THE JOGRNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 24, Issue ofAugust 25, pp. 17754-17761, 1993 Printed in U.S.A.

Expression, Purification, Crystallization, and Biochemical Characterization of a Recombinant Protein Phosphatase*

(Received for publication, March 22, 1993)

Shaoqiu ZhuoS, James C. Clemens, David J. Hakes, David Barford§, and Jack E. Dixonq

From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 and the PWalther Cancer Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2220

A protein phosphatase (PPase) from the bacterio- phage A was overexpressed in Eschericha coli. The re- combinant enzyme was purified to homogeneity yield- ing approximately 17 mg of enzyme from a single liter of bacterial culture. Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an acti- vator. The recombinant enzyme was active toward ser- ine, threonine, and tyrosine phosphoproteins and phos- phopeptides. Surprisingly, the bacterial histidyl phosphoprotein, N R ~ I , was also dephosphorylated by the A-PPase. The A-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phos- phatases, suggesting that the A-PPase will serve as a good model for structure-function studies. Crystalliza- tion of the recombinant purified A-PPase yie!ded mono- clinic crystals. The crystals diffract to 4.0 A when ex- posed to synchrotron x-ray radiation.

Kinases and phosphatases modulate the protein phospho- rylation “status” of a cell, which in turn governs numerous fundamental biological phenomenon such as cell division and development (Hunter, 1987; Walton and Dixon, 1993). Kinases have been classified according to their substrate specificity, being either tyrosine kinase, serinehhreonine kinases, or dual specificity kinases (which have both tyrosine as well as serine/ threonine kinase activities), Although the specificity of the ki- nases differ, they are all structurally related (Hunter, 1987).

Phosphatases have also been classified according to their substrate specificity. Members of the protein tyrosine phospha- tase family have an active site cysteine residue and mechanis- tically proceed through a thiol-phosphate enzyme intermediate (Guan and Dixon, 1991). Dual specificity phosphatases with activities toward serinehhreonine as well as tyrosine phos- phate were initially described in vaccinia virus, but other cell cycle proteins such as p8OCdcz5 also show dual specificity (Guan et al., 1991; Dunphy and Kumagai, 1991; Gautier et al., 1991; Millar et al., 1991). The dual specificity phosphatases are struc- turally related to the protein tyrosine phosphatases and also use an active site cysteine residue in catalyses.’

* This work was supported in part by Grant NJDDKD 18849 from the National Institutes of Health (to J. E. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This

with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked “aduertisement” in accordance

$ Recipient of a fellowship from the Scottish Rite Schizophrenia Re- search Foundation.

7 To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 5416 Medical Sci- ence I, Ann Arbor, MI 48109-0606. Tel.: 313-764-8192; Fax: 313-763- 4851.

K.-L. Guan and J. E. Dixon, unpublished data.

A number of protein phosphoseryUphosphothreony1 phos- phatases (PPasesI2 have been isolated and characterized (Bal- lou and Fischer, 1986; Cohen, 1989). These enzymes are clas- sified as Types 1 (PPase l), 2A (PPase 2A), 2B (PPase 2B), and 2C (PPase 2C). This nomenclature is based upon their sensi- tivity to natural occurring inhibitor 1 or 2 and on their prefer- ential specificity for dephosphorylation of the a or p subunit of phosphorylase kinase (Cohen, 1978; Ingebritsen and Cohen, 1983). Primary sequence analysis shows that PPase 1, PPase 2A, and PPase 2B are structurally related to one another (Fig. 1). Interestingly, several bacteria phages also have open read- ing frames, which encode proteins with structural similarity to PPase 1, 2A, and 2B (Cohen et al., 1988). The type 1 and 2 serinelthreonine protein phosphatase family is not structurally related to the tyrosine phosphatase family, which utilizes an active site cysteine in catalyses (Fischer et al., 1991).

The type 1 and 2 phosphatases are involved in many impor- tant biological processes such as the regulation of glycogen metabolism (Ingebritsen et al., 1983; Alemany et al., 1984) and muscle contraction (Chisholm and Cohen, 1988a, 198813). It was recently reported that PPase 1 and PPase 2A play a role in the cell cycle in fission yeast division and deletion of these two enzymes causes mitotic defects (Kinoshita et al., 1990). PPase 2A has also been shown to dephosphorylate and inactivate the p34cdc2-cyclin complex (Lee et al., 1991). Mutation of a PPase 1 gene blocks mitotic progression and terminates Drosophila de- velopment at an early stage (Axton et al., 1990; Dombradi et al., 1990; Gatti and Goldberg, 1991). The Drosophila retinal degen- eration C gene, which is required to prevent light-induced ret- inal degeneration, has also been identified to be a PPase (Steele et al., 1992). PPase 2B (calcineurin) was recently shown to be the target of the immunosuppressant drugs used to prevent host rejection in organ transplantation (Liu et al., 1991). Phos- phatases are also important in ion channel function. A recon- stituted epithelial chloride channel is shown to be closed upon the addition of PPase 2A and reopened by addition of Mg-ATP and the catalytic subunit of protein kinase A (Finn et al., 1992). Interestingly, PPase 2Ais found to be a target of the middle and small antigens of DNA tumor viruses (Pallas et al., 1990; Walter et al., 1990).

Although the type 1 and 2 PPases have been studied for a number of years, there is a limited amount of information avail- able on their structure and catalytic mechanism. In order to better understand the mechanism and structure of this family of enzymes, we have focused our attention on a PPase from the bacteriophage A. An open reading frame in the A genome (orf 221) was reported to encode a bacteriophage protein phospha- tase (A-PPase) (Cohen et al., 1988; Cohen and Cohen, 1989).

* The abbreviations used are: PPase, protein phosphatase; pNPP, p - nitrophenyl phosphate; PCR, polymerase chain reaction; PAGE, poly- acrylamide gel electrophoresis; DTT, dithiothreitol.

17754

A-Protein Phosphatase 17755

H P P l Y F I S P P l H P P 2 A A Y S I T 4 H C A L A l L A M B D A P P

H P P l Y F Z S P P l H P P Z A A Y S I T 4 H C A L A l L A M B D A P P

I F L S Q P I L L E L E A P I F L S Q P I L L E L E A P I L T K E S N V Q E V R C P L L M E E S N I Q P V Q T P I L R R E K T M I E V E A P M R Y Y E K I D G S K Y R N

" "_

8 9 8 9

7 9 8 3

1 2 4 46

1 2 0 120 1 1 3 1 1 0 1 7 3 71

H P P l -NRIYGFYDECKRRY-NIKLWKTFTDCFNC Y F I S P P l H P P 2 A A

-NRIYGFYDECKRRY-NIKLWKTFTDCFNC

YSIT4 T Q - V Y G F Y D E C L R K Y G N A N V W K Y F T D L F D Y T Q - V Y G F Y EECLNKYGSTTVWKYCCQVFDF

L A M B D A P P H C A L A l T E - Y F T F K Q E C K I K Y - S E R V Y E A C M E A F D S

- D G L S E R G N V N H W L L N G G G W F F N L D Y D K E I

H P P l K I F C C H G G - S P D L Q S M E Q I - - R R I M R P T g : V P D Q G L L C D Y F I S P P l K I F C C H G G - SPDLQSMEQI--RRIMRPTDVPDQGLLCD H P P 2 A A Q I F C L H G G - S P S I D T L D H I - - R A L D R L Q g V P H E G P M C D Y S I T 4 K I L C V H G G - S P E I R M L D Q I - - R V L S R A Q E V P H E G G F S D H C A L A l Q F L C V H G G - S P E I H T L D D I - - R R L D R F K g P P A F G P M C D L A M B D A P P A D E L P L I I E I V S K D K K Y V I C H A D Y P F D E Y & F G K P V D H Q Q

H P P l Q G W G E N D - - " - - - Y F I S P P l L G W G E N D - - - - - - - 2 4 1 H P P 2 A A GGWGISP-""" Y S I T 4 E A W Q V S P - - - - - - - H C A L A l G N E K S Q E H F S H N T V L A M B D A P P S Q N G I V K E I - - - - -

2 6 1 2 6 1 2 5 4 2 5 1 3 2 1 2 2 1

shaded. The full A-PPase sequence is shown. All other proteins are truncated. HPPl is human protein phosphatase 1 (Barker et al., 1990). YFISPPZ FIG. 1. Sequence similarity between A-PPase and type 1 and 2 PPase. Identical residues are blackened, and conserved replacements are

is a putative type 1 protein phosphatase encoded by the fission yeast dis2(+) gene (Ohkura et al., 1989). HPP2AA is human lung PPase 2A a catalytic subunit (Stone et al., 1988). YSIT4 is a suppressor of a HIS4 transcriptional defect in yeast and is closely related to PPase 2A(Arndt et al., 1989). HCALAl is human calcineurin Afrom human brain stem basal ganglia (Guerini and Nee, 1989). LAMBDAPP is the sequence of A-PPase (Cohen and Cohen, 1989).

The first 115 residues of this 25-kDa protein have 35% se- quence identity to the N-terminal region of PPase 1 and PPase 2A (Fig. 1). If conserved replacements are included in this com- parison, the similarity between sequences increases to 49%. This suggests that the A-PPase may have some catalytic prop- erties in common with the eukaryotic SerPThr phosphatases. Since the A-PPase has only 221 residues and is smaller than the eukaryotic Ser/Thr phosphatases, we felt it would serve as a good model for understanding the structure and mechanism of the PPase family. We describe the expression, purification, bio- chemical characterization, and crystallization of the recombi- nant A-PPase.

EXPERIMENTAL PROCEDURES Materials-Bio-Gel A-0.5m was purchased from Bio-Rad and Phenyl-

Sepharose fkom Sigma. Phosphorylated peptides were products of the Protein and Carbohydrate Facility, University of Michigan. ~ ~ 4 3 " ~ " " ~ was from Oncogene Science. Protein kinase A was the gift of Dr. M. Uhler, University of Michigan. Casein and p-nitrophenyl phosphate (pNPP) were from Fluka, and all other chemicals were from Sigma and Aldrich. NRTI and Che A were gifts from Dr. A. Ninfa and Dr. L. Ninfa, Wayne State University, Detroit, MI.

Construction ofpT7-7iLPP"The A-PPase coding sequence is present in the "arms" of gtl0 phage libraries. Two synthetic oligonucleotides (5"TTTCATATGCGCTATTACG and 5"TTTGAATTCTCATGCGCCT) were used as primers in the polymerase chain reaction (PCR, GeneAmp PCR Reagent Kit, Perkin-Elmer Cetus) utilizing DNA from a Agtl0 library as a template. One pl of the library was added t o 19 p1 of water,

and the mixture was heated to 100 "C for 5 min. Five pl of the heated sample were then used in the PCR reaction. The PCR cycle conditions were 94 "C (melt) for 1 min, 55 "C (anneal) for 1 min, and 72 "C (extend) for 1 min. The reaction was repeated for 30 cycles. The resulting PCR fragment, containing the entire coding sequence of the A-PPase, was digested with NdeI and EcoRI (sites present in the oligonucleotides) and ligated into NdeI- and EcoRI-digested pT7-7 producing the vector pT7- 7/LPP. The complete sequence of the A-PPase PCR product was deter- mined. A single nucleotide mutation resulting in A119T was discovered. The mutation probably arose due to PCR error. This mutation was corrected by site-directed mutagenesis, and both the wild-type enzyme and the A119T mutants were expressed, purified, and examined kinet- ically. There were no differences in the apparent KT,, or V,,,, values for the two enzymes from pH 6.5 to 8.5, suggesting that this mutation has no effect on catalysis. The mutation falls within a non-conserved region of the protein, and the corresponding residue in the phage 680-PPase is a serine (Cohen and Cohen, 1989).

Expression of A-PPase-The various pT7-7LPP clones were trans- formed into BLZl(DE3) cells for overexpression. Single transformed colonies were grown 16 h at 37 "C in 5 ml of 2 x YT media containing ampicillin (100 pg/ml). These cultures were then used to inoculate 1 liter of the same media, and the cultures incubated at room tempera- ture until the absorbance reached a value between 0.6 and 1.0 (600 nm). At this point, isopropyl-1-thio-P-o-galactopyranoside was added to the cultures to a final concentration of 0.4 mM and the cells were incubated 16 h at room temperature with shaking.

Purification of the Recombinant A-PPase-Isopropyl-1-thi0-p-n- galactopyranoside-induced cells from a 1-liter overnight culture were harvested by centrifugation at 4200 x g for 15 min and resuspended in 25 ml of 50 mM Tris-HC1 buffer, pH 7.5, containing 2 mM EGTA, 0.5 M

17756 A-Protein Phosphatase NaC1, and 20% glycerol. The cells were disrupted by passage through a French press at 1000 p. s. i. The lysates were then centrifuged at 145,000 g for 60 min at 4 "C, and the supernatants were applied to a gel filtration column.

Bio-Gel A-0.5m Chromatography-All procedures were carried out at 4 "C. Supernatant (25 ml) was loaded onto a Bio-Gel A-0.5m column (2.5 x 100 cm), which was equilibrated with 50 mhl Tris-HCI, pH 7.5, con- taining 1 mM EGTA, 0.5 M NaC1, and 20% glycerol. The column was eluted with the same buffer a t a flow rate of 10-12 ml/h. Fractions of 5.5 ml were collected and assayed for enzyme activity as described below. Fractions having high enzyme activity and purity (assessed by SDS- PAGE) were pooled, producing a total volume of approximately 60 ml.

Phenyl-Sepharose Chromatography-The pooled fractions from the Bio-Gel A-0.5m column were applied directly to a Phenyl-Sepharose column (2.5 x 10 cm), which had been equilibrated in 50 mM Tris-HC1, pH 7.5, containing 1 mM EGTA and 0.5 M NaCI. The column was then washed with 200-300 ml of the same buffer and subsequently 200 ml of 20 mM Tris, pH 7.5. The enzyme was eluted with 250 ml of 50 mw Tris-HCI in 50% glycerol, pH 7.5, a t a flow rate of approximately 2 mllmin. All procedures were carried out a t 4 "C. Fractions of 6 ml were collected and assayed as noted. Active fractions were pooled after as- sessing their purity (examination of SDS-PAGE) and stored at -20 "C in 50 mM Tris-HCI and 50% glycerol, pH 7.5. The enzyme maintains greater than 90% of its original activity for at least 6 months under these storage conditions. Sample for x-ray crystallization was concen- trated to >10 mg/ml by using Centriprep-10 (Amicon) at 4 "C and dia- lyzed for 16 h against a buffer of 10 mM Tris-HCI, pH 7.0,50 mM NaCI, 2 mM MnCI,, and 0.2% a-mercaptoethanol.

Preparation of ~~2PICasein-[32PITyr-casein was prepared by phos- phorylation of cy-casein (Fluka) with ~ ~ 4 3 " " ~ ' (Oncogene Science). Three hundred pl of reaction mixture, pH 7.4, with 50 mM Tris-HCI, containing 2.8 mg/ml casein, 1.3 mM [y-"PlATP, 13 mM DTT, 20 mM magnesium acetate, and 15 units of p ~ 4 3 " - " ~ ' were incubated at 30 "C for 4 h. The reaction was stopped by addition of trichloroacetic acid to final concentration of 20%. The phosphorylated casein was recovered as precipitate and was washed five times with 10% trichloroacetic acid. The precipitated ["2Plcasein was then dissolved in 0.5 ml of 50 mM Tris-HC1, pH 7.8, and extensively dialyzed against the same buffer overnight. Incorporation of ["2Pl was 0.01 mollmol of protein, and phos- phoamino acid analysis showed that only tyrosine was labeled. [3ZP1Ser- casein was prepared under the same conditions in a 1-ml reaction volume containing 50 mM Tris-HC1, pH 7.4, 2.8 mg/ml casein, 0.4 mM [y-32P]ATP, 20 mM DTT, 20 mM magnesium acetate, and 5 pg of the catalytic subunit of protein kinase A. Only serine was found to be phosphorylated following phosphoamino acid analysis.

Autophosphorylation of NR,, and Che A-Nine pg of NRll or 19.2 pg of Che A were autophosphorylated in 10 pl of 50 mM Tris-HC1, pH 8.0, containing 0.4 m~ [y-32PlATP, 60 mM KCl, and 5 mM MgC12. The reac- tion mixtures were incubated at 37 "C for 20 min and quenched by addition of 40 ml of 10 mM EDTA. Excess [Y-~~PIATP was removed by passing the 50-pl reaction mixture through a 1-ml centrifuge column of Sephadex G-25 equilibrated with 50 mM Tris-HC1, pH 7.8. The phos- phorylation yields were 12-30% for NR,,. The final concentrations of Che A and NR,, were 0.32 and 0.15 mg/ml, respectively.

Assay of Phosphatase Activity-The phosphatase activity was as- sayed in 1 ml of 50 mM Tris-HCl buffer, pH 7.8, containing 20 mM pNPP and 2.0 mM MnCl, with or without DTT. A-PPase (20-50 ng) were added to start the reactions. Increase of p-nitrophenol was monitored at 410 nm on a Beckman DU-64 Spectrophotometer a t 30 "C. The extinction coefficient of p-nitrophenol were determined a t various pH levels in order t o determine the micromoles of product produced. One unit of enzyme activity was defined as 1 pmol of pNPP hydrolyzed/min. The assays for [32PlSer-casein were performed in 300 pl of a solution con- taining 50 mM Tris-HC1, pH 7.8,0.08 mM MnCI,, 5 mM DTT, 0.089 pg/ml A-PPase, and different amounts of [52PlSer-casein a t 26 "C. Forty pl of the reaction mixture was quenched with trichloroacetic acid added to a final concentration of 20% at the times specified. The casein was recov- ered by centrifugation and washed with 100 pl of 10% trichloroacetic acid three times. The supernatants were combined and counted for The precipitated casein was dissolved in 200 p1 of 0.1 M NaOH and also counted for 32P. The same procedures were used to assay for dephos- phorylation of ["PlTyr-casein, except the enzyme concentration was 0.53 pg/ml. Dephosphorylation assays of phosphorylated peptides were carried out at 26 "C in 600 p1 of 50 mM Tris-HC1, pH 7.8, 2 mM MnCI, with an enzyme concentration of 1.5 pg/ml for Qr-phosphorylated pep- tide and 0.42 pg/ml for Thr-phosphorylated and Ser-phosphorylated peptides. The reaction was quenched by addition of 100 pl of reaction mixture to 100 pl of 50% trichloroactic acid at the indicated times.

Release of inorganic phosphate was determined by the molybdate assay (Fiske and Subbarow, 1925). The A-PPase used for all kinetic analyses had a specific activity between 1500 and 2500 unitsimg.

Typically a dephosphorylation assay of ["PINRII was carried out in 10-20 pl of 50 mM Tris-HC1, pH 7.8, containing 0.4 mM MnC1, and 6 mM DTT. The reaction was started by adding A-PPase. The mixture was incubated at 37 "C for 10 min and stopped by addition of 10 ml of SDS-PAGE loading buffer (100 mhl Tris-HC1, pH 8.0, containing 8 M urea, 0.4% SDS, and 10 mM EDTA). Samples were incubated at room temperature for 30-60 min and loaded on a 15% gel (Laemmli system). The gel was run a t constant voltage of 90 V for about 2 h to avoid hydrolysis by heating. After furation in 50% methanol for 20 min and 20% methanol for 20 min, the gel was sealed in a plastic bag and x-ray film used to detect the position of the radioactive proteins. Radioactive bands were cut from the gel and counted in scintillation fluid.

Crystallization-A sparse matrix method (Jancarik and Kim, 1991) was used to screen for initial crystallization conditions using the hang- ing drop vapor diffusion technique (McPherson, 1982). Protein at 10 mg/ml was mixed with an equal volume of precipitant solution and applied to silanized microscope coverslips and inverted over 1 ml of precipitant in a 24-well tissue culture tray (ICN). Trays were incubated at both 4 and 16 "C.

X-ray Diffraction-Single crystals were mounted in thin-walled glass capillary tubes (Charles Supper Co.) between plugs of mother liquor. X-ray data were collected on an Enraf Nonius FAST area detector at- tached to the National Synchrotron Light Source beamline X12C. The FAST area detector was controlled by the program, MADNES (Messer- Schmidt and Pflugrath, 1987). The wavelength was 1.0 A.

Protein Sequencing-Purified enzyme (0.1 mg) was precipitated by dialysis against distilled water and recovered by centrifugation. The material was then redissolved in 0.5 ml of 0.1% trifluoroacetic acid in 50% acetonitrile and subjected to N-terminal sequence analysis using an Applied Biosystems model 4704 ProteidPeptide Sequencer (Univer- sity of Michigan, Protein Sequencing Facility).

Other Methods-Protein concentration were determined by Coo- massie (Pierce Chemical Co.) (Bradford, 1976) and BCA(Pierce) (Smith et al., 1985) assays using bovine serum albumin as a standard. DNA sequences were determined by the dideoxy nucleotide termination method of Sanger (Sanger et al., 19771, using T7 DNA polymerase (Sequenase, U. S. Biochemical Corp.). SDS-PAGE was performed on 6.5 x 8 x 0.15-cm vertical slabs in 15% acrylamide with Laemmli system (Laemmli, 1970). Gels were stained for protein with Coomassie Blue R-250.

RESULTS

Expression and Purification of A-Protein Phosphatase (A- PPase)-Cohen and Cohen (1989) observed that infection of Escherichia coli with phage A g t l O resulted in the appearance of a Mn2+-dependent protein phosphatase activity in bacterial ex- tracts. Using two synthetic oligonucleotides as primers and a A g t l O cDNA library as a template, the complete coding se- quence of the A-PPase was obtained by PCR. The PCR fragment was inserted into a pT7-7 vector using NdeI and EcoRI restric- tion sites. Transformation of the recombinant plasmid into E. coli, BL21(DE3) cells, led to overexpression of a soluble A- PPase. The E. coli extracts prepared from the overnight culture contained a pronounced Mn2+-dependent pNPP phosphatase activity. Most of the high molecular weight impurities were removed by passing the cleared lysate through a size exclusion column. The protein phosphatase was further purified from the lower molecular weight contaminants by hydrophobic interac- tion chromatography using a phenyl-Sepharose column. The expression and purification procedures employed here are eas- ily repeatable steps, which produce pure protein in approxi- mately a 80% yield. Approximately 17 mg of the pure enzyme can be obtained from 1 liter of culture, with specific activities ranging from 2500 to 3800 unitdmg of protein. The phospha- tase activity was not particularly stable unless 50% glycerol was added to the storage buffer. The purification data from one of the preparations is shown in Table I. The purified enzyme was homogeneous, as shown by SDS-PAGE (Fig. 2). An esti- mated molecular weight of 25,000 was observed on SDS-PAGE. The calculated molecular weight of the A-PPase is 25,222.

A-Protein Phosphatase 17757 TABLE I

Purification of a protein phosphatase from bacteriophage A expressed in E. coli

s t ep Protein pNPP phosphatase activity

Purification T o t a l

mg 437

units x I O 5 77.3

unitslmg -fold 8 Crude extract 177 1 100

82 Bio-Gel A-0.5 m 76.3 932 5 95.2 Phenyl-Sepharose 17 61.4 3870 22 79.4

Yield Specific

kD 200 - 97.4 - 68.0 - 43.0 - 29.0 -

18.4 - 14.3 - 6.2 -

1 2

FIG. 2. SDS-polyacrylamide gel electrophoresis of purified A- PPase. Samples were denatured and reduced in loading buffer contain- ing 200 m~ ?tis-HC1, pH 6.8, 0.48 SDS, and 10 mM dithiothreitol. The proteins were separated by 15% SDS-PAGE and stained with Coo- massie Blue. Lane l , crude cell extract; lane 2, purified A-PPase.

Amino acid sequencing of the first 21 residues of the purified enzyme resulted in the observed sequence, MRYYEKIDG- SKYRNIWWGDL, corresponding to the expected sequence of the A-PPase.

Activation and Inactivation of A-PPase by Metal Zons-The activity of A-PPase toward pNPP, phosphoproteins, and phos- phopeptides was shown to be metal ion-dependent. The effects of metal ions on pNPP phosphatase activity are shown in Table 11. Of all the metal ions examined, only Mn2+ and Ni2+ were capable of activating the enzyme. Alkali metal ions, Mg2' and Ca2+, were neither activators nor inhibitors of the Mn2+ acti- vated enzyme activity. At a concentration of 0.2 mM, V3+, C f + , Fez+, Co2+, Pd2+, and Sn2+ salts inhibited the Mn2+-pNPP phos- phatase activity to varying degrees (838%). The salts of Sc3+, Yb3+, Cu2+, Zn2+, and Hg2' were potent inhibitors of the en- zyme, generally resulting in >90% inhibition of activity. Inhi- bition by Zn2+ was shown to competitive with Mn2+ (data not shown).

Nonmetal Zon Inhibitors of X-PPase--Almost all common oxyanions were found to inhibit the Mn2+-pNPP phosphatase activity as shown in Table 111. Among them, vanadate was the most potent inhibitor having an IC50 of 0.72 PM. Tungstate inhibited the enzyme with an IC50 of 58 p, while phosphate showed a value of 0.71 mM. Molybdate and arsenate were less potent, and their IC5@ were observed at concentration where precipitation with Mn2+ was observed. Sulfate and carbonate were also poor inhibitors. As was the case with type 1 and 2 protein phosphatases (Ballou and Fischer, 19861, fluoride in- hibited the A-PPase.

Inhibition of A-PPase by phosphate monoesters was also tested (Table IV). Phosphoenolpyruvate and adenosine 5'- monophosphate (AMP) were relatively good inhibitors with IC50 values of 2.7 and 6.7 mM, respectively. Surprisingly, phos-

TABLE I1 Activation and inhibition of A-PPase by metal ions

20 mMpNPP and metal ions as indicated a t 25 "C. Purified A-PPase (47 Activities were assayed in 1 ml of 50 mM Tris-HC1, pH 7.8, containing

ng) was added in each assay to start the reaction.

Activation Inhibition

Metal ions Activity Metal ions Activity

unitslmg 0.2 m~ Mg2' 0 0.2 m~ Mn2+ + 0.2 mM Mg2' 103 0.2 mM Ca2* 80 0.2 mM Mn2* + 0.2 m~ Ca2+ 101 0.2 mM Sc"+ 8 0.2 mM Mn2* + 0.2 mM Scz+ 6 0.2 mM V"+ 17 0.2 mM Mn2* + 0.2 mM \m+ 77 0.2 mMYb3* 0 0.2 mM Mn2* + 0.2 mM Yb3* 11 0.2 m~ Cr2+ 0 0.2 mM Mn2* + 0.2 mM C1.2' 81 0.2 mM Mn2* 1384 0.4 mM Mn2+ 100 0.2 mM Fez+ 0 0.2 m~ Mn2* + 0.2 m M Fe2* 92 0.2 m M COS' 37 0.2 mM Mn2+ + 0.2 mM Co2+ 62 0.2 mM Ni2+ 1148 0.2 m~ Mn2+ + 0.2 mM NiZ* 105 0.2 mM cu2* 0 0.2 mM Mn2* + 0.2 m~ Cu2* 2 0.2 mM Zn2* 0 0.2 mM Mn2* + 0.2 mM Zn2* 0 0.2 m~ Pd2* 20 0.2 m~ MnZ* + 0.2 mM Pd2+ 92 0.2 mM Hg2+ 0 0.2 m. Mn2* + 0.2 mM Hg2+ 0 0.2 mM Sn2* 0 0.2 m~ Mn2* + 0.2 mM Sn2* 59

%

TABLE 111 Inhibition of the Mn2+-stimulated pNPPphosphatase activity

by oxyanions The enzyme activity was assayed in 50 m~ Tris-HC1, pH 7.8, contain-

ing 20 mMpNF'P and 2 m~ Mn2* at 30 "C. Inhibitors were added to the assay buffer a t various concentrations.

Anions ICs0

r n M

Vanadate Tungstate 0.058 Phosphate 0.71 Molybdate 2.0 Arsenate >1.9 Fluoride 3.9 Sulfate 18 Carbonate >16

7.2 X 10-4

phoserine, phosphothreonine, and phosphotyrosine were not good inhibitors. Many inhibitors and stimulators of phosphoseryllphosphothreonyl phosphatases, such as histone H1, protamine, spermine, polylysines, and trifluperazine (Bal- lou and Fischer, 1986), did not have any effects on A-PPase activity. Specific and potent inhibitors of type 1 and type 2 protein phosphatases, okadaic acid (124 nM) and microcys- tin-LR (5 p), were also found to have no effects on the enzyme. The enzyme was not inhibited by the acid phosphatase inhib- itor, L-(+)-tartrate, at concentration of 5 mM.

Catalytic Properties of A-PPase-The physiological sub- strates for the A-PPase are unknown. The enzyme showed a broad range of activities toward phosphoproteins and phospho- peptides. The kinetic constants of some substrates tested are presented in Table V. As noted in Table V, the enzyme can dephosphorylate phosphoseryllphosphothreonyl- as well as phosphotyrosyl-containing substrates. The phosphotyrosyl peptide sequence corresponds to the substrate of the insulin receptor kinase (Pike and Krebs, 1986). The phosphoseryl and phosphothreonyl contain the recognition sites for kinase C

17758 A-Protein Phosphatase TABLE IV

Inhibition of the Mn"+-stimulated pNPP phosphatase activity of A-PPase by phosphate monoesters

The enzyme activity was assayed in 50 mM Tris-HC1, pH 7.8, contain- ing 20 mM pNPP and 2 mM Mn2+ at 30 "C. Inhibitors were added to the assay buffer a t various concentrations.

Compounds IC50

rnlw Phosphoenolpyruvate 2.7 Adenosine 5'-monophosphate 6.7 ~(-)-3-Phosphoglyceric acid 26 Pyridoxamine 5-phosphate 52 o-Phosphoethanolamine 63 Phosphoserine 90 Phosphorylcholine 110 Phosphotyrosine No inhibition a t 20 mM Phosphothreonine No inhibition a t 25 mM

(Heasley and Johnson, 1989; Pike and Krebs, 1986). All of these peptides were prepared by chemical synthesis, incorporating phosphate into the peptide in stoichiometric amounts. The phosphorylation of casein by p ~ 4 3 " - ~ ~ ' or the protein kinase A catalytic subunit incorporated [32P]phosphate to approxi- mately 1%. Phosphoamino acid analysis of 32P-labeled casein demonstrated that only seryl or tyrosyl phosphorylation oc- curred via protein kinase A or the v-abl kinase, respectively. Nevertheless, both of these proteins are good substrates for the enzyme. The A-PPase seems to have a preference for "protein" substrates and is capable of hydrolyzing both phosphotyrosine as well as phosphoserine-containing protein substrates. Al- though pNPP was dephosphorylated with a very high turnover rate, no hydrolysis could be detected for phosphoserine, phos- phothreonine, and phosphotyrosine under similar conditions. Because no naturally occurring substrates for the A-PPase have been described, we also examined the ability of the enzyme to hydrolyze ADP and ATP. Neither compounds were substrates (data not shown).

Characterization of Histidine Phosphatase Activity of the A- PPase-Several bacterial proteins important in gene regulation and chemotaxis undergo autophosphorylation (Stock et al., 1989). NRII and Che A are two representative proteins in this family, and they undergo autophosphorylation of a histidine residue (Ninfa and Bennett, 1991). Fig. 3 demonstrates the dephosphorylation of bacterial [32PlNR11 protein by A- PPase. The dephosphorylation of [32PlNRII by increasing con- centrations of the recombinant enzyme is shown in Fig. 3A. The enzyme catalyzed dephosphorylation of [32P1NRII was also monitored as a function of the time of incubation (Fig. 3B). Under the assay conditions used, NRII also undergoes a non- enzyme-catalyzed dephosphorylation. The rate of this reaction is also shown in Fig. 3B. The activity of the A-PPase catalyzed reaction was estimated to be 25 pmol/midmg at a substrate concentration of 180 nM. Due to the limiting amounts of L3'PI NRII, the enzyme concentration in the reaction mixture exceeds the substrate concentration making it difficult to establish that the A-PPase is indeed catalytic. Several observations, however, suggest that this is likely an enzyme-catalyzed reaction. The dephosphorylation of [32PlNRlr was dependent on the storage conditions of the phosphohistidine-containing protein. The highest histidine phosphatase activity was observed when fresh phosphorylated sample was used. Decreased dephospho- rylation could be detected after the phosphorylated [32PlNR~~ was frozen at -20 "C. Autophosphorylated NRll is known to be denatured when frozen a t -20 O C 3 This suggests that the structure of the substrate is important for the dephosphoryla- tion. Support for this suggestion was also obtained by examin-

A. Ninfa, personal communication.

ing the ability of the A-PPase to dephosphorylate the bacterial phosphohistidine-containing protein, Che A. Although Che A could be dephosphated, the apparent rate of dephosphorylation was significantly slower than the dephosphorylation of NRII (Fig. 4).

We also examined the metal dependence of the histidine dephosphorylation. Mn2+ and Ni2+ were the only two metal ions that were capable of activating the enzyme. No phosphatase enzyme activity was observed using [32P]NRII as substrate when Cu2+, Zn2+, Co2+, and Mg2+ were added to the reaction mixture. These results parallel those seen in Table 11 when pNPP was used as a substrate, suggesting that the hydrolysis of both substrates, pNPP and 132P1NR~~, has a similar metal dependence. The pH rate profile for dephosphorylation of N R I ~ was also examined (Fig. 5 ) . The optimum rate of hydrolysis is seen between pH 7.0 and 7.8, which is approximately the same pH optimum noted for pNPP. The dephosphorylation of Che A was also much slower than the dephosphorylation of NRII by A-PPase at all pH values examined (Fig. 5 ) .

Crystallization of A-PPase-The sparse matrix screening con- dition containing 0.1 M sodium citrate, 0.2 M ammonium ace- tate, 30% polyethylene glycol 4000, pH 6.3, at 16 "C yielded microcrystals within 2 days. Refinement of pH, salt, and pre- cipitant concentrations indicated optimal conditions of 0.1 M sodium citrate, pH 6.5, 24% polyethylene glycol 4000, 2 mM MnC12, and 0.3% p-mercaptoethanol. Under these conditions, crystals appear within 2 days and grow to a maximum size of 0.6 mm by 0.2 mm by 0.07 mm within a month. Crystals grow as either clusters or single crystals exhibiting a monoclinic morphology (Fig. 6).

X - r u ~ Diffraction of A-PPase Crystals-The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation. The unit cells dimensions were determined using the AUTOINDEX rou- tine of MADNES and confirmed by analysis of the diffraction images. The cell dimensions are a = 80.8 A, b = 193.2 A, c = 53.9

Systematic absences indicate that the crystal belongs to space group P21, with each asymmetric unit containing be- tween 4 and 8 molecules assuming a solvent content of between 71 and 42%, respectively, using the method of Matthews (1968).

DISCUSSION

A, a = 90.00, p = 93.30, y = 90.0".

Although a number of PPases have been isolated and char- acterized, there are limited amounts of data available regard- ing their catalytic properties. Few studies have been directed a t understanding their structure and function. The protein phos- phatase from the bacteriophage A contains only 221 amino acid residues, and for this reason it would appear to be a good model for structural studies of the PPases. Large amounts of the enzyme were obtained by the overexpression of the A-PPase protein in E. coli and the development and implementation of a simple and direct method of purification. The large quantities of pure enzyme have made crystallization efforts possible.

We were particularly interested in addressing the question: "is the A-PPase likely to be a good biochemical model for the type 1 and 2 PPases?" Biochemical studies reported here show that the A-PPase shares a number of properties with type 1 and 2 PPases. The A-PPase requires Mn2+ or Ni2+ for phosphatase activity, whereas Zn2+ and Cu2+ are inhibitory. These results mimic the metal ion dependence of calcineurin (Pallen and Wang, 1984). Indeed, all type 1 and 2 PPases can be activated by Mn2+ and inhibited by Zn2+ (Ballou and Fischer, 1986). Mg2+ has been only reported to activate type 2C PPase (Binstock and Li, 1979; Hiraga et ai., 1981; Mieskes et al., 1984; Pato and Adelstein, 1983). Unlike the other PPases, which can also use Co2+ as an activator, A-PPase is inhibited by Co2+. NaF is an non-competitive inhibitor of phosphoseryVphosphothreony1

A-Protein Phosphatase 17759

TABLE V Summary of the kinetic constants for the dephosphorylation of various substrates by A-PPase

K , and k,,, values were determined by Eadie-Hofstee plots (Eadie, 1942; Hofstee, 1959). Assay conditions were described under “Experimental Procedures.”

Substrates K“, kcat k c d L

Acetvl-RRLIEDAE (Yu)AARG-amide KR(+~)IRR-OH KRP(SD)QRHGSKY-amide [“P1Sei-cLein [32P1~r-casein pNPP Phosphoserine Phosphothreonine Phosphotyrosine

260 P M S”

4.3 1200 4 10 1000 68

0.13 0.23 0.32 0.067

7620 930 No detectable hydrolysis No detectable hydrolysis No detectable hydrolysis

SS’ PM” 0.017 0.36 0.066

0.21 1.8

0.12

0.00 0.20 0.40 0.60 0 20 40 60 80

lambda PPase (pg) Time (minutes)

FIG. 3. Phosphatase activity of A-PPase toward [32P]NEtI,. A , ”P-labeled NRII (10 pg/ml) was incubated at 37 “C for 10 min with different amounts of A-PPase in 20 ~1 of 50 mM ”is-HC1 containing 6 mM DTT and 0.4 mM MnCI2. The reactions were quenched by addition of equal volume of SDS-PAGE loading buffer, and the mixtures were subjected to electrophoresis and quantitated as described under “Experimental Procedures.” B, 110 pl of reaction mixture containing 50 mM Tris-HC1, pH 7.8,0.4 m~ MnCI,, 6 mM DTT, 180 nM [32P1NRII, and 0.05 mg/ml(1.8 p ~ ) A-PPase were incubated at 37 “C. At the time indicated, an aliquot of 10 pl was quenched with equal volume of gel loading buffer and subjected to electrophoresis as described under “Experimental Procedures.” Solid line indicates hydrolysis in the presence of A-PPase. Dashed line is autohydrolysis of I”ZP]NR1,.

PPases (Ingebritsen and Cohen, 1983; Shacter-Noiman and Chock, 1983), and it also inhibits the A-PPase. Compounds such as okadaic acid are effective inhibitors of the type 1 and type 2 PPases, but they do not affect the activity of A-PPase. This may suggest that the inhibition by okadaic acid may require amino acid residues found outside of the region of sequence similarity noted between the A-PPase and other PPases shown in Fig. 1 (or require residues that are not conserved in the A-PPase). Collectively, there are a number of similarities, as well as sev- eral differences, between the metal ion requirements and in- hibitors that affect the A-PPase and the mammalian protein phosphatases. One must therefore be cautious in concluding that the A-PPase will be a good model for all aspects of catalysis and inhibition properties of the mammalian A-PPases.

The A-PPase has a number of catalytic properties that are particularly interesting. The enzyme can hydrolyze phosphoseryllphosphothreonyl as well as phosphotyrosyl sub- strates. A similar dual substrate specificity has been demon- strated for a phosphoseryUphosphothreony1 PPase from bovine cardiac muscle (Chernoff et al., 1983). The k,,,/K, values noted in Table V clearly show that the enzyme prefers protein as

opposed to peptide substrates. This suggests that other resi- dues in addition to the phosphorylated amino acid are impor- tant for binding of enzymes and substrates.

The A-PPase was shown to dephosphorylate the phosphohis- tidyl-containing proteins, NRll and Che A, The rates of dephos- phorylation of Che A and NRI, by the A-PPase differed dramat- ically. Both NRII and Che A are protein kinases that catalyze transfer of y-phosphoryl group from ATP to the N-3 position of one of their own histidines (Weiss and Magasanik, 1988; Ninfa and Bennett. 1991; Stock et al., 1988; Hess et al., 1988). Among the family of related kinases, sequences surrounding the auto- phosphorylation sites are conserved (Stock et al., 1989). The major site of autophosphorylation of NRII is at His-139. Amino acids surrounding the site of phosphorylation are: GLApHEIK (Ninfa and Bennett, 1991). The autophosphorylation site of Che A is at His-48 (near the N terminus of the protein; Hess et al. (1988)). The sequence surrounding the phosphorylation site in Che A is RAApHSIK. We do not know if the differences in dephosphorylation rates of Che A and NRII are associated with differences in primary or three-dimensional structure. The fact that denaturation of NRII leads to a loss in dephosphorylation

17760 A-Protein Phosphatase

A B C D

N R I I

Che A

FIG. 4. Dephosphorylation of [82PlNFt~r and P2P1Che A by A- PPase. 20 pl of 32P-labeled NRIl (25 pg/ml) and che A (43 pglml) were incubated separately a t 37 "C for 10 min with 0.5 pg of A-PPase in 50 mM Ms-HCI, pH 7.8, containing 6 m w DTT and other additives as indicated. The reactions were quenched and analyzed as described in Fig. 4. Lune A, in 0.4 m~ MnClz and A-PPase was boiled for 10 min before addition; lane B, in 6 m~ EDTA, lane C, in 0.4 m~ MnC12; lane D, in 0.3 mM NiCI2.

PH 5.8 7.0 7.4 7.8 8.2 h-PPase - + - + - + - + - +

Che A

FIG. 5. Dephosphorylation of [82PlNRII by A-PPase at different pH. 20 pl of 32P-labeled NRII (25 pglml) and Che A (43 pg/ml) were incubated at 25 "C for 20 min with 0.5 pg of A-PPase in 50 mM Ms-HCI a t different pH containing 6 m~ D l T and 0.4 m MnCIz. At pH 5.8, 50 m~ sodium acetate-Ms was used instead of 50 mM Ms-HCI.

FIG. 6. Crystal of A-PPase. Conditions for crystal growth are de- scribed under "Results." Overall size is 0.6 x 0.2 x 0.07 mm.

rate suggests that three dimensional structure is likely to be important. The dephosphorylation of phosphohistidyl N R I I by A-PPase shows the same metal ions and optimal pH as noted for pNPP. This suggests that the mechanisms of the catalyses of the two substrates are likely to be similar.

Prokaryotic histidine protein kinases play very important roles in bacterial signal transduction processes. These enzymes effect rapid transient change in motility as well as long term global reorganizations of gene expression and cell morphology (Stock et al., 1989; Hess et al., 1988). More than 20 such signal transduction systems have been identified, and it is estimated that there might be as many as 50 regulatory systems present in E. coli (Stock et al., 1989,1990; Bourret et al., 1991). We have not shown that N R I 1 is the substrate for A-PPase in vivo. How- ever, the in vitro observation that demonstrates dephospho- rylation of NRIl by A-PPase raises the interesting possibility that the bacteriophage may use this enzyme to control aspects of host signal transduction mechanisms.

Acknowledgment-We are grateful to Elizabeth and Alex Ninfa for their gifts of Che A and NRII. We acknowledge Randy Stone for com- menting on the manuscript.

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