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). Since the amino-terminal fragment of NTRC (=12.5 kDa) did not adsorb to heparin-agarose, the large O ---12.5 carboxyl-terminal fragment (=42.5 kDa) and residual undi- gested NTRC were separated from it by adsorption to this FIG. 1. Labeling of NTRB or NTRC in the presence of [- matrix. After concentration by ammonium sulfate precipita- 32P]ATP (experiments 1-6) and partial tryptic digestion of NTRC-32P tion (70% saturation), the amino-terminal fragment was (lanes 7 and 8). Experiments 1-5: Phosphorylation reactions were further purified by sieving on a Superose 12 column. The first initiated with [y-32P]ATP (0.15 mM final concentration; 6650 nine amino acids of this fragment were determined by Edman cpm/pmol) in mixtures containing 29 nM, 58 nM, or 116 nM NTRB dimers (experiments 1-3, respectively); 2.5 AuM NTRC dimers degradation at the University of California San Francisco (experiment 4); or 116 nM NTRB and 2.5 AuM NTRC (experiment 5). Biomolecular Resource Center; they matched exactly the Samples were removed for electrophoresis at 2 min (a lanes, amino-terminal amino acid sequence of native NTRC. The experiments 1-5) or 10 min (b lanes, experiments 1-5). Samples were high yield of amino-terminal fragment upon partial proteoly- initially mixed with an equal volume of buffer appropriate for sis is consistent with its designation as a structural domain NaDodSO4/PAGE (15) and quick frozen. They were later subjected (10). to electrophoresis in a 10o polyacrylamide gel (15) without prior Assays. A standard phosphorylation reaction mixture con- boiling. After the gel was stained with Coomassie blue, it was dried tained 57 mM Tris-HC1 at pH 8.0, 40 mM KCI, 0.1 mM and then exposed to X-AR film (Kodak) for 18 hr at - 70'C with one EDTA, 4% (vol/vol) glycerol, and 0.8 mM dithiothreitol. intensifying screen. Inspection of the stained gel indicated that lane lb was underloaded. Experiment 6: After separate incubation of Concentrated ATP was made equimolar in MgCl2, and was NTRB and NTRC with [y-32P]ATP (experiments 3 and 4), the then diluted and added to carrier-free [y-32P]ATP (5000 Ci/ reaction mixtures were combined in a 1:1 ratio to give a final NTRB mmol; Amersham) to yield a 10-fold concentrated stock concentration of 58 nM and an NTRC concentration of 1.2 tiM; solution (1.5-5.0 mM; see figure legends). After addition of samples were removed for electrophoresis at 25 sec, 75 sec, and 9 min NTRC and then NTRB to the buffer, the reaction mixture 30 sec (lanes 6 a-c, respectively). The separate incubations were was warmed for 4 min at 37°C and the reaction was then carried out for 12.5 min (NTRB; experiment 3) or 1 min (NTRC; initiated by addition of 0.1 vol of stock ATP. At the times experiment 4) before the reaction mixtures were combined. Lanes 7 indicated 10-,ul samples were withdrawn and spotted onto and 8: After a standard phosphorylation reaction the assay mixture Whatman ET31 filters (2 cm square), which were washed was added to immobilized trypsin in the presence of excess unlabeled ATP. Two minutes later a sample was frozen and then subjected to with trichloroacetic acid, and the radioactivities of the electrophoresis in a 15% polyacrylamide gel as described above. This precipitates (TCA ppt.) were measured as described (14). gel was stained with Coomassie blue (lane 7) and subsequently Dephosphorylation of NTRC-32P generated in a standard exposed to film as above (lane 8) to visualize unlabeled as well as phosphorylation reaction was measured after adding an labeled fragments of NTRC. The apparent molecular masses (kDa) excess of unlabeled MgATP to the assay mixture to increase of the fragments were determined more precisely on other gels the ATP concentration by 20- to 25-fold (see figure legends); containing different percentages of acrylamide. Downloaded by guest on September 27, 2021 4978 Biochemistry: Keener and Kustu Proc. Natl. Acad. Sci. USA 85 (1988) detected phosphoserine by two-dimensional thin-layer elec- A B trophoresis (ref. 16; results not shown). 4- Because virtually all of the radiolabeled protein in a 4.0- NTRC-P dilution 0 reaction was NTRC-P (Fig. 1, experiments 0P NTRC-P phosphorylation u 04 3. 5 and 6), we quantitated it by using a standard filter-binding 1:1.1 0 dilution to 3.5- -4 assay that measures radioactivity precipitated by trichloro- 0. 1:1.5 1:1.1 acetic acid (ref. 14; see Methods). Using this assay, we HaI 1:3 confirmed published results (5) indicating that the initial rate cs 3.0- 2" ofphosphorylation ofNTRC was proportional to the amount 1:6 of NTRB (Fig. 2). Surprisingly, accumulation of NTRC-P, 1:10 2.5T, mm Ii I which ceased after 12-15 min, was also dependent on the 0 2 468 amount of NTRB (Fig. 2). Similarly, when we held NTRB 0 2' 4 6 constant but varied its activity by limiting the concentration Time, min Time, min Km greater than 0.5 mM), both ofATP (0.1-1.0 mM; apparent FIG. 3. Rates of dephosphorylation of NTRC-32P at different the initial rate of synthesis of NTRC-P and its accumulation dilutions. (A) NTRC-32P was synthesized in a standard phosphoryl- were proportional to the ATP concentration-i.e., to NTRB ation reaction mixture in which NTRC was 1.6,uM, NTRB was 58 activity (not shown). nM, and ATP was 0.5 mM (2530 cpm/pmol). After an incubation of Preliminary characterization of the kinase activity of 15 min the reaction mixture was diluted as indicated in the figure into NTRB indicated that the maximal rate of phosphorylation of 37°C standard buffer plus unlabeled MgATP to give a final ATP NTRC was -5 mol/mol of NTRB dimers per min at an ATP concentration of 13 mM. Samples were removed at various times and concentration of 2 mM. The maximal extent of NTRC precipitated with trichloroacetic acid. The apparent first-order rate constant for dephosphorylation was 0.14 min-' (01/2 = 5.0 min). phosphorylation was -1 mol/mol of NTRC dimers, which Assuming that there was 1 mol ofphosphate incorporated per NTRC was achieved with a large amount of NTRB (58 nM), high dimer, we calculate that the concentration of NTRC-32P was 0.5 ,AM concentrations of ATP (1.25 mM), and a subsaturating level before dilution. (B) NTRC-32P that had been separated from NTRB ofNTRC (0.19,M). The pH optimum for phosphorylation of and ATP was diluted as indicated into 37°C standard buffer plus 1 mM NTRC was -8.3. Neither the initial rate of synthesis of MgCl2. The apparent first-order rate constant for dephosphorylation NTRC-P nor its steady-state accumulation were affected by was 0.19 min-' (1/2 = 3.6 min). Prior to dilution NTRC-32P was 95 the metabolites glutamine or 2-oxoglutarate, by the fully nM (assuming one phosphate per dimer; 1895 cpm/pmol). uridylylated form of the PI, protein [PI, (UMP)4], or by synthesis of NTRC-32P for 15 min and then made several PII-(UMP)4 in combination with 2-oxoglutarate (data not mixture into excess unlabeled ATP. shown). dilutions of the reaction of NTRC-P ceased be- The amount of NTRC-32P decreased exponentially with time We suspected that accumulation at a rate that was independent ofdilution over the 9-fold range cause an opposing activity dephosphorylated it. To test for a such an activity in the presence of the kinase, NTRB, we assayed (Fig. 3A). We obtained the same result over 6-fold made two modifications of the assay. After allowing phos- range of concentrations with NTRC-32P that had been puri- phorylation (radiolabeling) of NTRC for a period of time, we fied free of NTRB and ATP as described in Methods (Fig. decreased the specific activity ofthe [y-32P]ATP substrate by 3B). As expected, reconstitution of the original reaction adding an excess of unlabeled ATP (see Methods); this made mixture by addition of NTRB and ATP to the purified the assay insensitive to subsequent kinase activity and NTRC-32P had no effect on the rate of dephosphorylation allowed us to determine whether label could be "chased out" (Fig. 4B), again indicating that neither was required. The of NTRC-32P. Alternatively, we destroyed the kinase sub- labeled product released from NTRC-32P was chromato- strate with ATPase and monitored loss of label from NTRC- graphically identified as inorganic phosphate (not shown). 32p. In both cases the amount of NTRC-32P decreased We will refer to this dephosphorylation reaction as the exponentially with time. The apparent first-order rate con- autophosphatase activity of NTRC-P. stant, -0.14 min- 1 at 37°C, was the same in the presence of Preliminary characterization ofthe autophosphatase activ- ATP (Fig. 3) as in its absence (not shown). To determine ity revealed that it was insensitive to variation ofpH between whether dephosphorylation was truly first-order and thus 3.8 and 9.5; the first-order rate constant was 0.17 (±0.01) occurring by an intramolecular mechanism, we allowed min-1 at pH values of 3.8, 5.5, 6.1, 6.9, 8.0, and 9.5. Autophosphatase activity was decreased to less than 4% at 10 pH 1.5, thus validating the use of trichloroacetic acid pre- cipitation of NTRC-32P. The autophosphatase activity was 8- also slowed to ~=12% at 4°C and was inhibited :=50% by

0. EDTA. 6- In addition to the autophosphatase activity of NTRC-P, there is regulated dephosphorylation of NTRC-P (5). When _0. added to a phosphorylation reaction the PI, protein (see 4. introduction) causes net dephosphorylation of accumulated NTRC-P (5). This could be due to inhibition of the kinase activity ofNTRB, resulting in net autophosphatase activity of NTRC-P, or it could be due to a more direct stimulation of dephosphorylation. We found that PI, did not inhibit the 0 10 20 30 initial rate ofphosphorylation of NTRC, indicating that it did Time, min not inhibit the kinase activity of NTRB (not shown). By contrast, PI1 did stimulate the rate of dephosphorylation of FIG. 2. Time courses of NTRC phosphorylation at different NTRC-32P in a "cold chase" experiment (not shown). It did concentrations of NTRB. Reactions were carried out at an NTRC not do so when ATP had been hydrolyzed, indicating that concentration of 1.1 ,uM dimers, an ATP concentration of 0.27 mM (2227 cpm/pmol), and the NTRB concentrations indicated in the ATP was required for regulated dephosphorylation (not figure. We calculate that the maximal extent of phosphorylation in shown). Dilution experiments of the sort done to study the this experiment was 0.36 mol of phosphate per mol of NTRC dimers. autophosphatase activity indicated that NTRB was also Downloaded by guest on September 27, 2021 Biochemistry: Keener and Kustu Proc. Natl. Acad. Sci. USA 85 (1988) 4979

required for regulated dephosphorylation (not shown). To A B confirm the requirements for regulated dephosphorylation we studied the dephosphorylation of purified NTRC-32P. To Native 4.2 obtain the regulated activity, which was considerably more C- rapid than autodephosphorylation, three components had to x uL I to 3.8 be added to purified NTRC-32P: PI, NTRB, and ATP (Fig. * Native

4). When any one of these components was omitted, the rate L . 0 of dephosphorylation was not stimulated above that of the U00' 3.4- autophosphatase (Fig. 4). We demonstrated chromatograph- N-term ically that the labeled product released in the PI1-stimulated v reaction was inorganic phosphate. We will refer to the 3.0- I42 PI,-stimulated reaction as regulated phosphatase activity (but O 10 20 I0 2 4, 6 see Discussion). Time, min Time, min Preliminary characterization of the regulated phosphatase in "cold chase" experiments revealed that it had a pH FIG. 5. Phosphorylation and dephosphorylation of the purified optimum of ==7.7 and was much more sensitive to changes in amino-terminal domain of NTRC. (A) Time courses for phosphoryl- pH than was the autophosphatase activity of NTRC-P (this ation of NTRC (1.2 uM) or an amino-terminal fragment of NTRC generated by limited digestion with trypsin and purified (estimated was also true of the kinase activity of NTRB). Regulated concentration 1 ,uM). Phosphorylation was measured at an NTRB phosphatase was unaffected by glutamine or by PII-(UMP)4, concentration of 58 nM and an ATP concentration of 0.15 mM (3220 which was added in amounts equal to those of free PI, (data cpm/pmol). (B) Time courses for autodephosphorylation of NTRC- not shown). We had several preliminary indications that or the phosphorylated amino-terminal fragment of NTRC. To regulated phosphatase activity was sensitive to or dependent initiate the reactions, portions of the reaction mixtures used for A on some aspect ofNTRC conformation that autophosphatase were withdrawn after 12 min of incubation and added to excess was not. For example, one mutant NTRC protein that we unlabeled ATP (final concentration 3.0 mM). Samples were then have studied [ntrC6J0 product (8)] was phosphorylated at a removed at various times for precipitation with trichloroacetic acid. normal rate and had autophosphatase activity; however, its The first-order rate constants for dephosphorylation of native NTRC dephosphorylation was not stimulated by PI, (not shown). and the amino-terminal fragment were 0.17 minm and 0.18 minm- Similarly, when we used heat-denatured wild-type NTRC respectively. (heated to 90'C for 5 min, then immediately chilled to 40C), Methods), radiolabel was found in the amino-terminal 12.5- the initial rate of phosphorylation and the rate of autode- kDa fragment and in residual undigested NTRC-32P but not phosphorylation were indistinguishable from those for unde- in the large (42-kDa) carboxyl-terminal fragment (Fig. 1, natured NTRC, but the heat-denatured protein was largely lanes 7 and 8). After the addition of excess unlabeled ATP, insensitive to PI,-stimulated dephosphorylation. radioactivity in the amino-terminal fragment decreased with It had been hypothesized that the amino-terminal domain time, as did that in the of NTRC, which is conserved in one member of a number of undigested protein (not shown). These two-component regulatory systems, might contain the site of results were consistent with the view that the amino-terminal phosphorylation ofthe protein (9, 10). We suspected from our fragment had autophosphatase activity. To further test this preliminary characterization of the autophosphatase activity idea, we purified the amino-terminal fragment as described in of NTRC-P that this domain might contain all of the deter- Methods and used the purified fragment as a substrate for minants necessary for autodephosphorylation as well. To test phosphorylation. The fragment was efficiently phosphoryl- these possibilities, we first determined whether NTRC was ated (Fig. 5), indicating that it alone is sufficient for recog- phosphorylated in its amino-terminal domain. When NTRC- nition by NTRB. After the addition of unlabeled ATP the 32p was subjected to partial proteolysis with trypsin (see fragment was dephosphorylated at the same rate as native NTRC (Fig. 5), indicating rigorously that it contained the A B autophosphatase activity. PII+ NTRB DISCUSSION 3.5- S 3.0- The NTRB protein of S. typhimurium is a protein kinase, as XPII+A Q * no t evidenced by its ability to phosphorylate itself. The NTRC to o addition 0. protein is a substrate for NTRB C.) 2.5- (5) and is phosphorylated 0. 2.5- 04 within its amino-terminal domain. An amino-terminal frag- F-0. II+ ment of NTRC (-12.5 kDa) was utilized as a substrate nearly 2.0- PII+ N RB NTRB NTRB +ATP as efficiently as native NTRC, which indicates that this E +ATP +ATP fragment contains all of the determinants necessary for 1.5 . 1.5 . I . ' recognition by NTRB as well as the target for phosphoryl- 0 2 4 6 8 0 2 4 6 8 10 ation; the remainder of NTRC, including its putative ATP- 1Time, min Time, min binding site, was dispensable for phosphorylation. Because phosphoserine was detected after partial acid hydrolysis of FIG. 4. Requirements for regulated (rapid) dephosphorylation of NTRC-P, we infer that NTRB is specific for serine and purified NTRC-32P. NTRC-32P that had been separated from NTRB potentially threonine. NTRB does not have easily recogniz- and ATP was added to complete or pairwise combinations of NTRB, able sequence similarities to eukaryotic protein kinases (11). P.,, and ATP in assay buffer, as indicated. Samples were removed at Another member ofthe NTRB set of regulatory proteins, the various times for precipitation with trichloroacetic acid. The initial CheA protein ofEscherichia coli, is also a protein kinase (17). concentrations of NTRC-32P used in A and B were 220 nM (4220 Purified NTRC-P autodephosphorylated with a first-order cpm/pmol) and 95 nM (1895 cpm/pmol), respectively. The concen- -1 tration ofMgATP (when the ATP was present) was 1.5 mM (A) or 1.0 rate constant of0.14-0.19 min at 37°C, which corresponds to mM (B), that of NTRB was 29 nM (A and B), and that of P., was 4.5 a half-life of 5.0-3.6 min. Neither phosphoserine nor any ,uM (A) or 5.0 yIM (B). The "no addition" control (B) contained 1 mM other acid-stable phospho amino acid is so uniformly labile MgCl2. Note that in A the best-fit lines for the PI, + NTRB and PI, between pH 3.8 and pH 9.5 as was NTRC-P (16), so we + ATP time courses are superimposed. conclude that the autophosphatase reaction is not simply due Downloaded by guest on September 27, 2021 4980 Biochemistry: Keener and Kustu Proc. Natl. Acad Sci. USA 85 (1988) to chemical instability of the phosphorylated residue. Like members of the NTRC set of regulatory proteins will also be the site of phosphorylation of NTRC, the site of autophos- phosphorylated within their amino-terminal domains by their phatase activity was localized to an =12.5-kDa amino-ter- partners (9, 10) and that these domains will have autophos- minal fragment. phatase activity. Genetic evidence is consistent with the view NTRC-P was subject to regulated dephosphorylation that that members of the NTRB set of proteins can modulate the required the PI, protein, NTRB, and ATP (summarized in activity ofmembers ofthe NTRC set other than their partners Fig. 6). The concentrations of PI, required for regulated with low efficiency-i.e., there is "cross-talk" between dephosphorylation of NTRC-P were =100 times higher than two-component regulatory systems (9). We have recently those required for efficient control ofthe activity ofadenylyl- used NTRB to phosphorylate another protein in the NTRC transferase (13); we do not know the significance of this. set, the SPOOA protein ofBacillus subtilis (J.K., D. Arnosti, Regulated phosphatase activity was distinguishable from the S.K., and M. J. Chamberlin, unpublished results), and we autophosphatase not only by its multiple requirements but have observed phosphatase activity of the phosphorylated also by its faster rate and relative sensitivity to pH. We will protein. be interested in determining the mechanism for regulated Note Added in Proof. The phosphorylated amino-terminal domain of phosphatase activity-in particular, whether NTRB func- NTRC is sufficient as a substrate for regulated dephosphorylation; as tions directly as a phosphoprotein phosphatase and is there- for native NTRC-P, this activity requires PI,, NTRB, and ATP. fore a bifunctional enzyme, or alternatively, whether the We thank Danny Szeto for expert technical assistance and Jack autophosphatase activity ofNTRC-P is utilized for regulated Kirsh, Donal Walsh, Sue Goo Rhee, Terry Leighton, Dave Popham, dephosphorylation. Like our regulated phosphatase, the and David Weiss for helpful suggestions during the course of the phosphoprotein phosphatase activity of the bifunctional en- work and for critical review of the manuscript. This work was zyme that controls the catalytic activity of isocitrate dehy- supported by Grant GM38361 from the National Institutes of Health drogenase in enteric bacteria is also ATP dependent (18). to S.K, As discussed above, a purified amino-terminal fragment of 1. Hirschman, J., Wong, P. K., Sei, K., Keener, J. & Kustu, S. NTRC is efficiently phosphorylated by NTRB and contains (1985) Proc. Natl. Acad. Sci. USA 82, 7525-7529. all of the determinants required for the autophosphatase 2. Hunt, T. P. & Magasanik, B. (1985) Proc. Natl. Acad. Sci. activity of NTRC-P. Presumably, the effect of phosphoryl- USA 82, 8453-8457. ation is transmitted from the amino-terminal domain of 3. Wong, P. K., Popham, D., Keener, J. & Kustu, S. (1987) J. of the protein to control its function Bacteriol. 169, 2876-2880. NTRC to the remainder 4. Gussin, G. N., Ronson, C. W. & Ausubel, F. M. (1986) Annu. in activating transcription. We think it likely that other Rev. Genet. 20, 567-591. 5. Ninfa, A. J. & Magasanik, B. (1986) Proc. Natl. Acad. Sci. ATP ADP USA 83, 5909-5913. 6. Stadtman, E. R. & Ginsburg, A. (1974) in The Enzymes, ed. NTRB Boyer, P. A. (Academic, New York), Vol. 10, pp. 755-807. 7. Chock, P. B., Schacter, E., Jurgensen, S. R. & Rhee, S. G. (1985) Curr. Top. Cell. Regul. 27, 3-12. 8. Keener, J., Wong, P. K., Popham, D., Wallis, J. & Kustu, S. (1987) in RNA Polymerase and the Regulation ofTranscription, NTRC NTRC-P eds. Reznikoff, W. S., Burgess, R. R., Dahlberg, J. E., Gross, C. A., Record, M. T., Jr., & Wickens, M. P. (Elsevier, New York), pp. 159-175. 9. Ronson, C. W., Nixon, B. T. & Ausubel, F. M. (1987) Cell 49, 579-581. 10. Drummond, M., Whitty, P. & Wooten, J. (1986) EMBO J. 5, 441-447. 11. Ronson, C. W., Astwood, P. M., Nixon, B. T. & Ausubel, F. M. (1987) Nucleic Acids Res. 15, 7921-7934. PII 12. Remaut, E., Stanssens, P. & Fiers, W. (1981) Gene 15, 81-93. ATP 13. Son, H. S. & Rhee, S. G. (1987) J. Biol. Chem. 262, 8690-8695. 14. Corbin, J. D. & Reimann, E. M. (1974) Methods Enzymol. 38, FIG. 6. Summary diagram of the kinase and phosphatase activ- 287-290. ities of NTRB and NTRC. NTRB acts as a protein kinase to catalyze 15. Laemmli, U. K. (1970) Nature (London) 227, 680-685. phosphorylation of NTRC at the expense of the y phosphate of ATP 16. Cooper, J. A., Bartholomew, M. S. & Hunter, T. (1983) Meth- (5). NTRC-P spontaneously dephosphorylates (autophosphatase). ods Enzymol. 99, 387-402. NTRC-P is also subject to regulated dephosphorylation that depends 17. Hess, J. F., Oosawa, K., Matsumura, P. & Simon, M. I. (1987) on NTRB, the PI, protein, and ATP. The diagram is not meant to Proc. Natl. Acad. Sci. USA 84, 7609-7613. imply that NTRB is mechanistically a phosphoprotein phosphatase; 18. LaPorte, D. C. & Koshland, D. E. (1982) Nature (London) 300, our experiments do not bear on this. 458-460. Downloaded by guest on September 27, 2021