of E1A protein function, we have mapped the phosphorylation sites of the 289R and 243R E1A proteins of Ad5 through the analysis of peptides generated by digestion with proteolytic enzymes. The results indicated the presence of at least three phosphorylation sites on serine residues common to both the 289R and 243R proteins.

MATERIALS AND METHODS3

RESULTS

Phosphoamino Acids of E l A Proteins-Fig. 2A shows the pattern on SDS-PAGE of E1A proteins immunoprecipitated from extracts of Ad5-infected cells labeled with [3ZP]orthophosphate using E1A-C1 antipeptide serum. Four major phos- phoprotein species were evident of nominal sizes of 52 and 48.5 kDa, and 50 and 45 kDa which had previously been shown to be encoded by the 13 S and 12 S E1A mRNAs, respectively (28, 29). With the mutant dl520, which produces the 12 S mRNA but not the 13 S message (33), only the 50- and 45-kDa species were immunoprecipitated (Fig. 2B). Pre-

M. L. Tremblay and P. E. Branton, unpublished results. Portions of this paper (including “Materials and Methods,” Fig.

4, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

6375

6376 Phosphorylation Sites of Adenovirus 5 E1A Proteins

parative polyacrylamide gels similar to those shown in Fig. 2 were used to purify E1A proteins which were then subjected to acid hydrolysis and analysis by TLC to identify the labeled phosphoamino acids. All of the radioactivity comigrated with free phosphate, incompletely hydrolyzed phosphopeptides, and the phosphoserine standard, and none was detected in the positions of phosphothreonine or phosphotyrosine (data not shown). These data were similar to those obtained in a previous study (41) and indicated that phosphorylation of E1A proteins takes place exclusively a t serine residues.

Tryptic Peptides of ElA Proteins-Fig. 1B shows the amino acid sequence of the 289R protein encoded by the 13 S E1A mRNA (4). As indicated, the 243R product of the 12 S mRNA lacks residues 140 to 185, but is otherwise identical to 289R. Fig. 1B also shows the expected trypsin cleavage sites. As summarized in Table I, 17 tryptic peptides should be produced of which 11 are common to both the 289R and 243R proteins, five unique to 289R and one unique to 243R. Seven of these peptides contain methionine residues, and of these, three are common to both 289R and 243R3, three unique to 289R3, and one unique to 243R. In order to analyze methionine-containing peptides, the 52-kDa 289R product from ["'S]methionine- labeled wt Ad5-infected cells was isolated from polyacrylamide gels, digested with trypsin, and the resulting peptides were separated by reverse-phase HPLC. Fig. 3A shows that five RsSS-labeled species (denoted as Ml-M5) were detected.

DOYAIW 1 2 3

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I I I 60

W I P E DPNEC AVSQI FPMV K U V O EGIDL LTPPP APGSP EPPHL SR- 7 0 I 00 90 IO0 . . '*

" _ -I C T 5 - l C- T7-C - - - --TI/TIB- 160 170 LOO 190 200

CHOCR schyh rlNTG D P D l l CSlrCI amG R W Y S W S E P EPEPE PEPLP k * *k

- ) T 9 4 C T 1 W C T l I d 1 T 1 2

ARPRI rpwL P A I U ) r p t s p V*ICC nsatd S C d q p s n t p W l h p wplc 110 220 210 2 4 0 250

k k'

* + * . . ( . . . . . -kTlld - T I S 1 CTlbl

P I R p v av'vq qrrqa V.CI= d l l n e pqqpl dlsck rprp 260

t ** 270 2 8 0

' *

FIG. 1. Organization and amino acid sequence of E1A proteins. The structure of E1A mRNAs and the sequence of E1A proteins were derived from the nucleotide sequence of cloned ElA cDNAs (2-9). A, E1A mRNAs and their protein products. The positions of splice sites including the corresponding amino acid residues of the 289R product, the number of residues encoded by each exon, and the reading frame ( 7 1 and =77/= ) of the mRNAs have been indicated. Shown at the top are the E1A functional domains described in the text (cf. 48-51). E, amino acid sequence of the 289R and 243R E1A proteins. The amino acid sequence of the 289R product is given and included are the positions of serine residues (*), the sites of trypsin cleavage ( 4 1, the location of tryptic peptides T1 to T16 (- or - - -), pertinent S. aurew V-8 protease cleavage sites ( + ), and the position of the sequence removed from the 243R 12 S product by splicing ( 4 ). Residues present in methionine- containing tryptic peptides have been presented in upper cave and those in other peptides in lower case.

wt dl520 "

A B -

FIG. 2. Separation of E1A proteins by SDS-PACE. KB cells infected with wt Ad5 or the mutant dl520 were labeled with ["PI orthophosphate and E1A proteins were immunoprecipitated using E1A-CI serum and separated by SDS-PAGE. A, wt Ad5. R, d1520. The positions of the 52- and 48.5-kDa proteins (289R) encoded by the 13 S mRNA and those of the 50- and 45-kDa species (243R) produced by the 12 S mRNA (27-29) have been indicated.

Barring coelution of peptides, these results appeared to indicate that two of the methionine-containing tryptic peptides failed to be eluted from the column. The M1 species was identified as peptide T1 because, as also shown in a previous study (26), it comigrated on TLC with a synthetic Met-Arg dimer (data not shown). Further information on the identity of peptides M2, M3, M4, and M5 was obtained from a similar analysis of the [%]methionine-labeled 50-kDa 243R product purified from cells infected with the mutant dl520 (Fig. 3B), which was found to lack peptides M3 and M5. These data indicated that these peptides were unique to the 289R product and that M2 and M4 appeared to be common to both the 289R and 243R proteins.

Identification of Methionine-containing Tryptic Peptides by Automated Edman Degradation-To identify tryptic peptides M2-M5, a mixture of [RsSS]methionine-labeled proteins obtained from wt Ad5-infected cells was digested with trypsin and the peptides separated by HPLC. Individual peptides with elution profiles identical to those shown in Fig. 3A were subjected to automated Edman degradation. As summarized in Table I methionine residues are present a t unique diagnostic sites in tryptic peptides of E1A proteins. Fig. 4 shows that M3 yielded radioactivity in cycles 8 and 14 and thus must be peptide T 7 which is comprised of residues 163-177 and is unique to the 289R. The small amount of radioactivity in cycle 1 may have been due to contamination by M4 (see below) or to the release of a small amount of peptide during the first sequencing cycle. Sequencing of peptide M4 yielded radioactivity in cycle 1, and because M1 had already been identified as peptide T1, these data indicated that M4 must be peptide T10 which is comprised of residues 209-215 and is common to both 289R and 24312. Peptide M5 released "S in cycle 4, and this position identified it as peptide T8 which is unique to the 289R product and comprised of residues 178-

Phosphoryhtion Sites of Adenovirus 5 EIA Proteins 6377

FIG. 3. Separation of tryptic peptides by HPLC. Cells infected with either ut Ad5 or dl520 were labeled with either [s6S]methionine, [32P]orthoph~s- phate, [36S]cysteine, or [3H]leucine, and the E1A proteins were immunoprecipitated and separated by SDS-PAGE as in Fig. 2. The regions in gels containing all four of the E1A protein species or the 289R 52-kDa protein (preparations from ut-infected cells), or the 243R 50-kDa species (preparations from dffi2O-infected cells) were excised, treated with trypsin, and the resulting peptides were analyzed by HPLC. [36S]Methionine-labeled 289R 52-kDa (A) and 243R 50-kDa (B) proteins. [3zP~O~hophosphate-labeled 52- (C) and 50- (0) kDa proteins. A mixture of E1A protein species labeled with Is6S]cysteine ( E ) or 13H]leucine (F).

r

1 , 1 20 SO d o 160 200

4 . , 40 $0 130 160 260

I ' 20 do I l O I L O 2 0

F R A C T I O N N U M B E R

205. All of these data were consistent with the results shown in Fig. 3, A and B with wt Ad5 and d1520. The data obtained with peptide M2 were more surprising as this material yielded radioactivity in cycle 7. Together with the data presented in Fig. 3, these results indicated that M2 must consist of two coeluting peptides, T4A which is unique to 289R and comprised of residues 106-155, and T4B which is unique to 243R and comprised of residues 106-139 linked to amino acids 186- 205. It may not be surprising that these two peptides have similar elution properties as they are of similar length (50 and 54 residues, respectively) and share a common sequence of 34 residues. Thus, all of the methionine-containing peptides have been identified with the exception of peptide T2 which is comprised of residues 3-97 from the region common to both 289R and 243R.

I d e n ~ f i c ~ t i o ~ of Tvptic F ~ s ~ ~ ~ p t ~ e s Isolated by HFLC-In previous studies in which tryptic phosphopeptides of E1A proteins were examined by TLC, one major and several minor species were detected (27,41), indicating that phosphorylation at several sites occurs. To identify these sites, tryptic peptides were prepared from 52-kDa 289R or 50-kDa 243R

proteins isolated from wt Ad5- or d~2O-infected cells which had been labeled with [32P]orthophosphate. Fig. 3, C and D show that both the 52- and 50-kDa proteins yielded two '*P- labeled tryptic peptides, a major labeled form designated P2 and a very broad heterogeneous peak designated P3. The material present at P1 was shown by TLC analysis to consist of free phosphate (data not shown). Neither P2 nor P3 comigrated with any of the methionine-containing peptides eluted from the HPLC column (compare with Fig. 3, A and 3). A previous study had suggested, through the generation of a mutation at Ser-219, that this residue could be the major phosphorylation site in both 289R and 243R (41). As summarized in Table I, serine residues are present at unique diagnostic sites in the tryptic peptides of E1A proteins. Se- quencing of material present in P2 (see Fig. 5A) indicated the presence of 3zP in cycle 4. T11 and T12 are the only peptides that contain serine in position 4. These two peptides differ in that T12 contains both cysteine and leucine whereas TI1 contains neither. An analysis of tryptic peptides from [35S] cysteine- or [3H]leucine-labeled E1A proteins (Fig. 3, E and F, respectively) indicated that no labeled peptide was eluted

6378 300

200

100

P U

E

- > > I- - - I- V

A

P2

I- R P T S P V S 1 1 3 8 5 b 7

L

P3

Phosphorylation Sites of Adenovirus 5 E l A Proteins

8 I

1

L - l

I 2 3 4 5 6 7 8 9 1 0 1 1 11 13 U IS 16 17 F C N S S T D S C D S G P S N T P

CYCLE NUMBER FIG. 5. Automated Edman degradation of phosphopeptides.

The 3ZP-labeled peptides P2 and P3, purified as in Fig. 3, were subjected to automated Edman degradation and the radioactivity released in each cycle was measured. The recoveries of 32P were 2% (P2) and 4% (P3) which are comparable to those obtained in previous studies with phosphate label (36, 42). The amino acid sequences of the identified phosphopeptides have been included in the figure. A, P2. B, P3.

in the position of P2 (compare Fig. 3, C and E, and D and F). Thus, P2 represented peptide T11 with Ser-219 as the major phosphorylation site. The precise profile of the material eluting as P3 (ie. fractions 110-160) was quite variable from experiment to experiment, but in all cases a broad heterogeneous series of peaks was observed. To analyze this material more carefully, fractions at various positions across the peak were reanalyzed by two-dimensional TLC. All fractions gave rise to a similar series of species which varied in their electro- phoretic mobilities but which had similar migration properties in the chromatography dimension (see Fig. 6B). This heterogeneous pattern was also observed when a complete tryptic digest of 32P-labeled E1A proteins was analyzed directly by TLC (see Fig. 6A). The positions of T2 and P2 on TLC were determined by separate analyses (data not shown and see below). An identical heterogeneous array of peptides was also observed when material from various areas of the P3 peak were separated by SDS-PAGE (as in Fig. 7A, below). These results suggested that P3 is comprised of a single peptide with differing numbers of phosphorylation sites. Variations in the level of phosphorylation at different sites could contribute to the heterogeneity of P3 on HPLC, SDS-PAGE, and TLC. However, other peptides have also been described in which the heterogeneous elution pattern on HPLC is caused by

other properties intrinsic to their composition (36, 42). The elution profiles on HPLC for tryptic peptides from a mixture of E1A proteins labeled with [35S]cysteine and 13H]leucine (Fig. 3, E and F, respectively) both show heterogeneously eluting material in a position similar to that of P3. The only tryptic peptide lacking a methionine residue and containing multiple serine residues as well as both cysteine and leucine is peptide T12 (residues 224-258) which is present in both the 289R and 243R products (see Fig. 1 and Table I). Two other observations support the idea that the entire P3 peak is comprised of the single heterogeneously eluting peptide T12. First, all of the P3 material was absent with the deletion mutant dZ313 (43), which lacks sequences coding for amino acids 220-289 (data not shown). Second, recleavage of 32P- labeled E1A tryptic peptides with chymotrypsin had no effect on the elution of P2 but resulted in a shift in the elution of the entire heterogeneous P3 peak (data not shown). Peptide T11 (P2) does not contain a chymotrypsin site, whereas peptide TI2 does, at Leu-249. T12 contains 5 serine residues (at positions 227, 228, 231, 234, and 237), of which several may be phosphorylated. To examine the phosphorylation sites directly, the entire P3 region obtained from 32P-labeled E1A protein was pooled and sequenced, and as shown in Fig. 5B, radioactivity was clearly detected in cycle 8. The presence of phosphoserine in this position in a peptide lacking methionine is unique to peptide T12 and suggests that Ser-231 is a site of phosphorylation. Over the course of three such sequencing experiments, cycles 4 and 14 consistently contained increased levels of radioactivity, indicating that Ser-227 and Ser-237 could be phosphorylated, at least at a low level. In addition the level of radioactivity in cycle 11 was higher than expected for the trailing portion of the cycle 8 peak, and indeed in some analyses of P3 a distinct peak of radioactivity has been observed at cycle 11. Thus, it is possible that Ser-234 is also phosphorylated.

Purification of Tryptic Peptide T2"The failure to elute tryptic peptide T2 (residues 3-97) from the HPLC column left the possibility that phosphorylation sites exist in this region of the 289R and 243R proteins. A number of other columns and solvent systems were examined but this peptide still was not recovered. It is possible that its large size and highly hydrophobic composition, especially at acid pH, ac- counted for its loss on the columns. As another approach to its isolation, tryptic peptides produced from [35S]methionine- or [32P]orthophosphate-labeled wt Ad5- or dl520-infected cells were either examined directly by SDS-PAGE or were first immunoprecipitated with the E1A-specific rat monoclonal antibody, R28, which was known to react with an epitope present in the region between residues 20 and 120 (34). Fig. 7A shows that when the complete tryptic digest of the 243R 50-kDa protein obtained from 32P-labeled dl520-infected cells was analyzed, three major species were present. In separate studies (not shown), purified P2 was found to migrate, along with free phosphate, at the gel dye front. Purified P3 (including various fractions across the P3 peak) migrated heterogeneously as a collection of phosphopeptides. The third, most slowly migrating species was also present in immunoprecipitates prepared from digests of a mixture of E1A species from 32P-labeled wt Ad5-infected cells (Fig. 7B) and from the 243R 50-kDa species isolated from dl520-infected cells labeled with either [32P]orthophosphate (Fig. 7C) or [35S]methionine (Fig. 70). Similar results were also obtained with the 289R 52-kDa protein isolated from wt Ad5-infected cells (data not shown). Thus, it appears that this species must represent peptide T2 and that at least one phosphorylation site. must exist between residues 3 and 97. To confirm that this species was indeed

Phosphorylation Sites of Adenovirus 5 E l A Proteins 6379

A

P2

P3 r""""""""""-~

w lor igin

FIG. 6. Analysis of phosphopeptides by TLC. A mixture of "P-labeled tryptic phosphopeptides or "P- labeled peptide P3 purified as in Fig. 3 were analyzed by two-dimensional TLC, as described under "Materials and Methods." The positions of migration of peptides T2 and P2 were determined in separate analyses using gel- or HPLC-purified material. A, EIA tryptic phosphopeptides. B, HPLC-purified P3.

the T2 peptide, samples prepared from [XsSS]methionine-labeled infected cells were immunoprecipitated, and the peptide was purfied by SDS-PAGE and sequenced. Fig. 8A shows that 3sS was recovered in cycle 13 of the sequencing reaction, thus proving that this species was peptide T2.

Identification of Phosphorylation Sites in Peptide 7'2-Be- cause of its large size, it was necessary to redigest peptide T2 with Staphlococcus aureus V-8 protease in order to generate subfragments which could be analyzed for the presence of phosphorylation sites. Fig. 1B shows the expected V-8 cleavage sites in T2 and Table I the predicted V-8 peptides of which two contain methionine residues. Fig. 9A shows the HPLC elution pattern obtained using T2 peptide precipitated from [RsS]methionine-labeled wt Ad5-infected cells and redi- gested with V-8 protease. As predicted, two labeled species, termed M6 and M7, were detected. These species must be peptides T2-V3 and T2-V9 (see Table I), although we have not determined which species is which. In a parallel analysis of peptides produced from E1A products isolated from ["PI orthophosphate-labeled wt Ad5-infected cells, one principal labeled peptide peak (termed P4) was detected which appeared to comigrate with M7 (see Fig. 9B). Peptide M7 is either T2- V3 (residues 15-25 with serine at position 4) or T2-V9 (residues 61-76 with serines a t positions 3 and 9). To identify the phosphorylation site(s), "'P-labeled P4 peptide was subjected to automated Edman degradation, and as shown in Fig. 8B, a significant amount of "P was detected in cycle 13, but not in cycles 4, 3, and 9. These results clearly identified phosphopeptide P4 as peptide T2-VlO (residues 77-97) and indicated that Ser-89 is a phosphorylation site. A small peak of ?'P was also released in cycle 20, indicating that Ser-96 could possibly be another site of phosphorylation.

DISCUSSION

The 289R and 243R E1A proteins have both been shown to possess a t least three sites of phosphorylation, one (Ser- 89) in the region encoded by the first exon of the 12 S mRNA and two in the exon 2 region of E1A polypeptides (Ser-219 and Ser-231). Some evidence was also obtained to suggest that serines at positions 96, 227, 234, and 237 might also be phosphorylated, although the levels of radioactivity detected made the identification difficult. Phosphorylation at various combinations of sites could account for the extensive array of E1A protein species. Other phosphorylation sites could exist that were undetected due to low levels of labeling or high sensitivity to phosphatases. The latter possibility seems less likely, however, as we4 have seen no differences in phosphorylation of E1A proteins isolated from cells treated in vivo prior to and during Ad5 infection with NaF and/or sodium orthovanadate which efficiently inhibit phosphoseryl as well as phosphotyrosyl phosphastases (44). It is also possible that phosphorylation sites exist in peptides which, like T2, fail to be eluted from the HPLC column. The only serine residues present in peptides not positively identified are those at positions 36, 156, and 283, but several lines of evidence (data not shown) suggest that these are not common sites. Low levels of phosphorylation could also take place at threonine residues. No attempt has been made to account for these sites as phosphothreonine was not evident in E1A proteins.

The data presented are consistent with those obtained with an Ad2 mutant by Tsukamoto et al. (41) who suggested that Ser-219 is the major phosphorylation site. The present study directly identifies Ser-219 as this site and eliminates the possibility that phosphorylation is at the neighboring Ser-221

' D. Takayesu, M. L. Tremblay, and P. E. Branton, unpublished results.

6380 Phosphorylation Sites of Adenovirus 5 E lA Proteins

R28

32p 3 3 - 50Kmix 50K 50K A B C D

A

‘ool 7 5 i

T2 35S-MET

n

P3 I P O 4 + P2”L II(

FIG. 7. Immunoprecipitation of peptide T2. Cells infected with dl520 or wt Ad5 were labeled with either [R2P]orthophosphate or [=S]methionine and a mixture of the E1A species or the 243R 50- kDa species alone was purified and digested with trypsin. In one case the digestion mixture was examined directly on a 15% polyacrylamide gel. Other samples were then immunoprecipitated with R28 rat monoclonal antibody and the precipitates were analyzed on a 15% polyacrylamide gel. A, complete tryptic digest from 32P-labeled BO-kDa protein from dl520-infected cells. Immunoprecipitates obtained with R28 using ”P-labeled E1A proteins ( E ) or 50-kDa protein (C), or “Ss- labeled 50-kDa protein (D). The positions of migration of purified phosphopeptides P3 and P2 determined from separate analyses of HPLC-purified material have been indicated.

residue. Our data differ in that the tryptic phosphopeptide analysis of Tsukamoto et al. (41) suggested that the Ad2 289R and 243R species are phosphorylated at different sites or a t sites resident on peptides that are unique to each species (41), whereas we have found that both Ad5 proteins are phosphorylated at the same sites in peptides common to both species. These differences could be explained by differences in trypsin digestion, although in our studies complete digestion was assured from the peptide pattern of human globin carrier protein. The differences could also have a more interesting basis, reflecting variations in the action of protein kinases or phosphatases on the 289R and 243R products under varying physiological conditions in cells during labeling. Tsukamoto et al. labeled E1A products late after infection in cells treated with cytosine arabinoside, whereas we labeled early after infection in the absence of drug.

Previous studies in our laboratory have shown a precursor- product relationship between the major 13 S and 12 S mRNA products (45), and we and others have suggested that phosphorylation is responsible for this “jump” in gel migration.”‘

M. L. Tremblay, G. E. Gerber, and P. E. Branton, unpublished

J. Culp and M. Rosenberg, personal communication. data.

> - B c L

200 4 - n

0 4

P4

T2- a OW + H I I C H G G V I T E E M A A S L I- 15 -

150

100.

n

CYCLE NUMBER

FIG. 8. Automated Edman degradation of purified peptide T2 and its S. aureua V-8 protease subcleavage product. A, peptide T2 labeled with [3sS]methionine was purified by SDS-PAGE as in Fig. 7 and subjected to automated Edman degradation (recovery 15%). The predicted amino acid sequence has been presented. B, peptide T2 labeled with 32P was purfied by SDS-PAGE and subjected to further digestion with S. aureus V-8 protease. Peptide P4 (see Fig. 9) was purified by HPLC and subjected to automated Edman degradation (recovery 2%). The deduced peptide sequence has been indicated.

Studies with various deletion mutants have suggested that the modification responsible maps between residues 20 and 120 (46), and thus, phosphorylation a t Ser-89 could be involved.

Of prime importance is the role of phosphorylation in the regulation of E1A function. Conversion of Ser-219 to an alanine residue appeared to have little effect on any of the E1A functions measured (41). Similarly the Ad5 mutant dl313 which lacks residues 220-289 encodes functional E1A products (43). Thus, the role of all of the phosphorylation sites in the portion of E1A proteins encoded by the second exon remains unclear. While this region may yet be shown to harbor an important functional domain, most of the known biological activity of E1A molecules resides in three domains encoded mainly by the first exon of 12 S mRNA and sequences unique to the 13 S message (see Fig. L4) in regions which are highly conserved among various adenovirus serotypes (19,20,44-49, and references therein). The Ser-89 (and Ser-96) phosphorylation site maps to a region between domains 1 and 2. A mutant lacking residues 86-120 was found to be capable of transformation in combination with an activated ras gene and of transcriptional activation (50). Thus, the role of this site

Phosphorylation Sites of Adenovirus 5 E l A Proteins 6381

A Y 6

B

-80

"60

-40

-20

I I I I I 20 40 60 80 100 120

-1oo'kg

-80

20 i o i o 100 1 io

P 4

F R A C T I O N N U M B E R FIG. 9. Separation by HPLC of the S. aureua V-8 protease

products of peptide T2. Peptide T2 was isolated from cells infected with wt Ad5 and labeled with either [Y3]methionine or [32P]orthophosphate as in Fig. 7. Purified T2 peptide was treated with S. aureus V-8 protease and the products were separated by HPLC. A, %- labeled material. B, 32P-labeled material.

is also unclear. It should be noted that these results were only semiquantitative and that enhancer repression, transformation with the Ad5 E1B gene, and other E1A functions were not tested. Phosphorylation may also inhibit biological activity or inactivate a negative regulatory element. We are cur- rently producing mutants which will allow us to examine E1A protein phosphorylation more carefully.

Acknowledgments-We would like to thank Arnie Berk for the gift of the rat monoclonal antibodies R7 and R28, Nick Jones for the Ad5 mutant d1520, Geoff Flynn for help with some of the amino acid sequencing, and Pierre Leblanc and Rob Morton for technical advice. We also thank Sylvia Cers for the preparation of cells and viruses.

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Identification of the Phosphorylation Sites in Early … of the Phosphorylation Sites in Early...

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