isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein...

Ž .Comparative Biochemistry and Physiology Part B 131 2002 103�112

Isolation and characterization of a protein-tyrosinekinase and a phosphotyrosine-protein phosphatase from

Klebsiella pneumoniae

R. Prenetaa, S. Jarraudb, C. Vincenta, P. Doubleta, B. Duclosa,J. Etienneb, A.J. Cozzonea,�

aInstitut de Biologie et Chimie des Proteines, Centre National de la Recherche Scientifique, Lyon, France´bDepartement de Microbiologie Medicale et Moleculaire, Faculte de Medecine R.T.H. Laennec, Lyon, France´ ´ ´ ´ ´ ¨

Received 7 August 2001; received in revised form 24 September 2001; accepted 8 October 2001

Abstract

Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in thereversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene andpurified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosinetriphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid contentrevealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the correspond-ing gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capableof cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it wasassayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, withno effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observationthat Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstratethat similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K.pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provideevidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of aregulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are bothinvolved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae,we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determinethe pathogenicity of bacteria. � 2002 Elsevier Science Inc. All rights reserved.

Keywords: Bacterial protein phosphorylation; Exopolysaccharides; Pathogenicity; Phosphotyrosine-protein phosphatase; Protein-tyro-sine kinase

� Corresponding author. IBCP-CNRS, 7 Passage du Vercors, 69367 Lyon Cedex 07, France. Tel.: �33-4-72-72-26-75; fax:�33-4-72-72-26-01.

Ž .E-mail address: [email protected] A.J. Cozzone .

1096-4959�02�$ - see front matter � 2002 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 6 - 4 9 5 9 0 1 0 0 4 9 0 - 0

( )R. Preneta et al. � Comparati�e Biochemistry and Physiology Part B 131 2002 103�112104

1. Introduction

In eukaryotes, a plethora of protein-tyrosinekinases and phosphotyrosine-protein phos-phatases that catalyze the reversible phosphoryla-tion of proteins on tyrosine residues, have beendetected and shown to play a key role in theregulation of various important biological func-tions, including signal transduction, growth con-

Žtrol and malignant transformation Fantl et al.,.1993; Hunter, 1995 . In prokaryotes, the presence

of protein-tyrosine kinase activities was sug-gested, much later than in eukaryotes, by thefinding of phosphotyrosine, first in the proteins of

Ž .Escherichia coli Cortay et al., 1986 then in theproteins of a series of other bacterial speciesŽ .Cozzone, 1993, 1997; Kennelly and Potts, 1996 .On the other hand, the occurrence of phosphoty-rosine-protein phosphatases was recently re-ported in a few examples, such as the YopH

Žprotein of Yersinia pseudotuberculosis Guan and.Dixon, 1990; Bliska et al., 1991 , the IphP protein

Žof Nostoc commune UTEX 584 Howell et al.,.1996 , the PtpA protein of Streptomyces coelicolor

Ž .Li and Strohl, 1996 , and the MPtpA and MptpBŽproteins of Mycobacterium tuberculosis Koul et

.al., 2000 . However, in bacteria, the biologicalsignificance of reversible protein phosphorylationon tyrosine is still unclear, essentially because, fora long time, no individual protein-tyrosine kinasewas characterized and no endogenous proteinsubstrate for a phosphotyrosine-protein phos-phatase was identified. The main exception so farreported concerns two proteins of Acinetobacterjohnsonii that harbor opposing activities: the Ptkprotein which has been recently demonstrated toautophosphorylate on several tyrosine residuesŽ .Duclos et al., 1996 , and the Ptp protein whichhas been identified as a phosphotyrosine-protein

Ž .phosphatase Grangeasse et al., 1998 . Moreover,in vitro experiments have shown that Ptp is ableto specifically dephosphorylate Ptk, which consti-tutes the first evidence for a reversible proteinphosphorylation reaction on tyrosine in bacteria.A similar pair of tyrosine kinase�phosphataseactivities, the Wzc�Wzb proteins, have also beendetected in another Gram-negative species, Es-

Ž .cherichia coli Vincent et al., 1999 .From these observations, it seemed interesting

to determine whether such reversible tyrosinephosphorylation systems were restricted to thebacterial genus Acinetobacter or Escherichia, or

else, were applicable to other types of bacteria aswell.

For that purpose we comparatively analyzedanother bacterial species, Klebsiella pneumoniae,which accounts for a significant proportion ofhospital-acquired urinary tract infections includ-ing pneumonia, septicemia and soft tissue infec-

Ž .tion Arakawa et al., 1995 . This species containstwo proteins, Yco6 and Yor5, which exhibit strik-ing sequence similarity with proteins Ptk and Ptpof A. johnsonii, and with Wzc and Wzb of E. coli,respectively. We checked whether such sequencerelationships were connected to functional ho-mologies. Proteins Yco6 and Yor5 are known toparticipate in the synthesis of the capsular

Ž .polysaccharide Arakawa et al., 1995 . Exo-polysaccharides are important virulence factorsand their role in forming a capsule that protectsthe pathogen from phagocytosis is well docu-

Ž .mented Podschun and Ullmann, 1998 . Yco6 isan inner-membrane protein that possesses anATP-binding domain and two predicted tran-smembrane segments, while Yor5 has an aminoacid sequence homologous to that of acid phos-phatases.

In this work, protein Yco6 was overproduced,purified to homogeneity and shown to possess anautophosphorylating activity on tyrosine. ProteinYor5 was also overproduced and purified, andfound to exhibit a protein phosphatase activitywith a strict specificity for phosphotyrosine. Thefunctional properties of these two proteins wereanalyzed and the phosphorylated form of Yco6was shown to be sensitive to dephosphorylationby Yor5, thus indicating that the Yco6�Yor5 pairof K. pneumoniae is a homolog of the Ptk�Ptppair of A. johnsonii and to the Wzc�Wzb pair ofE. coli.

2. Materials and methods

2.1. Bacterial strains and plasmids

ŽKlebsiella pneumoniae Chedid a gift from X..Nassif was used as template for PCR amplifica-

tion of the yco6 and yor5 genes. Escherichia coliBL21 and JM109 strains were used to propagateplasmids in cloning experiments. Escherichia coli

Ž .BL21 pREP4-groESL , used for expression ex-Žperiments, was previously described Amreim et

.al., 1995 ; it was a gift from I. Martin-Verstraete

( )R. Preneta et al. � Comparati�e Biochemistry and Physiology Part B 131 2002 103�112 105

Ž .Pasteur Institute, Paris . Plasmid vectors pQE30and pGEX-KT were purchased from Qiagen.

2.2. Culture media and growth conditions

E. coli strains were grown in LB or 2YT mediumat 37 �C. In the case of strains carrying drugresistance genes, the antibiotics kanamycin, ampi-cillin or tetracycline, were added to the mediumat concentrations of 25, 50, and 15 �g ml�1,respectively. Growth was monitored by measuringthe absorbance at 600 nm.

2.3. DNA manipulation

Small- and large-scale plasmid isolations werecarried out by the alkaline lysis method, andplasmids were purified by using cesium

Žchloride�ethidium bromide gradients Ish-.Horowicz and Burke, 1981 . Genomic DNA from

K. pneumoniae was prepared as describedŽ .Arakawa et al., 1995 . All restriction enzymes,calf intestine phosphatase, T4 DNA ligase andTaq DNA polymerase, were used as recom-

Ž .mended by the manufacturer Promega . Trans-formation of E. coli cells was performed as previ-

Ž .ously reported Dagert and Ehrlich, 1979 .

2.4. Construction of the yco6 and yor5 expressionplasmids

Total DNA from the K. pneumoniae strainserved as template in PCR amplification forpreparing the yco6 and yor5 genes with appropri-ate restriction sites at both ends.

For yco6 gene cloning , the sequences of thetwo primers were 5�-CGCGGATCCACTTCAA-TATCCAAAAAGCAACCAG-3� at the N-

Ž .terminus the BamHI site is italicized and 5�-TCCCCCGGGGGGGATTCTTCGTCCCCTTC-3�

Ž .at the C-terminus the SmaI site is italicized .The amplified fragment was digested with BamHIand SmaI restriction enzymes, and ligated intopGEX-KT vector, opened with the same enzymes,to yield plasmid pGEX-yco6.

For yor5 gene amplification, the followingprimers were used: 5�-CGC GGATCC TT-TAGTACAATATTAATTGTTTGCACAGGG-3�

Ž .at the N-terminus the BamHI site is italicizedand 5�-CGG GGTACCCTAAGCTTTTAATT-

ŽTCTCAGCCC-3� at the C-terminus the Kpn I.site is italicized . The synthesized DNA was re-

stricted by BamHI and KpnI, and ligated intoPQE30 vector, opened with the same enzymes.The resulting plasmid was termed pQE30-yor5.

In each case, the nucleotide sequence of thesynthesized gene was checked by dideoxynu-

Ž .cleotide sequencing Sanger et al., 1977 .

2.5. Purification of protein Yco6

Ž .E. coli BL21 pRep4-groESL cells were trans-formed with plasmid pGEX-yco6. Cells from thisstrain were used to inoculate 1 l of LB mediumsupplemented with ampicillin and kanamycin, andwere incubated at 37 �C under shaking until A600reached 0.7. Isopropyl-�-D-thiogalactopyranosideŽ .IPTG was then added at a final concentration of0.1 mM, and growth was continued for 2 h at 37�C under shaking. Cells were harvested by cen-trifugation at 3000�g for 10 min and suspended

Žin 12 ml of buffer A 10 mM sodium phosphate,pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glyc-

.erol containing 1 mM phenylmethylsulfonyl fluor-Ž .ide PMFS , and deoxyribonuclease I and ribonu-

clease A at a final concentration of 100 �g ml�1

each. Cells were disrupted in a French pressurecell at 16 000 psi. The resulting suspension wassupplemented with Triton X-100 at a final con-centration of 1% and centrifuged at 4 �C for 30min at 30 000�g. The supernatant was incubatedfor 30 min at 4 �C with glutathione Sepharose 4B

Ž .matrix Pharmacia Biotech , suitable for purifica-Ž .tion of glutathione-S-transferase GST fusion

proteins. The protein�resin complex was packedinto a column in view of washing and elution. Thecolumn was washed with 50 ml of buffer A con-taining 1% Triton X-100. Protein elution was

Žcarried out with buffer B 50 mM Tris�HCl, pH.8.0, 5 mM MgCl, 10% glycerol containing 0.1%

Triton X-100 and 10 mM glutathione. Elutedfractions were analyzed by gel electrophoresisŽ .Laemmli, 1970 . Fractions containing GST-Yco6

Žwere pooled and dialyzed against buffer C 20mM Tris�HCl, pH 8.8, 1 mM EDTA, 10% glyc-

.erol supplemented with 20 mM NaCl, and storedat �20 �C.

2.6. Purification of protein Yor5

E. coli JM109 cells were transformed withplasmid pQE30-yor5. Cells from this strain wereused to inoculate 1 l of LB medium supplementedwith ampicillin, and were incubated at 37 �C un-


der shaking until A reached 0.7. IPTG was600then added at a final concentration of 1 mM, andgrowth was continued for 2 h at 37 �C undershaking. Cells were harvested by centrifugation at3000�g for 10 min and suspended in 10 ml of

Žbuffer D 50 mM Tris�HCl, pH 7.4, 250 mM.NaCl, 10% glycerol containing 1 mM PMSF, and

deoxyribonuclease I and ribonuclease A at a finalconcentration of 100 �g ml�1 each. Cells weredisrupted in a French pressure cell at 16 000 psi.The resulting suspension was centrifuged at 4 �Cfor 30 min at 30 000�g. The supernatant was

2� Žloaded on a Zn -immobilized matrix Boeh-.ringer Mannheim , suitable for purification of fu-

sion proteins carrying a poly-histidine tag. Thecolumn was washed first with buffer D, then with50 mM imidazole in the same buffer for 5 min.Protein elution was monitored at 280 nm andeluted fractions were analyzed by gel elec-

ŽŽ .trophoresis Laemmli, 1970 . His-tagged Yor5was eluted at a concentration of 100 mM imida-zole. Fractions containing purified Yor5 were dia-lyzed against buffer E made of 50 mM Tris�HCl,pH 6.5, 100 mM NaCl, 1 mM EDTA, 10% glyc-

Ž .erol and 5 mM dithiotreitol DTT , and stored at�20 �C.

2.7. In �itro phosphorylation assay

Phosphorylation of approximately 3 �g of puri-fied GST-Yco6 protein was performed at 37 �C in10 �l of a buffer containing 25 mM Tris�HCl, pH7.0, 1 mM DTT, 5 mM MgCl , 1 mM EDTA, and2

�1 � 32 �10 �M ATP with 200 �Ci ml �- P ATP.After 10 min of incubation, the reaction wasstopped by addition of an equal volume of 2�

Ž .sample buffer Laemmli, 1970 , and the mixturewas heated at 100 �C for 5 min. One-dimensionalgel electrophoresis was performed as previously

Ž .described Laemmli, 1970 . After electrophoresis,gels were soaked in 16% trichloroacetic acidŽ .TCA for 10 min at 90 �C. They were stainedwith Coomassie Blue, and radioactive proteinswere visualized by autoradiography.

2.8. Analysis of the phosphoamino acid content ofproteins

Protein samples were separated by one-dimen-Ž .sional gel electrophoresis Laemmli, 1970 , then

electroblotted onto an Immobilon polyvinylideneŽ .difluoride PVDF membrane. Phosphorylated

proteins bound to the membrane fraction weredetected by autoradiography. The 32 P-labeledprotein bands were excised from the Immobilonblot and hydrolyzed in 6 M HCl for 1 h at 110 �C.The acid-stable phosphoamino acids thus liber-ated were separated by electrophoresis in the first

Ž .dimension at pH 1.9 800 V h in 7.8% aceticacid�2.5% formic acid, followed by ascendingchromatography in the second dimension in 2-

Ž .methyl-1-propanol�formic acid�water 8:3:4 . Af-ter migration, radioactive molecules were de-tected by autoradiography. Authentic phosphoser-ine, phosphothreonine and phosphotyrosine wererun in parallel and visualized by staining with

Ž .ninhydrin Duclos et al., 1991 .

2.9. Phosphatase assay

Acid phosphatase activity was monitored at 37�C by using a continuous method based on the

Ž .detection of p-nitrophenol PNP formed fromŽ .p-nitrophenyl phosphate PNPP . Rates of de-

phosphorylation were determined at 405 nm in areaction buffer containing 100 mM sodiumcitrate, pH 6.5, 1 mM EDTA, 0.1% �-mercaptoethanol, and PNPP at a concentrationvarying from 0.5 to 40 mM. The amount of PNPreleased was estimated by using a molar extinc-

�1 �1 Žtion coefficient � of 18 000 M cm Cirri et405.al., 1993 . The assay was optimized with respect to

protein concentration, time, and pH.Phosphotyrosine phosphatase activity was as-

sayed at 37 �C in a 50-�l reaction volume con-taining 10 mM O-phosphotyrosine as substrate, 1mM EDTA, 100 mM sodium citrate, pH 6.5, and1 �g of purified Yor5. After 15 min of incubation,the reaction was stopped by adding 150 �l of 25%

ŽTCA, then 50 �l of bovine serum albumin 10 mg�1 .ml . The precipitated protein was removed by

centrifugation, and the supernatant was used formeasurement of released inorganic phosphate byusing 1 vol of a mixture containing 1.2 M sulfuricacid, 0.5% ammonium molybdate, and 2% ascor-bic acid. Samples were heated at 56 �C for 15 minand the absorbance was measured at 750 nmŽ .Chen et al., 1956; Mustelin et al., 1989 .

2.10. Yco6 dephosphorylation assay

In vitro phosphorylation of approximately 1 �gof purified Yco6 protein was performed as de-scribed above. After 10 min of incubation, de-


phosphorylation of Yco6 was carried out with 1�g of purified Yor5, at 37 �C for 5�30 min in 30�l of buffer consisting of 100 mM sodium citrate,pH 6.5, and 1 mM EDTA. The reaction wasstopped by addition of an equal volume of 2�

Ž .sample buffer Laemmli, 1970 , and the mixturewas heated at 100 �C for 5 min. The Yco6 proteinwas separated by gel electrophoresis, treated withTCA and analyzed by autoradiography. The ra-dioactive bands were excised and their radioactiv-ity was counted in a liquid scintillation spec-trometer.

3. Results and discussion

The starting point of this study was the com-parative analysis of the amino sequence deducedfrom the nucleotide sequence of both the ptk

Žgene of Acinetobacter johnsonii Grangeasse et.al., 1997 and the wzc gene of Escherichia coli

with the different amino acid sequences deducedŽfrom the Klebsiella pneumoniae genome Arakawa

.et al., 1995 . By using the Swissprot database, wedetected a striking sequence similarity betweenproteins Ptk and Wzc, on the one hand, andprotein Yco6 of K. pneumoniae, on the otherhand. Indeed, the bestfit sequence alignmentsshowed that these three proteins exhibited over

Ž .25% identity and 37% high similarity Fig. 1 .Since proteins Ptk and Wzc were known to au-tophosphorylate on multiple tyrosine residuesŽ .Duclos et al., 1996; Vincent et al., 1999 , it wasof interest to also assay protein Yco6 for phos-phorylation. For that purpose it was necessary, ina preliminary step, to overproduce and purify thisprotein.

3.1. O�erproduction and purification of protein Yco6

The yco6 gene lacking the start codon wassynthesized by PCR, by using oligonucleotideprimers deduced from the yco6 gene sequenceŽ .Arakawa et al., 1995 . The amplified DNA wascloned in plasmid pGEX-KT previously digestedwith BamHI and SmaI restriction enzymes. Theresulting plasmid, termed pGEX-yco6, was able toencode a fusion protein consisting of Yco6 bear-ing glutathione-S-transferase at its N-terminus.This construct was used to transform competent

Ž .cells from the BL21 pREP4-groESL strain of E.coli. This strain overproduces the two chaperone

proteins GroES and GroEL and is suitable forthe overproduction of proteins that possess a highdegree of hydrophobicity and thus a tendency toaggregate, such as Yco6. Upon induction by IPTG,efficient overexpression of a 105-kDa protein,consistent with the calculated molecular mass ofthe fusion protein, was obtained in the solublefraction of cells.

The GST-Yco6 fusion protein was then purifiedto homogeneity in a single-step affinity chromato-graphic procedure on a glutathione-Sepharose 4Bmatrix. In these conditions, approximately 1 mgof GST-Yco6 protein was obtained from 1 l ofbacterial culture.

3.2. Autophosphorylation of Yco6 at tyrosine

For comparison with Ptk and Wzc, the GST-Yco6 protein was assayed for phosphorylation. Itwas observed that purified GST-Yco6 was signifi-

�cantly labeled in vitro in the presence of �-32 � Ž .P ATP Fig. 2a . The capacity of GST-Yco6 tophosphorylate in these conditions indicated thatit contains an intrinsic protein kinase activity thatcatalyzes its autophosphorylation. The phospho-amino acid content of the labeled protein wasdetermined after acid hydrolysis and two-dimen-sional analysis. In these conditions only acid-resistant phosphoamino acids were analyzed sincea number of other phosphorylated compoundssuch as phosphohistidine, phosphoarginine, orphosphoaspartate, are known to be labile in acidŽ .Duclos et al., 1991 . Only phosphotyrosine wasrevealed on the corresponding autoradiogramŽ .Fig. 2b which indicated that GST-Yco6 wasmodified exclusively at tyrosine residues. ProteinYco6, like Ptk and Wzc, contains a relatively

Ž .large number of tyrosine residues 24 in totalespecially in its C-terminal part, but the precisenumber and the location of the phosphorylationsites are still unknown.

To characterize the Yco6 protein further, dif-ferent attempts were made to obtain the Yco6protein in its native state, i.e. without GST at itsN-terminus, after cleavage by thrombin. The fu-sion protein was efficiently hydrolyzed but thenative Yco6 protein thus obtained had no moreautophosphorylating activity. This loss of activitymight be related to the aggregation of the Yco6protein, due to its high degree of hydrophobicity.


Fig. 1. Comparison of proteins Yco6, Wzc and Ptk. The alignment of the amino acid sequence of Yco6 with that of the prokaryoticprotein-tyrosine kinases Wzc from E. coli and Ptk from A. johnsonii is presented. The amino acids that are identical in the threesequences are indicated by asterisks. The residues common to Yco6 and one of the other two prokaryotic protein-tyrosine kinases areunderlined. These various identical or common amino acids are shaded.


Ž .Fig. 2. a GST-Yco6 autophosphorylation assay. Approxi-�mately 3 �g of purified GST-Yco6 were incubated with �-

32 �P ATP. The protein was analyzed by SDS-PAGE, gels weresoaked in 16% TCA and either stained with Coomassie BlueŽ . Ž . Ž .lane 1 or submitted to autoradiography lane 2 . b . Phos-phoamino acid content of GST-Yco6. Protein GST-Yco6

� 32 �labeled with �- P ATP was analyzed by SDS-PAGE andelectroblotted onto an Immobilon PVDF membrane, thenexcised and hydrolyzed in acid. The phosphoamino acids thusliberated were separated by electrophoresis in the first dimen-

Ž .sion 1st and ascending chromatography in the second dimen-Ž .sion 2nd . After migration, radioactive molecules were de-

Ž .tected by autoradiography. Authentic phosphoserine P-Ser ,Ž . Ž .phosphothreonine P-Thr and phosphotyrosine P-Tyr were

run in parallel and visualized by ninhydrin staining.

The fusion protein GST-Yco6 was therefore usedin all subsequent experiments.

3.3. O�erproduction and purification of Yor5

A further search in the Swissprot database re-vealed, in addition, a high similarity between boththe phosphotyrosine-protein phosphatases Ptp ofAcinetobacter and Wzb of Escherichia, and a pro-tein, termed Yor5, from K. pneumoniae. Thecomparative analysis of the amino acid sequencesof these three proteins showed that they were27% identical and 34% similar over their entire

Ž .length Fig. 3 . In particular they all appeared toŽ .contain the CX R S�T motif which is encoun-5

tered in the N-terminal part of numerous low-MrŽ .acid phosphotyrosine phosphatases PTPases ,

namely eukaryotic phosphatases, and which isconsidered to be the major signature of this type

Ž .of enzyme Cirri et al., 1993; Zhang et al., 1995 .From this observation, it seemed worth analyz-

ing protein Yor5 in more detail, especially itsenzymatic activity on dephosphorylatable sub-strates. For this, it was first necessary, as previ-ously done for protein Yco6, to overproduce andpurify this protein. The oligonucleotide primerscorresponding to the 5� and 3� ends of the yor5

Ž .gene Arakawa et al., 1995 were prepared withthe appropriate restriction sites at both ends. Theyor5 gene lacking the start codon ATG was thensynthesized by PCR and cloned in the expressionvector pQE30 from E. coli, previously digestedwith the restriction enzymes BamHI and KpnI.The resulting plasmid pQE30-yor5 allowed pro-duction of the Yor5 protein with an N-terminaladdition of 11 amino acids, including six his-tidines. It was used to transform competent cells

Ž )of strain BL21 pREP4-groESL of E. coli. Uponinduction with 1 mM IPTG, a relatively high levelof a 17-kDa protein, consistent with the calcu-lated molecular mass of the fusion protein 6His-Yor5, was obtained in the soluble fraction ofcells. The fusion protein was then purified tohomogeneity in a single-step chromatographicprocedure by using a Zn2�-immobilized matrixgenerally used for purifying His-tagged proteins.In these conditions, approximately 4 mg of pureprotein was obtained from 1 l of bacterial culture.

3.4. Phosphotyrosine-protein phosphatase acti�ity ofYor5

The phosphatase activity of 6His-Yor5 was firstassayed for its ability to cleave p-nitrophenyl

Ž .phosphate PNPP . It was observed that the pro-tein could efficiently hydrolyze this synthetic sub-strate at an optimum pH value of 6.5. The corre-sponding kinetic constants, K and V , mea-m maxsured at 37 �C, were 1.81 mM and 11.2 �molmin�1 mg�1, respectively. These values are in thesame range as those previously reported for eu-karyotic low-Mr PTPases such as bovine heart

Ž .phosphatase Zhang and Van Etten, 1990 .Further experiments were performed to mea-

sure the in vitro activity of Yor5 on individualphosphorylated amino acids. Yor5 was shown toquantitatively release inorganic phosphate from


Fig. 3. Comparison of proteins Yor5, Wzb and Ptp. The alignment of the amino acid sequence of Yor5 with that of the prokaryoticphosphotyrosine-protein phosphatases Wzb from E. coli and Ptp from A. johnsonii is presented. The amino acids that are identical inthe three sequences are indicated by asterisks. The residues common to Yor5 and one of the other two prokaryotic PTPases areunderlined.

Fig. 4. Dephosphorylation of Yco6 by Yor5. Purified Yco6� 32 �was phosphorylated in vitro with �- P ATP. The labeled

protein was incubated with Yor5 for various times, as indi-cated, then separated by gel electrophoresis, treated with 16%TCA, and revealed by autoradiography. The amount of ra-dioactivity incorporated in Yco6 was counted in a liquid

Ž . Ž .scintillation spectrometer. � without Yor5; � with Yor5.

phosphotyrosine but had no effect on either phos-phoserine or phosphothreonine. This result isconsistent with a strict specificity of the dephos-phorylating activity of Yor5 for phosphotyrosine,which is a general property of the low-Mr

Ž .PTPases Cirri et al., 1993; Zhang et al., 1995 .

3.5. Endogenous substrate for Yor5

At this stage, two proteins of K. pneumoniaeharboring opposing activities had been identified:the Yco6 protein which is able to autophosphory-late on tyrosine residues, and the Yor5 proteinwhich possesses the characteristics of a phospho-tyrosine-protein phosphatase. In view of a possi-ble regulation of bacterial physiology by re-versible protein phosphorylation on tyrosine, itwas then of special interest to check whetherYor5 could utilize Yco6 as an endogenous subs-trate and catalyze its dephosphorylation.

For this, the purified Yco6 protein was first�radioactively labeled in the presence of �-

32 �P ATP, then incubated in the presence of Yor5.The results presented in Fig. 4 show that, in theseconditions, Yco6 was rapidly and extensively de-


Fig. 5. Protein dephosphorylation assay. Protein GST-Yco6�and protein GST-Wzc were first phosphorylated with �-

32 �P ATP. Each phosphoprotein was incubated in a dephos-phorylation buffer at 37 �C for 30 min either in the absenceŽ .lanes 1 and 4 or in the presence of 5 �g of purified Yor5 for

Ž . Ž .5 min lanes 2 and 5 and for 10 min lanes 3 and 6 . Proteinswere then analyzed by SDS-PAGE, gels were soaked in 16%TCA and radioactive bands were revealed by autoradiography.

phosphorylated by Yor5. These data provide evi-dence that Yor5 can use Yco6 as an endogenoussubstrate and support the concept that the enzy-matic activity of the phosphorylatable kinase Yco6is regulated by the dephosphorylating activity ofYor5.

The possibility that Yor5 could dephosphory-late other phosphorylatable proteins, as Wzc, wastested. For that purpose, Wzc from E. coli was

� 32 �labeled in vitro in the presence of �- P ATP,then assayed for dephosphorylation by using Yor5from K. pneumoniae as protein phosphatase. Itappeared that Yor5 could also dephosphorylate

Ž .protein Wzc Fig. 5 .Together, these data bring evidence, for the

first time, that an autophosphorylating protein-tyrosine kinase, Yco6, is present in K. pneumo-niae cells. In addition, they show that K. pneumo-niae also harbors a phosphotyrosine-protein phos-phatase, Yor5, with the same biochemical charac-teristics as those of several low-M acid phospho-rtyrosine-protein phosphatases, namely of eukary-otic origin, previously described by other authorsŽ .Cirri et al., 1993; Zhang et al., 1995 . Here again,this is the first evidence for an enzyme of thistype in K. pneumoniae cells. Of particular interestis the further finding that protein Yor5 can de-phosphorylate in vitro protein Yco6 which thusappears as a specific endogenous substrate forYor5. This observation supports the existence, tobe tested, of a regulatory mechanism of bacterialphysiology operating by reversible protein phos-

phorylation on tyrosine. In particular, since pro-teins Yco6 and Yor5 are both involved in thesynthesis of capsular polysaccharides essential to

Žthe virulence of K. pneumoniae Podschun and.Ullmann, 1998 , it can be suggested that re-

versible protein phosphorylation on tyrosinewould play a role in the pathogenicity of bacteria.Further experiments are, however, needed tocheck the validity of this hypothesis.

Acknowledgements

Thanks are due to Elisabeth Vaganay andMylene Riberty for expert assistance. This work`was supported by the Centre National de laRecherche Scientifique, the University of Lyon,

Žthe Ministere de la Recherche contract FNS`.2000 Microbiologie and the Societe Ezus-Lyon 1´ ´

Ž .contract 482.022 .

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isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein...

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