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Phosphorylation by Cellular Casein Kinase II Is Essential For Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6570-6574, July 1992 Biochemistry Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P (protein phosphorylation/casein kinase il/transcripfion) SAILEN BARIK* AND AMIYA K. BANERJEE Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-5069 Communicated by W. K. Joklik, April 3, 1992 ABSTRACT We have previously shown that phosphory- kinases revealed that they were distinct and different from latfon of vesicular stomatitis virus (VSV) phosphoprotein P by each other. In this communication, we report the purification cellular protein kinase activity is an essential prerequisite for its and detailed characterization of the cellular protein kinase transcriptional function. We have now purified this protein and show that it is a single enzyme with properties identical kinase by monitoring its ability to phosphorylate bacterially to that ofcellular casein kinase II (CKII). Phosphorylation of expressed, unphosphorylated P protein. Biochemical studies the phosphate-free P protein (PO) by purified CKII and L showed that the kinase is indi uihable from casein kinase kinase in vitro fully restored transcriptional activity of P H, a ubiquitous cyclic AMP-independent protein kinase pres- protein. ent in a wide variety ofeukaryotic cells and tissues. Functional VSV transcription could be reconstituted with viral L protein, N-RNA template, and P protein phosphorylated by either MATERIALS AND METHODS purified cellular protein kinase or purified casein kinase Il. The Viral (VSV, New Jersey serotype, Ogden subtype) L protein unusual role of casein kinase II in the transcription process of and N-RNA template and bacterially expressed P protein of a nonsegmented negative-strand RNA virus would have im- the same serotype were purified as described (3). CKII, portant implications in host-virus interactions and antiviral purified from bovine testis, was a kind gift from Edwin G. therapy. Krebs and David Litchfield (Howard Hughes Institute, Se- attle). Oligopeptides were synthesized in a Beckman 990B The transcription complex of vesicular stomatitis virus automated solid-phase peptide synthesizer and purified (VSV), a prototype rhabdovirus, consists of the following: through HPLC. Dephosphorylated casein, heparin, and prot- the single-stranded, nonsegmented, and negative-sense (anti- amine were purchased from Sigma. Rabbit anti-human CKII message sense) genome RNA of =11 kilobases, tightly antibody (Upstate Biotechnology, Lake Placid, NY) was wrapped with the nucleocapsid protein, N (N-RNA tem- raised against a 23-mer synthetic peptide corresponding to plate); the large protein, L; and the phosphoprotein, P. The residues 70-91 of the a (catalytic) subunit of human CKII L and P proteins together constitute the viral RNA- coupled to keyhole limpet hemocyanin. dependent RNA polymerase that transcribes the N-RNA Purification of Po Kinase (PK). The P protein phosphory- template to produce viral mRNAs (reviewed in ref. 1). lating activity (PK) was purified essentially as described for Genetic and biochemical evidence suggested that L protein CKII (4) except that the fractions were monitored by their may encode the ribonucleotidyl polymerase activity, while ability to phosphorylate bacterially expressed P protein (PO) phosphoprotein P acts as a transcription factor or a transac- as substrate. In brief, the postribosomal supernatant (S100) of tivator of the L protein. baby hamster kidney (BHK) cell extract (10 g of protein) in The precise mechanism of transactivation by phosphopro- buffer A [50 mM Tris-HCl, pH 7.5/0.1 mM EDTA/5% tein and the role of its phosphorylation status in this process (vol/vol) glycerol/0.02% NaN3/1 mM dithiothreitol] contain- have been the subject of intensive research in the past few ing 50 mM NaCl was loaded onto a 20-ml DEAE-cellulose years. We have recently cloned and expressed large quanti- column. After washing with 60 ml of the same buffer, the ties of phosphate-free P protein in Escherichia coli (2). The column was developed with a linear gradient of 0.1-0.4 M VSV transcription reaction mixture reconstituted in vitro NaCl in buffer A (total vol, 120 ml; collected in 60 fractions). with purified L protein, N-RNA template, and unphosphor- The activity peak (at 'z0.2 M NaCl) was loaded directly onto ylated P protein (P0) was found to be defective. Addition of a 3-ml phosphocellulose column, which was then washed uninfected cell extract to the transcription reaction mixture with 6 ml of buffer A plus 0.2 M NaCl and developed with a or prior phosphorylation of the P protein by the cell extract gradient of 0.2-1.0 M NaCl in buffer A (total vol, 18 ml; restored transcription, suggesting an essential role ofcellular collected in 60 fractions). The pooled fractions (at -0.6 M protein kinase-mediated phosphorylation in transcriptional NaCl) were dialyzed against buffer A plus 0.2 M NaCl and activity of P protein (3). The phosphorylated product (P,) of rechromatographed onto a second phosphocellulose column the cell kinase reaction was further phosphorylated by an L (1 ml) in a similar manner. The pooled peak was dialyzed protein-associated kinase to produce the fully phosphory- against buffer A plus 10 mM potassium phosphate (pH 7), lated form (P2). Thus, it was proposed that complete activa- chromatographed on a 0.5-ml hydroxylapatite (Bio-Rad) col- tion of P protein is mediated through a cascade phosphory- umn, and finally concentrated by batch elution in hydroxyl- lation pathway involving two protein kinase activities-the apatite exactly as described (4). cell kinase and the L kinase-acting sequentially (3). Sub- Protein Kinase Assay. Standard protein kinase reaction strate specificity and other biochemical parameters ofthe two mixtures (20 dul) contained 20 ng of kinase, 1-5 ,ug of The publication costs of this article were defrayed in part by page charge Abbreviations: PK, unphosphorylated P protein kinase; CKII, payment. This article must therefore be hereby marked "advertisement" casein kinase II; VSV, vesicular stomatitis virus. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6570 Downloaded by guest on September 29, 2021 Biochemistry: Barik and Banerjee Proc. NatL Acad. Sci. USA 89 (1992) 6571 appropriate substrate (P0 or casein), 100 juM ['y32P]ATP (or used as substrate. These elution parameters and our earlier GTP of the same concentration and specific activity, where demonstration that PK activity can utilize both ATP and GTP indicated), 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM as phosphate donor (3, 4) suggested that this activity may be MgCl2, and 1 mM dithiothreitol. Reaction mixtures were very similar to cellular CKII (7). We demonstrate (see below) incubated at 320C for 15 min, terminated by addition of SDS that the properties of this preparation are indeed identical to sample buffer, and analyzed by SDS/PAGE (5) followed by those of CKII. autoradiography. When peptide C (Arg-Arg-Arg-Glu-Glu- Both PK and CKII Phosphorylate Po and Casein. Since PK Glu-Thr-Glu-Glu-Glu) was used as substrate (6), 10 ,ul of the and CKII were originally purified by using P0 and casein as reaction mixture was spotted on a 2-cm2 piece of Whatman substrates, respectively, it was important to determine P81, which was washed five times (=30 min) in 150 mM whether the substrates and enzymes could be interchanged. H3P04, dried, and assayed for 32P radioactivity in a liquid Fig. 2 shows that both protein kinases can phosphorylate scintillation counter. For peptide P (Glu-Glu-Glu-Ala-Ser- both the substrates, although both ofthem phosphorylated Po Asp-Ser-Asp-Ala-Asp), an identical procedure was followed much more efficiently than casein. Heparin, which inhibits except that the P81 paper was substituted by Whatman DE81 PK in the crude extract (data not shown), inhibited both paper, which was carefully washed with 0.5 M Na2HPO4. It purified PK and CKII, while protamine stimulated both was necessary to use a cationic matrix for peptide P since the enzymes -3-fold. Cyclic AMP had no effect on either en- lack of basic residues in this peptide precluded its binding to zyme activity (data not shown). Essentially identical results the anionic P81 paper (data not shown). However, unreacted were obtained by using casein and Po as substrates in separate [y-32P]ATP also bound to DE81 and could only be removed kinase reactions (data not shown). We have previously by extensive washing with high salt, which also resulted in shown that unphosphorylated Po protein elutes from DEAE- some loss of the bound peptide (see Table 2). cellulose at 0.17 M NaCl (3), whereas the product of its phosphorylation by the crude cell extract P1 eluted at 0.27 M NaCl (3). Phosphorylation of P0 by purified PK or CKII in RESULTS vitro produced a similar chromatographic shift of P protein, Purifcation of the Cellular Protein Kinase Phosphorylating indicating that phosphorylation by both enzymes produces a Po. We have previously shown that extracts of uninfected similar structural alteration in P protein. BHK-21 cells contained a potent protein kinase activity that Biochemical Properties of PK and CKII Are Essentially phosphorylated bacterially expressed phosphate-free P pro- Identical. Results of a detailed study of the biochemical tein (P0) in vitro and that this phosphorylation was essential parameters of the two enzymes, such as Mg2+ optima, for transcriptional activation of P protein (3). To purify the apparent Km ofATP and GTP, stimulation by protamine, and putative protein kinase, we used an extract similar to the inhibition by heparin and 2,3-diphosphoglyceric acid (4, 7), starting material and subjected it to chromatographic proce- are listed in Table 1. It is clear that the two enzymes, purified dures.
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