Protein Kinase Induction in Escherichia Coli by Bacteriophage T7 (Early T7 Protein/Serylphosphate/UV Mappikig/Ribosomal Proteins) H

Home , Polyphosphate kinase, T7 RNA polymerase

Proc. Nat. Acad. Sci. USA Vol. 71, No. 2, pp. 586-589, February 1974

Protein Kinase Induction in Escherichia coli by Bacteriophage T7 (early T7 protein/serylphosphate/UV mappikig/ribosomal proteins) H. J. RAHMSDORF, S. H. PAI, H. PONTA, P. HERRLICH, R. ROSKOSKI, JR.*, M. SCHWEIGER, AND F. W. STUDIERt Max-Planck-Institut far Molekulare Genetik, Berlin-Dahlem, Germany; and * Department of Biochemistry, The University of Iowa, Iowa City, Iowa; t Brookhaven National Laboratory, Upton, New York Communicated by Fritz Lipmann, October 11, 1973

ABSTRACT After bacteriophage T7 infection, a protein MgSO4, 50 AM phosphate, and 2 mM of each amino acid. Cells kinase (EC 2.7.1.37; ATP:protein phosphotransferase) at = 0.25 were infected at a of infection of activity can be demonstrated in E. coli in vivo by sodium OD600 multiplicity dodecyl sulfate-polyacrylamide gel electrophoresis and 10, and surviving cells were measured. Survivors were in all ex- autoradiography. Cell-free extracts catalyzed the transfer periments less than 3% at 3 min after infection. The cells were of the terminal phosphoryl group of [-y_32P]ATP to endoge- harvested on frozen TMA buffer [10 mM Tris * HCl (pH 7.5)-10 nous protein acceptor or to added histone. The bond be- mM MgCl2-22 mM NH4Cl-5% glycerol-i mM dithiothrei- tween phosphate and protein shows the characteristies of serine phosphate: it is stable in 1 N HCl (1000) and cleaved toll, and washed once with TMA buffer. They were stored in by 1 N KOH (370) and by alkaline phosphatase treatment. liquid nitrogen. Phage stocks were purified by CsCl gradient Moreover, after partial acid hydrolysis, radiophosphate centrifugation. migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme ac- Phosphorylation of Proteins In Vivo. Two minutes after tivity in uninfected cells is negligible. Ultraviolet irradia- infection, 200-Isl aliquots of the noninfected or infected cul- tion of the phage genome prevents the appearance of the tures were mixed with 5 1Ci of [82P]orthophosphate and incu- protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and bation was continued for another 7 min. The cell pellet was its gene maps in the early region (promoter proximal to treated with 50 Il of sodium dodecyl sulfate (SDS) buffer [1% gene 1). Ribosomal proteins are phosphorylated in vivo and SDS, 1% mercaptoethanol, 10% glycerol, 20 mM EDTA, 50 are substrates in vitro. Enzyme activity in vitro is not mM Tris . HCl (pH 6.8)] at 1000 for 2 min. Treatment with changed by addition of cyclic AMP or cyclic GMP. RNase (100 Ag/ml) at 200 for 30 min followed. The samples Bacteriophage systems have been used extensively to study were then subject to SDS slab-gel electrophoresis (9); the gels the regulation of gene expression (1, 2). One particular aspect were dried and placed on x-ray film overnight. of these systems is the rapidity of changes in the pattern of Assay for Protein Kinase In Vitro. Brij-lysozyme cell ex- gene expression; phage T7 induces a whole series of control tracts (10) were used as enzyme sources. The incubations were mechanisms within 4-5 min (2, 3). This rapidity of change in carried out at 300 for 5 min in a total volume of 0.1 ml. The gene expression prompted our search for group transfers after incubation mixtures contained 30A]. of cell extract, 3 mg/ml of phage infection (4-6). A phage-induced group transfer to type II histone, and either (1) 15 mM sodium phosphate (pH components of the existing host machinery of gene expression 7.0), 0.01 MMgCl2, 0.14 mM ATP (35.7 Ci ['y32-P]ATP/mol) would allow both a fast and an economical control. or (2) 25 mM Tris HCl (pH 7.0), 5 mM MgCI2, 0.1 mM In our previous work on phage T7, we reported that labeled EDTA, 44,AM, ATP (114 Ci of [32P]ATP/mol). The incuba- phosphate is incorporated into ribosomal protein after T7 in- tions were started by the addition of enzyme (cell extract) fection (6, 23). A bacteriophage T7-induced protein kinase and stopped by addition of 1.5 ml of 10% trichloroacetic acid. (EC 2.7.1.3.7) is the subject of the present paper. The enzyme The precipitates were boiled in trichloroacetic acid for 15 min, is an early T7 gene product. The gene maps promoter-prox- chilled in ice for 10 min, and collected on glass-fiber filter imal to gene 1 (RNA polymerase). The kinase transfers phos- discs. The discs were washed 5 times with 10mlof 10% trichloro- phate from 'ylabeled ATP to seryl residues in protein. Its acetic acid and once with 10 ml of methanol. After the filters activity is not affected by the addition of adenosine 3':5'- were dried, radioactivity on the filters was measured by liquid cyclic monophosphate (cAMP) or guanosine 3': 5'-cyclic scintillation spectrometry. In selected experiments, the 10% monophosphate (cGMP). trichloroacetic acid suspensions of precipitate were heated to METHODS 900 for 20 min, cooled to ambient temperature, and centrifuged at 2500 X g. These precipitates were washed twice in Stains used were described in ref. 7 and legends to Figs. 1 ethanol-ether (3:1) at 370 for 30 min and then for 10 min, and 3. collected again by centrifugation, and air dried. The precipi- Growth of Bacteria and Phage. Escherichia coli B,,1 were tates were taken up in 0.5 ml of NCS solubilizer (Nuclear grown at 300 in TG medium (8) supplemented with 50 ,MI Chicago) for liquid scintillation counting. Chemicals. [7y-32P]ATP and [32P]orthophosphate were pur- Abbreviations: SDS, sodium dodecyl sulfate; PEI, polyethyl- chased from Amersham Radiochemicals. E. coli alkaline phos- eneimine; cAMP, adenosine 3':5'-cyclic monophosphate; cGMP, phatase and pancreatic DNase and RNase were purchased guanosine 3':5'-cyclic monophosphate. from Boehringer Mannheim. Pronase and type II calf-thymus 586 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Protein Kinase Induction 587

histone were products of Sigma Chemical Co., St. Louis. Control Polyethyleneimine PEI-cellulose thin layers were a product of T7 am 23 Brinkmann. T7 arn 23 Pronase RESULTS T7 cm 23 UV Incorporation of [32P]OrthophosphaMt into Protein In Vivo. To demonstrate protein phosphorylation in vio, control and Control a- T7 am 23 '.4. C 44It N T7-infected cells were grown in the presence of-labeled phos- T7 am 23 UV w phate. The cells were grown in a low-phosphate medium (high T7 orn, 23 CAM ..r je, ...... specific activity); the cells were harvested at specific times (7 in shown in Fig. 1) after infection, lysed Control aI >a min the experiment T7 am 23 _ _ i P H with SDS, treated with RNase, and subjected to polyacryl- T7 om 23/UV cells _4nI W amide gel electrophoresis. The location of the labeled phosphate was determined by autoradiography. Control 14C The formation of phosphoprotein in the control cells was Control 32p modest (Fig. 1). However, there is a considerable increase in T7 am 23 14C the number of labeled bands and in the extent of labeling in T7 om 23 32p T-; 14C .. 4VZ__,.t* I the T7-infected cells. The latter is also demonstrated by the 32p difference in total hot trichloroacetic acid-precipitable, labeled T7' material. 3-4 times more label is precipitated from homoge- T7 an 342o nates of T7-infected cells than from control cells (not shown). T7 H3 The labeled bands detected in gels appear to represent phos- T7V ; V .. ;' CS'>- phate-carrying proteins since the bands disappear upon Pro- *JV cells nase digestion (Fig. 1). ® C----H FIG. 1. Incorporation of [32Plorthophosphate into proteins Phosphorylation In Vitro. The characteristics of protein in vivo after T7 infection. Aliquots (50-200 Al) of culture were labeling in vitro were studied by measurement of the incorpora- labeled and analyzed on 10% slab gels as described in Methods. tion of radiophosphate from ['y-82P]ATP into various acceptor Individual sets were subjected to electrophoresis on the same slab substances by cell homogenates. Incorporation was observed gel with equal amounts of culture in each sample. Where indi- in cell homogenates from T7-infected cells with or without cated, UV treatment of phage or bacteria in sets I-III was for added histone (Table 1). Among those proteins tested, histone 10 min under the conditions of ref. 20; the bacteria used in set V is the best acceptor protein, better than albumin, RNase, were treated before infection with 6 min of UV under the condi- and washed ribosomes from E. coli (not shown). The addition tions of ref. 9. Samples were labeled with 25 A&Ci/ml of 32PO4 or of cAMP (Table 1) or cGMP does not alter the amount of I'4C]aminoacids where indicated. Label was present between 2 preparations, and 7 min after infection for sets I-IV, and 0-10 min in set V. phosphate incorporation. With these enzyme All samples were treated with RNase; sample 3 of set I was also the incorporation is linear for the first 6 min (not shown). treated with Pronase. Sample 4 of set II was infected in the Properties of the Bond Between Phosphate and Protein. presence of 75 pg/ml of chloramphenicol. T7 strains: am23 and Because of the lack' of definitive evidence for a bacterial am342a are amber mutants in gene 1 (7, 9). The amber peptide protein kinase (11), extensive studies on the nature of the link of T7 am23 is very small and only the ligase and two peptides between phosphate and protein were undertaken. The radio- of the kinase are detected in the 10% gel (sample 3 of set IV). activity was not solubilized by DNAse, RNAse, or 10% tri- H3 is a deletion mutant in the kinase gene (22). chloroacetic acid (90°, 20 min) treatments that destroy DNA or RNA (Table 2). Radioactivity was not solubilized by To further substantiate the identity of the phosphoprotein ethanol-ether, which would extract labeled phospholipids. bond, the trichloroacetic acid precipitate that had been ex- The following criteria are consistent with the phosphorylation tracted with ethanol-ether was treated with 2 N HCl for 20 hr of an acceptor serine residue: stability in 1 N HCl at 1000 for 1 at 110°. About 15% of the label migrated with standard serine hr, cleavage by 1 N KOH at 370 for 1 hr, and cleavage by al- phosphate on PEI-cellulose thin layers (Fig. 2), the rest of the kaline phosphatase treatment (Table 2). label migrated with orthophosphate. Under the conditions of TABLE 1. General characteristics of the protein kinase partial acid hydrolysis used, about half of the liberated serine reaction in vitro phosphate would be hydrolyzed. This convenient thin-layer method does not resolve serine phosphate from threonine 82p Incorporation, pmol phosphate. Addition Control T7 The T7-Induced Protein Kinase Is a Phage Gene Product. None 2.7 17.9 Protein synthesis is required for the appearance of protein Histone 4.4 37.7 kinase, as indicated by the observation that chloramphenicol Histone plus cAMP (1.4 uM) 4.1 35.0 (75 lig/ml) inhibits the appearance of the phosphorylating ac- Casein 3.3 20.3 tivity in vivo (Fig. 1). Inactivation of the phage genome by UV Histone without incubation 3.3 3.3 irradiation also prevents the appearance of protein phosphorylation. On the other hand, UV irradiation of the host genome E. coli B.-, were harvested 7 min after infection with T7 am23. sufficient to inactivate the expression of host genes does not Brij-lysozyme extracts were prepared. Assay mixture 1 was alter the appearance of kinase activity (Fig 1). used, with the additions shown. The background was not sub- tracted. Histone concentration: 3 mg/ml; casein concentration: 3 The T7-Induced Protein Kinase Is an Early T7 Protein. mg/ml. The position of the protein kinase gene in the early or control Downloaded by guest on September 24, 2021 588 Cell Biology: Rahmsdorf et al. Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 2. Properties of the phosphoprotein bond ! -1 c PM --}i .1-.1-:- --: 77 - 600 .- _ _ ,.7 , --I _ I Treatment 11P Incorporation, pmol

u _-500 -I-- None 32.0 I - --- 'I-t- . Fa-!- --f 1 NKOH 3.6 L----1- ~ I.I I are 1NHCl,1000 30.0 Alkaline phosphatase 6.4 _. -300 DNase 34.0 i RNase 31.8 nn Pronase 2.8

Enzyme incubations were carried out with incubation mixture 2, and the reaction product was precipitated with 2 ml of 10% trichloroacetic acid. The precipitates were extracted with ethanol- ether after the hot trichloroacetic acid treatment. The precipitates tfront kSer P -I sPi torigin (duplicates) were treated with 100 jsl of 1 N KOH, 100 Al of 1 N FIG. 2. Identification of ["2P]serine phosphate in the reaction HCI, 200 jl of 50 mM Tris-HCl (pH 8.0) containing 5,4 of alka- product. Labeled phosphoprotein was synthesized in vitro with line phosphatase, and 200 ul of 50 mM Tris-HCl (pH 7.0) con- incubation mixture in 0.1 ml incubation volume. The product taining 10 Asg of DNase, RNase, or Pronase. The incubations was then treated with hot trichloroacetic acid and extracted with were for 1 hr at 370; HCl treatment was at 1000. Controls were ethanol-ether as described in Methods. The precipitate was suspended in Tris HCl buffer without enzyme and carried treated with 1 ml of 2 N HCl at 1100 for 20 hr. Then 5-jul aliquots through the same procedure. At the end of the incubation, pro- were chromatographed on PEI-cellulose thin layers with 0.25 tein was precipitated with 2 ml of ice-cold 10%o trichloroacetic acid ,smol of carrier o-phosphoserine and 1 /Amole of K2HPO4 with the and centrifuged at 2500 X g. The precipitate was resuspended in following solvent: 0.15 M KCl in 1 M acetic acid adjusted to pH trichloroacetic acid and centrifuged. It was then dissolved in 0.5 3.5 with concentrated NH4OH. Phosphate and phosphoserine ml of NCS solubilizer, and the radioactivity was measured. were developed with phosphomolybdate reagent (21). The position of radioactivity was determined with a Packard Radiochromato- that time after infection, only four T7-specific proteins. Thus, gram scanner. the phosphorylated proteins are host proteins present at the time of infection. Also, coelectrophoresis shows that all but one peptide band migrate differently (Fig. 1). region of T7 (2) was suggested by the fact that the kinase is Previously it was shown that ribosomal proteins become induced by T7 gene 1- mutants (Fig. 1). These mutants fail phosphorylated Other components of the to synthesize the T7-specific RNA polymerase and late T7 (6). protein-synthe- sizing machinery do not appear to be phosphorylated. In proteins. The position of the kinase gene was determined by Ouchterlony tests with sera directed against host RNA poly- three methods. First, the activity occurs 4 min after infection merase, initiation factor 3, and elongation factor G, no phos- (Fig. 3). The T7 translational repressor appears between 2.5 phate label was found in the precipitates (not shown). and 3 min; the gene-1 product, phage RNA polymerase, appears at 5 min; and the phage-induced DNA ligase occurs after DISCUSSION 6 min (12, 7). These data indicate that the protein kinase gene The properties of protein kinases have been described from a is located promoter-proximal to gene 1. large number of eukaryotic organisms (13, 14). Their role in Also by the UV-irradiation technique of transcriptional the regulation of enzyme activity by catalyzing the phos- unit mapping (2), the kinase gene was mapped to the left of phorylation of specific serine residues is also well documented gene 1. At a given dose of UV irradiation, the capacity to (14). We find little, if any, protein kinase activity in uninfected direct the synthesis of phage RNA polymerase after infection E. coli. This observation agrees with the finding that the levels is more reduced than the capacity to direct the synthesis of of phosphoserine and phosphothreonine in E. coli are at least kinase (not shown). By the calculation of UV irradiation hits an order of magnitude less than in eukaryotic organisms (yeast, per 1000 nucleotides and by comparison with the UV sensi- rat liver, and muscle) (15). Preliminary reports on a protein- tivities of various known T7 genes, the right end of the kinase phosphorylating activity in prokaryotes (16, 17) may find gene appears to be at 7% of total T7 genome. an explanation in the existence of a polyphosphate kinase The third method involves the use of deletion mutants. that is stimulated by histone (18). We tend to believe that The deletions H3 (Fig. 1) and 5L (22) eliminate in vivo phos- there is no protein kinase in E. coli. The background of phorylation. Both deletions map to the left of gene 1. phosphorylated protein in uninfected cells may represent Phosphorylation by Various T7 Mutants. For the experi- metabolically active phosphoryl-enzyme intermediates, e.g., ments shown, a T7 gene-i mutant had been used. The succinate thiokinase (EC 6.2.1.4), nucleoside diphosphokinase reason was that gene-i mutants, like T7 +, induce large (EC 2.7.4.6), alkaline phosphatase (EC 3.1.3.1), and poly- nucleotide ligase (EC amounts of protein kinase. Cells infected with T7K, a dele- 6.5.1.1). The protein tion mutant in kinase, contain no kinase activity. Also, the kinase found after T7 infection, thus, is the first deletion mutants H3 and 5L induce no kinase activity (Figs. seryl protein kinase reported in a prokaryotic organism. The 1 and 3). protein kinase catalyzes the transfer to a serine or threonine residue in a polypeptide chain and is different from the protein Identification of Phosphorylated Proteins after T7 Infection. acyl kinase found in Caulobacter crescentus (11). The product Gene-i mutants induce the phosphorylation of at least 12 formed in the latter case is cleaved by 1 N HCI at 1000 for 1 different proteins (Fig. 1). However, these mutants form, at hr (11), but not by alkaline phosphatase. The phosphoserine Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Protein Kinase Induction 589

15 OOC phorylated proteins is unknown. We have, however, reported cpm previously that ribosomal proteins are phosphorylated in vivo and in vitro (6). That host proteins serve as substrates becomes clear through the observation that T7 gene-1 mutants induce only four proteins, but about 12 phosphorylated bands were detected. Moreover, at least three out of these four T7-specific proteins are definitely not phosphorylated. The role that protein phosphorylation plays in phage infection is not known. Kinase mutants appear to develop nor- 10 000 mally. Since host proteins are the substrates of phosphorylation, the kinase may allow the phage to rescue some essential host functions while inactivating others. We favor the idea that phosphorylation adapts host proteins to the altered conditions of an infected cell. We thank J. Kodrzynski and G. McGovern for skillful technical assistance R.R. Jr. was supported by the National Science Found- ation Grant GM-2591. The portion of this work carried out at 5000 :1000 Brookhaven National Laboratory was under the auspices of the U.S. Atomic Energy Commission. , ~~~ 1. Mathews, C. K. (1971) Bacteriophage Biochemistry (Van Nostrand Reinhold Co., New York). 2. Schweiger, M. & Herrlich, P. (1974) in Current Topics in Microbiology and Immunology (Springer-Verlag, Berlin), in press. 3. Studier, F. W. (1972) Science 176, 367-376. 4. Sauerbier, W., Schweiger, M. & Herrlich, P. (1971) J. Virol. 8, 613-618. 5. Rahmsdorf, H. J., Roskoski, R., Ponta, H., Pai, S. H., /10 15 Herrlich, P. & Schweiger, M. (1973) Hoppe-Seyler's Z. Time ofter infection (min) Physiol. Chem. 354, 1232. 6. Rahmsdorf, H. J., Herrlich, P., Pai, S. H., Schweiger, M. & FIG. 3. Appearance in ViVO of kinase after infection with bac- Wittmann, H. G. (1973) Molec. Gen. Genetics 127, 259-271. teriophage T7. E. coli B.,-, were infected with T7+ or T7 am23. 7. Scherzinger, E., Herrlich, P., Schweiger, M. & Schuster, H. At various times, 5-ml aliquots of the cultures were harvested on (1972) Eur. J. Biochem. 25, 341-348. 2.5 ml of frozen TIVIA buffer; the cells were washed and disrupted 8. Echols, H., Garen, A., Garen, S. & Torriani, A. (1961) J. by the Brij-lysozyme method. Kinase was determined with incu- Mol. Biol. 3, 425-438. bation mixture 2. T7 RNA polymerase and T7 ligase assays were 9. Studier, F. W. (1973) J. Mol. Biol., 79, 237-248. to refs. 10. Godson, G. N. & Sinsheimer, R. L. (1967) Biochim. Biophys. according 7 and 19, respectively. (O---) kinase in cells Acta 149, 476-488. infected with T7 am23; (- )kinase in cells infected with T7K. 11. Agabian, N., Rosen, 0. M. & Shapiro, L. (1972) Biochem. (Similar results were obtained with T7 =m342, H3, and T7 am3S42, Biophys. Res. Commun. 49, 1690-1698. 5L; these mutants are described in ref. 22. ) (A- - -iA) phage RNA 12. Schweiger, M., Herrlich, P., Scherzinger, E. & Rahmsdorf, polymerase (T7K); (a--- -O) DNA ligase (T7K). H. J. (1972) Proc. Nat. Acad. Sci. USA 69, 2203-2207. 13. Tao, M., Salas, M. L. & Lippman, F. (1970) Proc. Nat. Acad. Sci. USA 67, 408-414. bond, including the one observed here, shows the inverse 14. Holzer, H. & Duntze, W. (1971) Annu. Rev. Biochem. 40, characteristics. The product of the T7 kinase reaction is not a 345-374. polyphosphate, which is also cleaved by 1 N HCl for 1 hr at 15. Rask, L., Walinder, O., Zetterquist, 0. & Engstr6m, L. (1970) Biochim. Biophys. Acta 221, 107-113. 100° (18). An adenyl-tyrosine residue, like those found in E. 16. Kuo, J. F. & Greengard, P. (1969) J. Biol. Chem. 244, 3417- coli RNA polymerase and E. coli glutamine synthetase (14), is 3419. stable in 1 N KOH at 37° for 1 hr and is not a substrate for 17. Schechter, S. L., Li, H.-C. & Krulwich, T. A. (1972) Abstr. alkaline phosphatase; it is thus distinguished from the chief Annual Meeting American Soc. Microbiol. p. 176. found in our enzyme reaction. 18. Li, H.-C. & Brown, G. G. (1973) Biochem. Biophys. Res. product Commun. 53, 87.5-881. The kinase activity appears within 4 min after infection 19. Herrlich, P. & Schweiger, M. (1973) in Methods in Enzy- with bacteriophage T7. The early appearance, the UV- irradi- mology, eds. Moldave, K. & Grossman, L. (Academic Press, ation data, and the data obtained with deletion mutants indi- New York), Vol. 30, in press. cate that the kinase is the product of a T7 gene that is located 20. Herrlich, P., Rahmsdorf, H. J. & Schweiger, M. (1973) in to the left of 1. The kinase "Immunopathology," Advances in the Biosciences, eds. gene activity disappears again vrery Raspe, G. & Bernhard, S. (Pergamon Press-Vieweg, Braun- SOOI1, which could be caused by an e~xtreme lability of the schweig), in press. enzvme. In vitro, however, the enzyme is remarkably stable. 21. Dittmer, J. C. & Lester, R. L. (1964) J. Lipid Res. 5, 126- The disaIppearanlce of kinase, more likely, is regulated by the 127. phage. A\s for protein kinases from eukaryotic organisms, 22. Studier, F. W. (1973) J. Mol. Bio7. 79, 227-236. 23. Rahmsdorf, H. J., Herrlich, P., Tao, M. & Schweiger, M. histone is a good acceptor substrate for T7-induced kinase. (1973) in "Cell-Virus Interactions," Advances in the Bio- The exsistence of endogenlous acceptor substrates is shown in sciences, eds. Raspe, G. & Bernhard, S. (Pergamon Press- vivo (Fig. 1) and in vitro (Table 1). The identity of most phos- Vieweg, Braunschweig), Vol. 11, pp. 219-232. Downloaded by guest on September 24, 2021