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Protein Kinase and Phosphoprotein Phosphatase Activities of Nitrogen

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Protein Kinase and Phosphoprotein Phosphatase Activities of Nitrogen Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4976-4980, July 1988 Biochemistry Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: Roles of the conserved amino-terminal domain of NTRC (two-component regulatory systems/transcriptional regulation/glutamine synthetase/metabolic sensing) J. KEENER AND S. KUSTU* Department of Microbiology and Immunology, University of California, Berkeley, CA 94720 Communicated by Daniel E. Koshland, Jr., February 29, 1988 ABSTRACT The NTRC protein (ntrC product) of enteric Studies of Ronson et al. (9) indicated that the NTRB- bacteria activates transcription of nitrogen-regulated genes by NTRC system is likely to be a paradigm for a number of a holoenzyme form of RNA polymerase that contains the ntrA two-component regulatory systems in a wide variety of product (oa') as a factor. Although unmodified NTRC will eubacteria. One component of these systems, for which bind to DNA, it must be phosphorylated to activate transcrip- NTRB is an example, is thought to sense environmental tion. Both phosphorylation and dephosphorylation of NTRC stimuli and transmit information to the second component, occur in the presence of the NTRB protein (ntrB product). We for which NTRC is an example. This second component here demonstrate rigorously that it is the NTRB protein that is would then bring about an appropriate regulatory response. a protein kinase by showing that NTRB can phosphorylate Amino acid sequence identities among the proteins in these itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC- two-component systems occur in the carboxyl-terminal re- P) is capable of autodephosphorylation with a first-order rate gions of the proteins in the NTRB set (9) and the amino- constant of 0.14-0.19 min-' (t112 of 5.0-3.6 min) at 3rC. In terminal domains of the proteins in the NTRC set (9, 10). addition, there is regulated dephosphorylation of NTRC-P. By We here define the protein kinase and phosphoprotein contrast to the autophosphatase activity, regulated dephos- phosphatase activities of the NTRB and NTRC proteins of phorylation requires three components in addition to NTRC-P: Salmonella typhimurium. Though the NTRC protein has the PI, regulatory protein, NTRB, and ATP. NTRC is phos- sequences readily recognizable as an ATP-binding site (11) phorylated within its amino-terminal domain, which is con- and the NTRB protein does not, it is nevertheless the NTRB served in one partner ofa number oftwo-component regulatory protein that is a protein kinase. Surprisingly, phosphorylated systems in a wide variety of eubacteria. A purified amino- NTRC (NTRC-P) is capable of autodephosphorylation. It is terminal fragment of NTRC (-12.5 kDa) is sufficient for also subject to regulated dephosphorylation in the presence recognition by NTRB and is autodephosphorylated at the same of the PI, protein, and we compare these two phosphatase rate as the native protein. activities. NTRC is phosphorylated within its amino-terminal domain, which has all of the determinants required for Together with a holoenzyme form of RNA polymerase phosphorylation by NTRB and for autodephosphorylation as containing the ntrA product (a"4) as o- factor, the NTRC well. protein (ntrC product) of enteric bacteria activates transcrip- tion of a number of genes in response to availability of combined nitrogen (refs. 1-3, see ref. 4 for review). Ninfa and METHODS Magasanik discovered that activation of transcription was Protein Purifications. All procedures were performed at correlated with phosphorylation ofNTRC, which occurred in 4°C in a standard buffer that contained 10 mM Tris titrated to the presence of the NTRB protein (ntrB product) and ATP pH 8.0 with HCl or acetic acid, 50 mM KCl, 0.1 mM EDTA, (5). Consistent with genetic studies, preliminary biochemical 5% (vol/vol) glycerol, and 1 mM dithiothreitol. Protein studies indicated that the balance between phosphorylated concentrations were determined by the BCA (bicinchoninic and unphosphorylated forms of NTRC was controlled by the acid) method (Pierce), after dialysis of samples into buffer PI, regulatory protein (5). This protein, which has been lacking dithiothreitol; bovine serum albumin was used as studied extensively by Stadtman, Rhee, and their colleagues, standard. has no enzymatic activity but rather functions as a protein NTRB was overproduced from the A phage PL promoter allosteric effector of the bifunctional enzyme adenylyltrans- (12) in a plasmid identical to one described in ref. 1 (pJES 45), ferase/adenylyl-removing enzyme to control the degree of except that the ntrC524 allele was introduced to prevent covalent modification and thereby the catalytic activity of synthesis ofNTRC (8). Two hours after heat induction NTRB glutamine synthetase (6, 7). Thus, PII, which is present in constituted -0.05% of total cell protein. After disruption of large amounts under conditions of excess nitrogen availabil- cells (175 g of wet paste) in a French pressure cell and ity, has two coordinated effects-it causes a decrease in centrifugation, NTRB was precipitated with ammonium sul- transcription of the ginA gene, which encodes glutamine fate (0-45% saturation). It was then chromatographed on a Q synthetase, and it causes a decrease in glutamine synthetase Sepharose fast-flow column (180 ml; Pharmacia), from which catalytic activity (reviewed in ref. 8). Under conditions of it eluted at -250 mM KCI in standard buffer (gradient from 50 limiting nitrogen availability, PI, is covalently modified by a to 500 M KCI). Active fractions were pooled, brought to 550 metabolic sensing protein that uridylylates it, and the fully mM KCI, and applied directly to a phenyl-agarose column uridylyated form has effects that are essentially opposite to equilibrated at the same KCl concentration (15 ml; Bethesda those of the free form (6, 7). Abbreviations: NTRB and NTRC, products of ntrB and ntrC genes, The publication costs of this article were defrayed in part by page charge respectively; NTRC-P, phosphorylated NTRC; TCA ppt., trichloro- payment. This article must therefore be hereby marked "advertisement" acetic acid precipitate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4976 Downloaded by guest on September 27, 2021 Biochemistry: Keener and Kustu Proc. Natl. Acad. Sci. USA 85 (1988) 4977 Research Laboratories). After extensive washing followed by samples were removed for precipitation with trichloroacetic a 150-ml gradient decreasing the KCI concentration to 50 mM, acid as above. Alternatively, dephosphorylation was mea- NTRB was step-eluted with 20%o (vol/vol) glycerol in standard sured after destroying the ATP in a phosphorylation assay buffer that lacked KCl. The active fractions were applied to a mixture with ATPase (3.25 units/ml final concentration; Mono Q FPLC column (1 ml; Pharmacia), and NTRB was Sigma). Dephosphorylation of purified NTRC-32P was mea- eluted at -250 mM KCl (shallow KCI gradient of 410 sured in the assay buffer described above in the presence of mM/ml). Only the earliest, most-purified, fractions, which the additional components indicated. contained -30% of the total activity, were pooled, concen- trated and fractionated by molecular size on a Superose 12 column (90 ml; Pharmacia). NTRB was eluted at a position RESULTS corresponding to a molecular mass of =70 kDa (i.e., as a Purified NTRB alone was labeled in the presence of [- dimer) and was greater than 95% pure as assessed by 32P]ATP (Fig. 1, experiments 1-3), indicating that it was a NaDodSO4/PAGE. Small samples (23 ttg/ml = 290 nM protein kinase. Purified NTRC alone was not labeled (Fig. 1, dimers) were stored at - 70'C. During purification NTRB was was heavily labeled in the presence of assayed by its ability to stimulate expression from the ginA experiment 4). NTRC promoter in a coupled transcription-translation system (8). NTRB (Fig. 1, experiments 5 and 6), indicating that it was a NTRC was purified essentially as described (1) from an substrate for NTRB. Interestingly, when prelabeled NTRB overproducing strain bearing a plasmid that lacked a func- was added to NTRC, not only was NTRC labeled but much tional ntrB gene. Briefly, NTRC was precipitated with of the label was lost from NTRB within 25 sec (Fig. 1, ammonium sulfate (0-35% saturation) and was then chro- experiment 6 versus experiment 2). This appears consistent matographed on DEAE-agarose (Bio-Rad), heparin-agarose with the possibility that phosphorylated NTRB is an inter- (Bethesda Research Laboratories), and Superose 12. The mediate in the phosphorylation of NTRC, but we have not purified protein (0.68 mg/ml = 6.2 ttM dimers) was greater tested this possibility further. To confirm that only the 'y than 95% pure. phosphate of ATP was transferred to NTRC, we used Phosphorylated NTRC (NTRC-32P), generated in a stan- [a-32P]ATP in place of the y-labeled nucleotide and demon- dard phosphorylation reaction (see below), was separated strated chromatographically that [a-32P]ADP was released from NTRB and ATP by chromatography on heparin-agarose (not shown). After partial acid hydrolysis of NTRC-P, we at 40C. After the column was washed with 30 vol of standard buffer containing 100 mM KCI, NTRC-32P (and NTRC) were 1 2 3 4 5 6 7 8 eluted at 1 M KCI. Radiolabeled fractions were pooled and a b a b a b a b a b a b c dialyzed twice against 200 vol of standard buffer for 15 min. Both unmodified protein and PI,-(UMP)4 (13) were - ---NTRC PI, ---42.5 generous gifts from Sue Goo Rhee (National Institutes of NTRC--- U-- Health). Limited Proteolysis. Proteolysis of NTRC was carried out NTRB--- __ for 2-5 min at 370C with immobilized trypsin that had been treated with tosylphenylethyl chloromethyl ketone (125 units/ ml gel; Sigma); the trypsin was diluted with Sephadex G-25 (Pharmacia).
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