Phosphorylation of adenovirus E1A proteins by the p341cdc2 protein kinase

VIROLOGY

189, Ill-120

(1992)

Phosphorylation

of Adenovirus

ElA Proteins

D. J. DUMONT’ Moiecufar

Virology and lmmunoiogy

Programme,

Depaflmenr

Received December

AND

by the ~34’~”

Protein

Kinase

P. E. BRANTON*

of Parhoiogy,

McMasTer

12, 199 1; accepted

University,

Hamilton, Ontario, Canada 181\1325

March 20, 7992

Adenovirus early region 1A (El A) products are phosphorylated nuclear oncoproteins which appear to derive transforming activity largely through interactions with cellular proteins including the tumor suppressor plOSIRb-I and cyclin A (pGO”p’), a regulatory subunit associated with ~34~’ and the related protein kinase ~33~“. We have identified several sites of phosphorylation on El A proteins previously and showed that phosphorylation at Ser-89 alters electrophoretic mobility significantly and affects El A-mediated transforming activity to some extent. We now report that both Ser-89 and Ser-219, the major ElA phosphorylation site, were phosphorylated in vitro by ~34~’ purified from HeLa cells. We also found that ElA proteins seemed to be phosphorylated at the highest levels in vivo in mitotic cells which express maximal levels of ~34~’ kinase activity. Thus, in addition to forming complexes with p60cwA, a regulator of phosphorylation, El A proteins seem to P34 cdczand related kinases, and pl05/Rb-7 which exhibits cell cycle-dependent be substrates for p34c”z. These data suggested that a link could exist between phosphorylation, cell cycle progression, 0 1992 Academic Press, Inc. and the regulation of transforming activity of ElA proteins.

ElA products and p34 cdc2 It is widely believed that p34cdc2, which exhibits maximal kinase activity during mitosis, regulates progression through the cell cycle by phosphorylating critical protein substrates (Norbury and Nurse, 1989). ElA proteins induce DNA synthesis and mitosis (Zerler et a/., 1987; Howe et a/., 1990) and thus affect processes which regulate the cell cycle. Second, ElA products interact with ~60, which has been identified as cyclin A, a regulatory protein associated with ~34’~” and the related kinase ~33’~‘~ (Giordano et al., 1989; Pines and Hunter, 1990; Tsai et a/., 1991). Finally, Table 1 shows that the amino acid sequences surrounding serine residues 89 and 2 19 resemble the consensus sequence for sites phosphorylated by ~34’~‘~. Such sites typically contain proline, often followed by a polar residue and a basic amino acid immediately downstream of the target serine or threonine residue (Cisek and Corden, 1989; Heald and McKeon, 1990; Hill et al., 1990; Kipreos and Wang, 1990; McVeyetal., 1989; Morgan eta/., 1989; Peteret al., 1990; Shenoy et al., 1989; Ward and Kirschner, 1990; reviewed in Moreno and Nurse, 1990; Pearson and Kemp, 1991). Thus, it seemed possible that ElA proteins might be substrates for ~34~~~‘and that such phosphorylation might be of functional significance. In the present report we show that ~34~~~~does appear to phosphorylate ElA polypeptides at both serine residues 89 and 219.

INTRODUCTION Products of early region 1A (El A) of adenovirus type 5 (Ad5) are involved in transactivation of viral and cellular genes (Berk eta/., 1979; Jones and Shenk, 1979a,b; Nevins, 1982; Kao and Nevins, 1983; Stein and Ziff, 1984), repression of enhancer activity (Borrelli et al., 1984; Hen eta/., 1985; Velcich and Ziff, 1985; Velcich et a/., 1986; Stein and Ziff, 1987; Jelsma et al., 1989), induction of DNA synthesis and mitosis (Zerler et al., 1987; Howe eta/., 1990), and in cooperation with other oncogenes such as Ha-ras or AdS-El B, cellular transformation (Graham et al., 1974; Land et a/., 1983; Ruley, 1983). Ad5 ElA encodes two major early proteins of 289 and 243 residues (289R and 243R) that are identical except for 46 internal residues (Perricaudet et a/., 1979). Both are phosphorylated at several common sites (Tsukamoto et a/., 1986; Tremblay et a/., 1988, 1989; S. Whalen, D. J. Dumont, and P. E. Branton, manuscript in preparation), including Ser-219, which is the major phosphorylation site, and Ser-89, which induces a large shift in gel mobility and increases the efficiency of E 1A-mediated transformation of baby rat kidney cells by two- to threefold (Dumont et al., 1989 and unpublished data; Smith et a/., 1989). There are a number of reasons to postulate an interaction between

MATERIALS AND METHODS Cell culture and viruses Human HeLa cells were cultured on 100 mm diameter plastic dishes in N minimal essential medium sup-

’ Present address: Samuel Lunenfeld Research Institute, Mount Sinal Hospital, Toronto, Ontario, Canada M5G 1X5. ’ To whom reprint requests should be addressed at Department of Blochemistty, McGill Unlverslty, McIntyre MedIcal Sciences Buildlng, 3655 Drummond Street, Montreal, Quebec, Canada H3G lY6. 111

0042-682’2192

$5 00

CopyrIght 0 1992 by Academic Press. lnc All rights of reproduction I” any form reserved

112

DUMONT

AND BRANTON

plemented with 10% fetal calf serum, as described previously (Rowe et al., 1983; Yee and Branton, 1985). Monolayer cultures were infected with Ad5 at 35 plaque-forming units per cell, as described previously (Rowe et al., 1983). For cell cycle experiments, subconfluent cultures were treated with hydroxyurea (10 mlVI)or with nocodazole (50 ng/mI) in fresh medium for 48 hr, as described previously (Bacchetti and Whitmore, 1969; Zieve et al., 1980). Cells were harvested by scraping except for mitotic cells which were collected by shaking. The mitotic indices for cells treated in these fashions were consistently 30-40, 0, and 95% for untreated, hydroxyurea-treated, and nocodazoletreated cultures, respectively (data not shown). Radioactive labeling Mock-infected and Ad5-infected cells were normally labeled from 10 to 12 hr postinfection with 100 &i of [35S]methionine (Amersham Corp.; 1300 Ci/mmol) in 2 ml of medium lacking methionine, or from 8 to 12 hr postinfection with 2.5 mCi of [32P]orthophosphate (New England Nuclear; 3000 Ci/mmol) in 2 ml of phosphate-free medium. Antisera and immunoprecipitation

of El A-proteins

El A proteins were immunoprecipitated using 5 ~1of the mouse monoclonal antibody M73 (Harlow et al., 1985) as described previously (Tremblay et al., 1989). In the case of cell cycle experiments, equal amounts of protein were immunoprecipitated. Precipitates were analyzed by SDS-PAGE using 9% polyacrylamide gels, as described previously (Rowe et a/., 1983). Phosphorylation p34=d=*

of ElA proteins and histone Hl by

The 289R ElA protein was produced in and purified from f. co/i, as previously described (Ferguson et a/., 1984; Egan et a/., 1987). Histone Hl was purchased from Sigma. p34cdc2was purified from cells by immunoprecipitation using an anti-peptide serum raised against the predicted carboxy terminus (Simanis and Nurse, 1986). Cells were combined with lysis buffer(20 mM Tris-HCI, pH 7.4, containing 5 mM EDTA, 100 mM NaCI, 1% Triton X-l 00, 1 mM PMSF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mn/l NaF, 1 mM EGTA, 1 mlLlp-glycerophosphate, and 5 pg/ml of leupeptin and pepstatin), and lysates were cleared by centrifugation and immunoprecipitated with 5 ~1 of serum. Precipitates were washed sequentially with lysis buffer, lysis buffer containing 2 n/l NaCI, lysis buffer, and finally HB buffer (25 mM MOPS, pH 7.2, containing 15 rnn/l EGTA, 15 mM p-nitrophenyl phosphate, 60 mlVI /3-glycerophosphate, 15 m/l/l MgCI,, 1

mM DTT, 0.1 mlVI sodium orthovanadate, 1% Triton X-100, and 5 pg/ml of leupeptin and pepstatin). Immunoprecipitates were resuspended in 10 ~1 of HB buffer and combined with 5 kg of either histone Hl or El A-289R along with 1 mn/r ATP containing 50 &i of [T~~P]ATP(New England Nuclear, 10.0 mCi/ml; sp act 3000 CilmM). Reaction mixtures were incubated at 37” for 10 min and stopped by addition of an equal volume of double-strength SDS-PAGE sample buffer. Equal aliquots were analyzed by SDS-PAGE. Preparation of tryptic peptides Regions of polyacrylamide gels containing labeled El A proteins were excised from polyacrylamide gels and, after extensive washing with 10% glacial acetic acid in 20% methanol, distilled water, and 50 mM ammonium bicarbonate (pH 8) the gel pieces were crushed and the mixture was digested with TPCK-trypsin (10 mg/ml), as described previously (Tremblay et al., 1988). The peptides were oxidized with fresh performic acid according to Hirs (1967) prior to analysis. Separation of peptides by RP-HPLC Tryptic peptides were separated by HPLC using procedures adapted from Tremblay et al. (1988). Briefly, peptides were dissolved into 500 ~1of 20% formic acid in double distilled H,O and the mixture was passed through a 0.22-p filter. Samples were separated at room temperature on a Waters dual pump HPLC system with a 600E controller using a 4.6 X 250-mm UItrasphere ODS (Cl 8) reverse phase column which had been preequilibrated with solution A (5% formic in water). Peptides were eluted from the column by a linear 1 to 63% gradient of solution B (5% formic acid in ethanol) for 95 min at a flow rate of 1 ml/min. Detection of labeled peptides was achieved using an on-line LB507A isotope detector (Berthold). Separation of peptides by thin layer chromatography (TLC) Peptides were dissolved in 5 ~1 of lo/o ammonium carbonate (pH 8.9) and spotted on cellulose thin layer plates (Polygram CEL 300) along with 1 ~1 of tracking dye (0.1 O/OXylene Cyanol and 0.1% Orange G) and electrophoresis was performed at 0”. The plate was dried, rotated 90”, and subjected to ascending chromatography using A/-butanol:pyridine:formic acid:water (75:50: 15:60 by volume). The positions of labeled peptides were determined by radiography.

PHOSPHORYLATION

OF ElA

TABLE 1 TRYPTICPEPTIDESAND PHOSPHORYLATIONSITESOF El A PROTEINS~ Peptide number Tl T2 T3 T4A” T4Bd T5” T6C T7” T8” T9 TlO Tll T12 T13 T14 T15 T16

Phosphorylatior? site

Amino acid resrdues

l-2 s-97- ____..._.___..._.__.-..--.p,b,pGS (89) PGPPHLS (96) RQPE 98-l 05 10&155- --______.....____...___ --GFpps (132) DDED 106-l 39/186-205------------GFPPS 1566161 162 163-177 i 78-205 206-208 209-215

(1 32) DDED

224-258.-------------------------REINS

(227) s (228) TD-

2, ,?-223-.._...___....___.____...RR,DTS (2, 9) PVSR S (231) CDS (234) GPS (237) NTPP

259-262 263 264-285 286-289

a Tryptic peptrdes were derived from the known sequence of the Ad5 289R protein (Perricaudet et al., 1979). b Phosphorylation sites previously identified Included Ser.89 and Ser.96 rn 12, Ser.21 9 In Tl 1, and one or more serines between residues 227-237 rn T12 (Tsukamoto et al., 1986; Tremblay et al., 1988, 1989). Phosphorylation at Ser-132 wrthin T4/VT4B has also been demonstrated (Whalen et al., in preparation). c Peptide unique to the 289R ElA product. d Peptrde unique to the 243R ElA product.

PROTEINS

BY ~34’~”

113

Tl2 peptide was not apparent in the experiment shown in Fig. 1, Our recent studies have identified another phosphorylation site at Ser-132, present within peptides T4A and T4B of 289R and 243R, respectively, which exists in the context of a casein kinase II consensus sequence (Whalen et al., in preparation). Peptide T2 contains at least two phosphorylation sites located at serine residues 89 and 96 (Tremblay et al., 1988, 1989). Figure 1 shows that at least seven closely migrating species exist which, for several reasons, we believe all represent the T2 peptide. First, all were labeled in viva by [32P]orthophosphate in the slower but not the faster migrating El A protein (data not shown), as is characteristic of phosphorylation at Ser-89 (Dumont et al., 1989). Second, Ad5 mutant d/l 104, which lacks residues 48-60 of peptide T2, yielded phosphopeptide patterns in which the migration of all species was uniformly altered (data not shown). And finally, none of these species were detected in tryptic digests of 32P-labeled ElA proteins produced by mutant AD89A (Dumont et a/., 1989; Tremblay et a/., 1989) in which the codon for Ser-89 had been altered to that of alanine (Whalen et al., in preparation). It therefore appeared that all of these species correspond to peptide T2. Multiple species of a single phosphopeptide could represent phospho isomers or partial trypsin digestion products (Boyle et a/., 1991). Previous studies had suggested that the latter could exist because of the

Phosphoamino acid analysis by TLC Proteins were subjected to acid hydrolysis and phosphoamino acids were separated by electrophoresis at pH 3.5 in pyridine:acetic acid:0.5 M EDTA:ddH,O (5:50:2:945 by volume) on cellulose TLC plates (Polygram CEL300), as described previously (Tremblay et al., 1988). The positions of nonradioactive marker phosphoamino acids were detected by staining with ninhydrin.

RESULTS Phosphotylation

sites on El A proteins

Previous studies showed that phosphorylation of Ad5 El A proteins in infected human KB cells occurs at several sites. The major site was identified as Ser-2 19 present within tryptic peptide Tl 1 (see Table 1) and, as shown in Fig. 1, found to migrate on TLC as a single 32P-labeled species (Tsukamoto et a/., 1986; Tremblay et al., 1988). Phosphorylation of the site(s) between residues 227 and 237 in tryptic peptide T12 (Tremblay et a/., 1988) occurs at very low levels in infected KB cells (Whalen et al., in preparation) and thus labeled

FIG. 1. Tryptic phosphopeptides of ElA proteins labeled in viva in Infected KB cells. KB cells were Infected with it Ad5 and labeled with [3’P]orthophosphate. ElA proterns were purified by rmmunoprecipitation and SDS-PAGE, and tryptrc peptides were prepared from labeled El A proteins excised from the gels were analyzed by TLC, as described under Materials and Methods. The posrtrons of phosphopeptrdes Tl 1, T2, and T4AA4B (see Table 1) have been rndrcated.

114

DUMONT

Mol

A6 UT HU

CD N UT

E HU

AND BRANTON

FG N N+peD

A UT

BC HU N

D UT

E HU

FG N N+DeD

92.5-

69-

69-

ElA

46-

46-

30-

30-

18-

18-

Hl

FIG. 2. In vitro phosphorylation of histone Hl and ElA proteins by immunoprecipitates containing ~34’~“. Extracts from untreated cells (UT) and hydroxyurea- (HU), or nocodazole- (N) treated cells were precipitated with anti-peptide serum specific for ~34~~~‘and the precipitates were incubated with [y3’P]ATP as described under Materials and Methods. (A) SDS-PAGE of proteins in reactron mixtures stained by Coomassie blue. Positions of molecular weight markers (X10m3) have been indicated at the left of the figure (Mel). Lanes A-C, histone Hl; lanes D-F, El A-289R; lane G, El A-289R incubated with a precipitate prepared in the presence of 10 rg of synthetic peptide. (B) Autoradiogram of the gel shown in Fig, 1A. 3eP present in histone H 1 excised from lane C was found to be about 100 times that in lane B, whereas with El A protein (lane F vs lane E) the difference was about fivefold.

presence of Pro-99 just downstream of the cleavage site at Arg-97 (Tremblay et a/., 1989). It is also possible that additional previously unidentified phosphorylation sites exist within the T2 peptide. Whatever the reason, the generation of all labeled species appeared to depend on phosphorylation at Ser-89, or at least on the presence of a serine residue at this position. Phosphorylation

of El A proteins

by ~34’~” in vitro

To determine if p34”“’ was capable of phosphorylating ElA proteins in vitro, El A-289R synthesized in and purified from Escherichia co/i was incubated with [r3*P]ATP and ~34~(“‘, which had been purified by immunoprecipitation using a specific anti-peptide serum (Simanis and Nurse, 1986) from unsynchronized HeLa cells, or those blocked either in mitosis or Gl/S using nocodazole or hydroxyurea, respectively. As a control, histone H 1 was also used as substrate. Figures 2A and 2B (lanes A-C) show that, as found previously (Moreno

and Nurse, 1990) ~34’~” kinase activity with histone as substrate was highest during mitosis and considerably lower in Gl/S. Figures 2A and 2B (lanes D-F) show that while 289R was phosphorylated in all cases, the specific activity was highest using ~34’~” purified from mitotic cells. Incorporation of 32P into ElA protein was largely blocked in immunoprecipitates prepared in the presence of the peptide against which the p34cdcZ specific serum was raised (lane G), indicating that phosphorylation of 289R was dependent upon p34% Analysis by TLC following acid hydrolysis indicated that with histone Hi, serine and threonine residues were phosphorylated at approximately equal levels, whereas with ElA protein, phosphorylation was almost exclusively (over 95%) on serine (data not shown). Histone was also phosphorylated to a considerably higher specific activity than 289R. This difference may have been due to the number of phosphorylation sites available in each substrate and/or the ef-

PHOSPHORYLATION

OF ElA

fects on protein conformation of denaturation and renaturation during purification of El A proteins. Of some interest was the presence in Fig. 26 of a slower-migrating labeled El A species in samples incubated with ~34”~‘~ from mitotic (lane F) and unsynchronized (lane D) cells, but not hydroxyureatreated cultures (lane E). The 289R El A protein purified from f. co/i migrated on SDS-PAGE primarily as a single species with a nominal molecular mass of 48,500, although an ElA breakdown product of about 40,000 was also present (Ferguson et al., 1984) and found to be phosphorylated (see Fig. 2A, lanes D-G). Previous studies indicated that the 48.5K species is a precursor for a slower migrating 52K El A species (Branton and Rowe, 1985) which is produced largely as a result of phosphorylation of 48.5K at Ser.89 (Dumont et a/., 1989; Smith et a/., 1989). Thus it seemed likely that one of the sites phosphorylated in vitro by ~34’~” was Ser-89. p34@ phosphorylates peptides residues 89 and 219 in vitro

containing

serine

To identify the sites of phosphorylation, tryptic peptides from the faster (52K) and slower (48.5K) migrating E 1A proteins labeled in vitro using ~34’~” purified from mitotic cells (i.e., Fig. 2, lane F) were analyzed by TLC and reverse-phase high performance liquid chromatography (RP-HPLC). Fig. 3A’ shows that only one 32P-labeled species corresponding to peptide Tl 1 was apparent following TLC of the faster migrating ElA protein. Similarly, analysis by RP-HPLC (Fig. 3A) also indicated (in addition to an initial peak previously identified as free phosphate) only one labeled peptide eluting at 30 to 35 min and shown previously by mutational analysis and automated Edman degradation to correspond to peptide Tl 1 containing the major phosphor-y lation site at Ser.21 9 (Tsukamoto et a/., 1986; Tremblay et al., 1988). Figure 3B’ shows that the slower migrating ElA protein yielded three major labeled peptides on TLC, of which one again corresponded to peptide Tl 1. The other two migrated in the positions of the two major species of the multiple T2-related phosphopeptides which contain the principal phosphorylation site at Ser-89. Recent studies have shown that some T2 peptide elutes from RP-HPLC columns at 85 to 90 min as a heterogeneous peak (Whalen et a/., unpublished) and these species were also detected by RP-HPLC analysis of the peptides from the slow migrating ElA protein labeled in vitro (Fig. 38). No 32P was detected in any of the other ElA phosphopeptides. The phosphopeptides labeled in vitro were confirmed as Tl 1 and T2 in a mixing experiment in which they were shown to comigrate on TLC with Tll and

PROTEINS

115

BY ~134~~~”

the two principal T2 peptides obtained from ElA proteins labeled in viva with [32P]orthophosphate (data not shown). Thus the data suggested that purified ~34’~” is able in vitro to phosphorylate serine residues 219 and 89 which are present within consensus sequences typical of other substrates phosphor-ylated by ~34’~“. ElA proteins are phosphorylated cycle-dependent fashion

in vivo in a cell

To determine the pattern of phosphorylation of El A proteins in viva at different stages of the cell cycle, Ad5-infected cells which had been blocked either in Gl/S or mitosis or left unsynchronized were labeled with either [32P]orthophosphate or [35S]methionine, and ElA proteins were immunoprecipitated and analyzed by SDS-PAGE. Figure 4 shows that although approximately equal amounts of El A products were synthesized during the labeling period (Fig. 4, lanes A-C’), phosphor-ylation of El A proteins was considerably higher in mitotic cells which contain the highest levels of p34 cdc2kinase activity (Fig. 4, lanes A-C). TLC analyses of El A tryptic peptides from samples shown in Fig. 4 (lanes B and C) were performed. However, even though peptides were generated from El A proteins derived from equal amounts of cell extract, and such samples were processed in parallel, it was difficult to produce such preparations in a strickly quantitative fashion. Thus, a rigorous comparison of peptide patterns was not possible. However, Fig. 5 indicated that peptide Tl 1 containing Ser-219, peptide T2 containing Ser-89, and two other species which we have found to correspond to peptides T4A and T4B containing Ser132 (Whalen et a/., in preparation) were all phosphorylated at seemingly higher levels in mitotic cells (Fig. 5B) than in Gl/S (Fig. 5A) cells, although clearly some phosphorylation of most of these peptides was apparent in the latter. The potential significance of these data and the problems in their Interpretation will be discussed below. DISCUSSION Substrates of ~34’~” appear to include products of both viral (McVey et al., 1989; Morgan et al., 1989; Shenoy et al., 1989) and cellular (Hill et a/., 1990) oncogenes. Phosphor-ylation by ~34’~” is believed to affect DNA binding activity of SV40 large T antigen (McVey et al., 1989) and protein tyrosine kinase activity of pp60c-‘” (Morgan et al., 1989; Shenoy et al., 1989) and thus is of regulatory importance. In the present study, purified ~34~~~~was shown to phosphorylate Ad5 E 1A proteins in vitro at serine residues of two tryptic peptides, Tl 1 and T2. These pep-

116

DUMONT A - 5% formic B - 5% formic

acid/water acldlethanol

AND BRANTON

%B 70%

1

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(I

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B

A - 5% formic E - 5% formic

CDlll 1200

0.0

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20.0

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50.0

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cdc?,ElA

acidlwater acid/ethanol

60.0

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90.0

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I PH 6.9

of ElA proteins phosphorylated in vitro. The 48.5K and 52K ElA proteins shown in Fig. 2 were excised, FIG. 3. Tryptic phosphopeptides digested with trypsin, and the resulting phosphopeptides were resolved by reverse-phase HPLC or TLC. (A) (48.5K) and (B) (52K): analysis by RP-HPLC showing 32P detected with time of column elution (in minutes) as a function of the 9/o solution B (5% formic acid in ethanol) in the gradient. (A’) (48.510 and (B’) (52K): analysis by two-dimensional TLC and autoradiography; direction of electrophoresis, pH 8.9; direction of chromatography, Chr; origin, 0

tides are known to be phosphorylated in viva at Ser2 19 and Ser-89, both of which exist within potential substrate consensus sites for this protein kinase. While other serines in these peptides may have been the actual targets, this possibility seems remote. No other serine was present within sequences remotely resembling ~34’~‘~ substrates. Using an El A mutant containing Ala-89, we have found that phosphorylation at Ser.89 is probably necessary for phosphorylation to take place at any site within peptide T2 (Whalen et al., in preparation). In addition, Ser-219 was previously shown by automated Edman degradation to be the only phosphorylated residue in peptide Tl 1 (Tremblay et al., 1988). The existence of a consensus sequence

cannot guarantee use of that site by the corresponding kinase in viva. Such was the case with Ser-2 19 which exists within a sequence that also resembles the consensus site for cyclic AMP-dependent protein kinase (reviewed in Pearson and Kemp, 1991). However, Tsukamoto et a/. (1986) showed using cell mutants that this kinase does not act at Ser-219 in viva. In the present studies, some correlation between data from in vitro experiments using purified ~34’~‘~ and that on El A protein phosphorylation in viva was observed. Results obtained using the inhibitors nocodazole and hydroxyurea suggested that ElA proteins are phosphorylated in mitotic cells at much higher levels than in those in Gl/S. These data strengthened the

PHOSPHORYLATION

A B C UT HU N

OF ElA

PROTEINS

117

BY ~34’~“~

gested somewhat increased levels of phosphorylation of Tl 1 and T2 during mitosis; however, this analysis was not really quantitative and clearly some phosphorylation of most sites occurred in Gl/S. To definitively address this question, quantitative experiments which

A’ B’ C’ UT HU N

A

69-

ElA 46-

-48.1

30Chr.

18-

PH 8.9

B

FIG. 4. Phosphorylation of ElA proterns In infected KB cells. KB cells were treated with nocodazole (N), hydroxyurea (HU), or left untreated (UT) immediately after infection with Ad5, and 48 hr laterthey were labeled with [32P]orthophosphate or [35S]methionine, as described under Materials and Methods. ElA proteins were rmmunoprecrpitated from extracts containrng equal amounts of protein and analyzed by SDS-PAGE. The positions of the molecular weight markers are Indicated on the left; lanes A-C: %labeled proteins from UT (A), HU (B), or N (C) treated cells; lanes A-C’: %-labeled proteins from UT (A’), HU (B’), or N (C’) treated cells,

possibility that El A products are substrates for ~34’~” in viva, as this kinase is generally most active in mitotic cells. However, some difficulty exists in the interpretation of these data. One concern is that drugs which affect the cell cycle and cell metabolism could alter the rate of synthesis, stability, or phosphorylation of ElA products. Previous studies have shown that increased levels of ElA proteins are produced following treatment with cytosine arabinoside which blocks cells in S-phase (Gaynor et al., 1982). In addition, cycloheximide selectively increases the stability of the more highly phosphorylated forms of ElA proteins (Branton and Rowe, 1985). Thus, comparisons of levels of El A proteins labeled in drug-treated cells (Fig. 4) may be inappropriate. Comparison of the phosphopeptides of El A products labeled in viva in these experiments sug-

pH 8.9

FIG. 5. Tryptic phosphopeptides of El A proteins labeled in viva. ElA proterns labeled with [32P]orthophosphate in cells treated wrth hydroxyurea (HU) or nocodazole (N) were excused from the gel shown in Fig. 4 (lanes B and C) and treated with trypsin. Aliquots of peptides prepared from equal amounts of E 1A protein were analyzed by TLC [(A) HU; (B) N].

118

DUMONT

employ methods other than drug treatment to examine the cell cycle need to be designed. Nevertheless, it seems likely that Ser-219 and Ser89 are substrates for a cdc2 kinase. Genes encoding several members of a family of protein kinases with substrate specificities similar to ~34~~~~have been cloned (Tsai eta/., 1991). Because purified p34cdc2was able to phosphorylate El A products in vitro and is highly active in mitotic cells, this may be the kinase involved in phosphorylation of Ser-219 and Ser-89, However, it is also quite possible that one or more of the other members of this kinase family act on ElA products. In addition, even though these sites are present within apparent ~34~~~~consensus sequences, other kinases have been found to utilize substrates composed of somewhat similar sequences (Hunter, 1991; Pearson and Kemp, 1991). The strongest evidence that ~34~~~~IS involved is clearly the ability of purified enzyme to act in vitro at these sites. The functional significance of such phosphorylation is uncertain. Although it is the major phosphorylation site, mutagenesis of Ser-219 did not appear to affect adenovirus replication or E 1A-mediated transformation (Tsukamoto eta/., 1986; Smith eta/., 1989). Phosphorylation of Ser-89 induces a major shift in mobility of El A products on SDS-PAGE (Dumont eta/., 1989; Smith et al., 1989). In addition, the ability of ElA products to cooperate with either Ad5 El B or activated p2 1c-Ha-ras in the transformation of primary rat cells was reduced about threefold in mutants containing alanine (Dumont et a/., 1989 and unpublished results) or glycine (Smith eta/., 1989) in place of Ser-89. Further large scale studies have indicated that even though this reduction was modest, it was nonetheless entirely reproducible and statistically significant (D. J. Dumont, unpublished results). Transformation and the induction of cellular DNA synthesis by ElA products appear to result from the formation of complexes with several cellular proteins (Yee and Branton, 1985; Harlow et al., 1986; Egan et a/., 1988), including pl05/Rb- 1, the product of the Rb- 1 tumor suppressor gene (Whyte eta/., 1988; Egan eta/., 1989; Howe et al., 1990), and p60-cyclin A, which probably associates with El A proteins indirectly via an interaction with the ~107 El A-binding protein (Ewen et a/., 1992; Faha et a/., 1992; Devoto et a/., 1992). plO5/ Rb-1 appears to play a role in regulating entry into Sphase and it seems that phosphorylation, possibly by p34Cd”” or a related kinase, may control this activity (Buchkovich et a/., 1989; Chen et al., 1989; DeCaprio et al., 1989). p60-cyclin A is a regulatory subunit of p34cdc*and ~33”~~~(Giordano et a/., 1989; Pines and Hunter, 1990; Tsai et al., 1991). It has been known for some time that protein kinase activity is associated

AND BRANTON

with Ad5 El A products (Lassam et a/., 1979; Branton et al., 1981), and it now appears that such activity is due to the presence of ~33’~~~(but not ~34~~~9which probably associates with E1A products via an interaction with its regulatory subunit p60-cyclin A, which itself binds to the ~107 El A-associated protein (Tsai et al., 1991; Giordano eta/., 1991; Hermann eta/., 1991). ElA proteins therefore form complexes with a kinase of the ~34~~~~family (~33”~~?, with substrate of such kinases (p105/Rb- 1 and probably plO7), and with a kinase regulatory subunit (p60-cyclin A). By acting as substrates for such kinases, ElA proteins could promote the formation of complexes which function to alter or regulate the activity of cell cycle kinases or facilitate their access to important substrates. Previous studies indicated that unphosphorylated E1A products synthesized in E, co/i form complexes with many of these cellular proteins at low efficiency (Egan et al., 1987). Thus, phosphorylation at Ser-89 or other sites could play a role in the regulation of complex formation. Further studies with appropriate ElA mutants may help to clarify the functional significance of ElA protein phosphorylation. ACKNOWLEDGMENTS We thank Drs. Chris Norbury and Paul Nurse (Oxford University) for the generous gift of anti-peptide serum specific for the carboxy terminus of ~34’~“. We also thank Dennis Takayesu for help with the HPLC analysis and Sylvia Cers and Monica Graham for cell culture work. This work was supported through grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. P.E.B. was a Terry Fox Career Research Scientist of the NCIC.

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