evidence for protein-tyrosine-phosphatase cysteine-phosphate

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for BiochemistrJl ’ and Moleculal ’ Biology, Inc

Evidence for Protein-tyrosine-phosphatase Cysteine-Phosphate Intermediate*

Vol. 266, No. 26, Issue of September 15, PP. 17026-17030,1991 Printed in U. S. A .

Catalysis Proceeding via a

(Received for publication, February 4, 1991)

KunLiang Guan and Jack E. Dixon From the Department of Biochemistry and the Walther Cancer Institute, Purdue University, West Lafayette, Indiana 47907

A recombinant protein-tyrosine-phosphatase has been expressed in Escherichia coli and purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using affinity chromatography. When the phosphatase was allowed to react with 32P- labeled substrates and then rapidly denaturated, a 32P- labeled phosphoprotein could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transient formation of a 32P-labeled phosphoprotein was observed, and the 32P-labeled protein disappeared as substrate was consumed. In the presence of ”P- labeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosine- phosphatase. Site-directed mutagenesis of a catalytically essential cystine residue (position 215) in the recombinant protein resulted in an inactive enzyme, and no phosphoprotein was formed. The 32P-labeled phosphoprotein showed a maximum lability between pH 2.5 and 3.5 and was rapidly decomposed in the presence of iodine. These properties, along with additional site-directed mutations, suggest that the protein- tyrosine-phosphatase forms a covalent thiol phosphate linkage between CysZ1‘ and phosphate.

Protein tyrosine phosphorylation is a mechanism utilized by many growth factors to modulate normal cellular prolifer- ation (1). Tyrosine kinases can also function as oncogenes leading to unrestrained cell growth and tumorigenesis. Evi- dence supports the idea that key steps in the cell cycle are also regulated by tyrosine phosphorylation (2-4). The level and duration of tyrosine phosphorylation within a cell would appear to be governed by both tyrosine kinases and protein- tyrosine-phosphatases (PTPases).’ The PTPases fall within two general families. The receptor-like PTPases have an extracellular domain, a single transmembrane sequence, and generally two duplicated cytoplasmic PTPase domains. Mem- bers of this family include CD45 (5), LAR (6), and several newly identified PTPases (7-9). The other family of PTPases are the nonreceptor-like proteins that have a single catalytic domain. PTPases corresponding to this family have been

* This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 18849 from the National Insti- tutes of Health and by funds from the Walther Cancer Institute. This is Journal Paper 12920 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PTPase, protein-tyrosine-phosphatase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pNPP, p-nitrophenyl phosphate; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid; RCMM lysozyme, reduced, carboxymethylated, and maleylated lysozyme.

purified and characterized from a number of sources (10-21). Tonks et al. (22) first reported the purification to homogeneity of a 35-kDa protein-tyrosine-phosphatase (PTPase 1B) from human placenta that was subsequently sequenced by Char- bonneau et al. (23) and demonstrated to share amino acid sequence identity to the cytoplasmic domain of CD45. De- duced PTPase sequences corresponding to the human placenta (24, 25) and rat brain (26) PTPases as well as a T-cell PTPase (27) have been reported. Recently, this family of phosphatases was extended to include a PTPase from Yersi- nia, a pathogenic bacterium that is the causative agent of the plague or black death (28). Interestingly, the Yersiniu PTPase appears to be an essential virulence determinant in the bacteria (29).

Alkaline and acid phosphatases have been studied in detail (30, 31). Hydrolysis of phosphate ester bonds by alkaline phosphatase proceeds via a phosphoserine intermediate (32), whereas some acid phosphatases catalyze a similar reaction that involves the formation of a phosphohistidine covalent enzyme intermediate (31). Detailed mechanistic studies similar to those noted above for alkaline and acid phosphatases have not been undertaken with the PTPases; however, the importance of thiol reagents in preserving PTPase activity has been reported by numerous investigators (22, 28, 33, 34). Reagents that show selective reactivity toward thiol residues within the protein also have a profound effect upon enzyme activity (33). In addition, site-directed mutagenesis has doc- umented a catalytically essential cysteine residue in both receptor-like (7, 34) and nonreceptor-like PTPases.

To develop a more detailed understanding of the catalytic mechanism employed by the PTPase, we have developed a system to obtain a high level of PTPase expression in bacteria (35). The recombinant phosphatase and several site-directed mutants have been purified via affinity chromatography. Using these mutants, we present evidence that Cys215 func- tions in the hydrolysis of tyrosine phosphate-containing substrates and that this cysteine residue forms a covalent thiol phosphate bond. The invariant amino acid sequence surrounding the active site (HCXAGXXR) of PTPl and other PTPases supports the idea that this family of enzymes are all likely to form a thiol phosphate intermediate during catalysis.

EXPERIMENTAL PROCEDURES

Construction of Expression Plasmids-Cloning of cDNA for rat brain protein-tyrosine-phosphatase has been described previously (26). The 1.7-kilobase EcoRIIBclI fragment encoding PTPase was subcloned into EcoRIIBamHI-digested M13mp18. Single-stranded DNA was prepared and used as the template for oligonucleotide- directed mutagenesis. Mutations were confirmed by DNA sequencing. The mutated PTPase cDNA in M13mp18 was digested with EcoRI and filled in by the Klenow fragment of DNA polymerase I, followed by digestion with SalI (SalI is located in the M13mp18vector adjacent to the 3’-end of the cDNA insert). This 1.7-kilobase EcoRIISalI fragment was purified from agarose gel and ligated with SmaIISalI-

17026

PTPase Catalysis: A Cysteine-Phosphate Intermediate 17027

digested vector pT7-7. The resulting plasmids were used to express PTPase in Escherichia coli strain BL21(DE3) pLysE (26). TO purify the PTPase rapidly, the 1.7-kilobases EcoRI/SalI fragment (this EcoRI site is from vector pT7-7 and adjacent to the 5'-end of the PTPase-coding sequence) was subcloned into the EcoRI/SalI sites of vector pGEX-KG (35). This construction produces an in-frame fusion protein with glutathione S-transferase. Each individual mutant was subcloned into vector pGEX-KG employing the same scheme as described above.

Expression and Purification-The numbering system used here defines the initiator methionine of PTPl (26) as residue 1. The expression of PTPU323 (U refers to the introductions of a stop codon at the numbered amino acid), PTPU285, PTPU323 C121S, PTPU323 H214Q, and PTPU323 C215S was performed essentially as described (35). Fusion protein was purified by glutathione-agarose affinity chromatography and cleaved by thrombin. Briefly, 1 liter of bacteria induced to express the fusion protein was collected by centrifugation, resuspended in 10 ml of PBST (20 mM sodium phosphate, pH 7.3, 150 mM NaC1, 1% Triton X-loo), and lysed by passing through a French press twice. The bacterial lysate was centrifuged a t 15,000 X g for 10 min a t 4 "C. To 1 ml of the lysate (equivalent to 100 ml of original culture) was added 2 ml (50%, v/v) of glutathione-agarose. After absorbing for 30 min a t 4 "C, the agarose beads were washed four times with 15 ml each of PBST at 4 "C. The agarose beads were further washed once with 15 ml of thrombin cleavage buffer (50 mM Tris, pH 8.0, 150 mM NaC1, 2.5 mM CaC12) a t room temperature. The washed agarose beads were resuspended in 2 ml of thrombin cleavage buffer containing 6 pg of thrombin and shaken gently for 20 min a t room temperature. Finally, the agarose beads were removed by centrifugation, and the supernatant was collected. The protein was examined by SDS-PAGE. Protein concentration was determined by the method of Bradford (36).

Activity Assay-Both phosphorylated Raytide (Oncogene Science) and p-nitrophenyl phosphate (pNPP) were used to assay the phosphatase. Raytide was phosphorylated on a tyrosine by p43'-ab' protein- tyrosine kinase (Oncogene Science). Raytide is a gastrin analogue having an M, of 2116. Although the exact sequence of Raytide is not available, it is phosphorylated only on tyrosine residue(s) by ~ 4 3 ' ~ " ~ ' . It is more efficiently phosphorylated by the p43'ab' kinase than is angiotensin. Phosphorylation was routinely performed in 30 p1 containing 5 pg of Raytide, 50 mM HEPES, pH 7.5,lO mM MgC12, 0.067% 8-mercaptoethanol, 0.03 mM ATP, 150 pCi of [Y-~'P]ATP, and 2 units of p43'-ab' for 30 min at 30 "C. The reaction was stopped by the addition of 120 pl of 10% phosphoric acid. Free ATP was separated by applying the sample onto a square (2 X 2 cm) of P-81 paper and extensively washing with 0.5% phosphoric acid. Raytide was eluted from the P-81 paper by 2 X 1 ml of 0.5 M (NH,),CO, and lyophilized. Dephosphorylation was performed in 20 pl containing 50 mM imidazole, pH 7.5, 0.1% P-mercaptoethanol, and -5 nM each phosphorylated Raytide and PTPase for 10 min a t room temperature. The release of 32P from Raytide was quantitated by a previously published method (7). Hydrolysis of p N P P by the PTPase was carried out as described (22, 28).

Affinity Binding to Thiol-phosphorylated RCMM Lysozyme-Seph- arose bearing thiol-phosphorylated RCMM lysozyme was a gift of Dr. Thomas S. Ingebritsen (Iowa State University). Approximately 0.3 ml of affinity resin was packed in a small disposable column (0.6 X 5 cm). The column was saturated in loading buffer (10 mM imidazole, p H 7.4, 0.05% P-mercaptoethanol). Twenty pg of purified PTPU323 or PTPU323 C215S was diluted in 300 p1 of loading buffer and applied to the column a t a flow rate of 3 ml/h. The sample was reloaded three times before washing with loading buffer at a flow rate of 12 ml/h. After a ten-column volume of loading buffer, the PTPase was eluted with 10 mM imidazole, pH 7.4, 0.05% P-mercaptoethanol, and 250 mM NaCl a t a flow rate of 12 ml/h. Enzymatically active PTPU323 bound to the column and was eluted with 250 mM NaCl in 95% yield. A similar percent of protein corresponding to the PTPU323 C215S mutant was bound to the column and selectively eluted. The protein was visualized by SDS-PAGE analysis.

Trapping the Phosphoenzyme-The trapping experiment was performed as follows. Enzyme, usually 1 pl(0.5 pg/pl), was quickly mixed with 9 pl of 50 mM imidazole, pH 7.5, 0.1% P-mercaptoethanol, 10 nM phosphorylated Raytide at room temperature. An equal volume of SDS sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol) was added seconds after the addition of enzyme. The trapped sample was then analyzed by SDS-PAGE without heating. 32P-Labeled protein was visualized by autoradiography. To determine the percentage of phosphoenzyme formed with "P-labeled Raytide, a wet SDS-

polyacrylamide gel was exposed to x-ray film after electrophoresis. The PTPU323 band was cut from gel. Enzyme was eluted in 1 ml of solution containing 0.1% SDS, 0.5 M urea, and 5 mM NaHCOs overnight a t 25 "C and analyzed by scintillation counting. When competition experiments were performed, either sodium vanadate or pNPP was premixed with phosphorylated Raytide before the addition of enzyme. The chase experiment was performed as follows. Two pl of enzyme (0.5 pg/pl) was added into 8 p1 of solution, which would give a final 10 pl of reaction mixture containing 50 mM imidazole, pH 7.5, 0.1% 0-mercaptoethanol, and 100 nM '"P-labeled Raytide. Ten s after the addition of enzyme, an equal volume of SDS sample buffer was added to quench the reaction or the reaction was incubated a t 37 'C for a given time before the addition of SDS sample buffer. The samples were analyzed by SDS-PAGE.

When ["PIpNPP was used in trapping, 1.78 nmol (68 pg) of PTPU323 was mixed in a final volume of 0.8 ml of solution containing 20 mM ["PIpNPP, 50 mM imidazole, pH 7.5, and 0.1% P-mercaptoethanol. One-hundred pl of 20% SDS was added 10 s after the addition of enzyme, followed by the addition of 100 pl of 0.5 M Tris, pH 8.5. Trapped PTPU323 was separated from [?'P]pNPP on a Sephadex G- 25 column (1 X 45 cm) equilibrated with 50 mM Tris, pH 8.5, 0.2% SDS, and 100 mM NaCl at a flow rate of 6 ml/h.

Synthesis of P2P/pNPP-[:'2P]pNPP was synthesized by a modified procedure outlined by Zhang and VanEtten.' Five mCi of '>Pi (supplied in 5 pl; ICN) was mixed with 5 ml of acetonitrile and rotary- evaporated at 50 "C. Crystalline phosphoric acid (50 mg, 0.5 mmol) was added to the 50-ml round-bottom flask containing acetonitrile (7.5 ml), p-nitrophenol (0.14 g, 1 mmol), triethylamine (0.21 ml), and the 5 mCi of 32Pi. After all solids dissolved, 0.06 ml (0.6 mmol) of trichloroacetonitrile was added, and the reaction proceeded at 37 "C for 2 h. Solvent and unreacted trichloroacetonitrile was removed by rotary evaporation at 50 "C. The remaining residue was dissolved in 10 ml of H20 and adjusted to pH 5-6 by HC1. Cyclohexylamine (0.3 ml) was added to the aqueous phase after extraction three times by ether. Water was removed by rotary evaporation. p N P P was recrys- tallized from 10 ml of 90% ethanol and finally dissolved in 0.7 ml of H'O. ["PlpNPP had an initial specific activity of 9440 cpm/nmol.

Characterization of Chemical Properties of Phosphoenzyme-The pH stability of the phosphoenzyme intermediate was determined. Approximately 11 pg of enzyme was mixed in 220 pl of solution containing 5 mM imidazole, pH 7.5, 0.05% @-mercaptoethanol, and 10 nM phosphorylated Raytide a t room temperature; 25 p1 of 20% SDS was added to quench the reaction 10 s after the addition of enzyme. To 22.5 p1 of this reaction mixture was added 2.5 pl of pH stock buffer (the pH stock buffers were 2 M sodium acetate for pH 2-6, 2 M Tris for pH 7-10, and 1 M HCI for pH 1). After incubation for 45 min a t 37 " C , each sample was passed through a Sephadex G- 25 column and analyzed by SDS-PAGE. The amount of phosphoenzyme was quantitated by scanning the autoradiography with a den- sitometer.

To determine the stability of phosphoenzyme in the presence of bromine, iodine, pyridine, and hydroxylamine, the phosphoenzyme intermediate was resolved by SDS-PAGE and transferred to Nytran. The Nytran filters were soaked in 250 mM Tris, pH 7.0, containing one of four chemicals (bromine, iodine, pyridine, and hydroxylamine) for 10 min a t 37 "C. Then these treated Nytran filters were briefly rinsed in 50 mM Tris, pH 7.0, and autoradiographed.

RESULTS AND DISCUSSION

CYS''~ Is Essential for Protein-tyrosine-phosphatase Actiu- ity-We recently reported the cloning and expression of a recombinant rat brain PTPase in E. coli (26). The recombinant enzyme was shown to be specific for the hydrolysis of phosphate from phosphotyrosine. Several Ser/Thr-containing phosphoproteins were not substrates for the enzyme. Because mechanistic studies often require considerable concentrations of pure enzyme, we have utilized a vector expression system where the PTPase is produced as a glutathione S-transferase

' Z.-Y. Zhang and R. L. VanEtten (Department of Chemistry, Purdue University) have recently isolated a low molecular weight phosphotyrosyl protein phosphatase from bovine heart. This soluble enzyme, which shares no significant sequence homology to PTP1, also appears to utilize a cysteine residue in the formation of a thiol phosphate enzyme intermediate (46).

17028 PTPase Catalysis: A Cysteine-Phosphate Intermediate

fusion protein (35). The fusion protein can be rapidly purified by affinity chromatography on glutathione-agarose. Proteo- lytic degradation was a significant problem when the full- length (50-kDa) PTPase was expressed in E. coli as a fusion protein. However, we have recently cloned a PTPase from yeast consisting of 335 residues corresponding to the amino- terminal region of rat brain PTP1.3 This suggested that the -300 residues contained within the amino terminus of PTPl were sufficient for catalytic activity. Based upon this finding, the first 322 amino acids of PTPl were cloned behind the coding sequences corresponding to glutathione S-transferase and expressed in E. coli. The presence of 5 glycine residues following the thrombin cleavage site allows efficient cleavage of the fusion protein and allows the recovery of the PTPase by a single affinity chromatography step (Fig. JA). Approxi- mately 20 mg of pure protein can be obtained per liter of bacterial culture.

Several investigators have noted that thiol reagents appear to be essential for maintaining PTPase activity (33, 37) as well as the activity of the low molecular weight phosphatase characterized from bovine heart (38). This observation suggests the possibility that a thiol-containing amino acid may be involved in catalysis. Sequence comparison of several PTPases shows 2 highly conserved cysteine residues (Cys'" and Cys2I5 of PTP1). Using site-directed mutagenesis, these 2 cysteine residues were changed to serine. Another invariant histidine residue (position 214) was also modified by site- directed mutagenesis (H214Q). A deletion mutant was made by introducing a stop codon a t amino acid 285. Each of these proteins was expressed as fusion proteins in the pGEX-KG vector system and purified by glutathione-agarose affinity chromatography. Cleavage of the affinity-purified proteins with thrombin resulted in a highly purified protein as shown by SDS-PAGE analysis (Fig. JA). Each recombinant protein appeared as a single Coomassie Blue staining protein with the anticipated molecular weight. Activities of the purified proteins were determined using either pNPP or phosphorylated Raytide as a substrate. The percent activity of the various catalysts were compared to PTPU323, which was arbitrarily set at 100%. The protein containing only 284 residues was 70% as active as PTPU323, suggesting that -150 residues may be eliminated from the carboxyl terminus of the native PTPase while still retaining enzymatic activity. This observation also establishes that the "catalytic" domain of the protein resides in the first 284 residues. Since some acid phosphatases (31) proceed via a phosphohistidine intermediate (31)) modifying the invariant in the putative active site would address the possibility that the PTPase also proceeded via a similar intermediate. This alteration (H214Q) reduced the catalytic activity of the enzyme by 95%, suggesting that plays a role in catalysis, but that it is not an essential residue required for PTPase activity. Mutagenesis of CYS'~' and CyS2I5 had dramatically different effects on enzyme activity. The C121S mutation reduced the activity to 44%) whereas the C215S mutation resulted in a complete loss in activity. Although the C215S mutation altered only a single atom within the protein, we felt that it was important to demonstrate that the inactive protein was still capable of binding substrate. Both the PTPU323 and C214S mutant enzymes bound to a thiol-phosphorylated RCMM lysozyme affinity column and were eluted with 250 mM NaCl (data not shown), suggesting that the binding of substrate has not been altered by these modifications. Collectively, these observations underscore the importance of CYS"~ in catalysis and suggests that the single oxygen for sulfur substitution that

:I K. L. Guan and J. E. Dixon, unpublished results.

A M 1 2 3 4 5

97- - 67- - 43- - " - - - 30- - 14 -

211 - 102 - 69 - 46-

. -0..

28 -

c 1 2 3 4 5

D l 2 3 4 5

FIG. 1. Expression and purification of PTPl mutants and analysis of formation of 32P-labeled phosphoprotein. A, SDS- PAGE of affinity-purified PTPase mutants. Lane M, molecular mass standards (in kilodaltons); lane 1, PTPU323 C121S; lane 2, PTPU323 H214Q; lane 3, PTPU323 C215S; lane 4, PTPU285; lane 5, PTPU323. B, autoradiography of PTPU323 "P-labeled phosphoprotein electro- phoresed by SDS-PAGE. Micrograms of PTPU323 are indicated on the top. The far right lane contains 0.5 pg of PTPU323, which was heated to 100 "C for 3 min prior to incubation with "P-labeled Raytide. C, effects of vanadate andpNPP on formation of phosphoenzyme intermediate. Identical amounts of enzyme (0.5 pg) and '*P- labeled Raytide (10 nM) were used in each trapping reaction in the presence or absence of vanadate or pNPP. Lane 1, no vanadate or pNPP; lanes 2 and 3, 1 and 10 mM sodium vanadate, respectively; lanes 4 and 5 , l and 10 mM pNPP, respectively. D, transient formation of "'P-labeled phosphoprotein. The phosphoenzyme disappeared when "P-substrate was consumed. Enzyme and substrates were incubated as described under "Experimental Procedures" for the times specified. The reactions were stopped by the addition of SDS. Lane I, no enzyme added; lane 2, 10 s after the addition of enzyme; lanes 3-5,2, 10, and 20 min, respectively.

has taken place in the "mutant" protein led to a total loss in enzymatic activity. The results noted with the C215S mutant are consistent with observations reported for CD45 (7), LAR (34), and the Yersinia phosphatase (28)) which also document the importance of the corresponding cysteine located in a structurally similar position in catalysis.

Phosphoprotein Is Formed-Mixing the recombinant enzyme and "'P-labeled b y t i d e followed by the addition of SDS resulted in the appearance of a "'P-labeled protein that could be visualized by SDS-PAGE (Fig. 1B). The intensity of the radiolabeled protein having the anticipated molecular weight by SDS-PAGE increased with increasing concentrations of

PTPase Catalysis: A Cysteine-Phosphate Intermediate 17029

2000 17 19 21 22 24 26 28

1800

1600

1400

lux)

6 1000

800

600

400

200

0 0 5 IO 15 20 25 30 FRACTION (0.75mlhuk)

FIG. 2. Chromatography of 32P-labeled phosphoprotein and ["'PIpNPP on Sephadex G-25 column. Counts/minute (minus background) of each fraction (0.75 ml) are indicated on the y axis. Fraction numbers are indicated on the x axis. Fractions 17, 19, 21, 22, 24, 26, and 28 were concentrated and analyzed by SDS-PAGE. Insert, autoradiograph of SDS-polyacrylamide gel. Fraction numbers are indicated on the top. Only the initial portion of the peak corresponding to ['*P]pNPP is shown.

recombinant phosphatase. Heat inactivation of the PTPase prior to incubation with the radiolabeled substrate led to a loss in its ability to incorporate the isotope (Fig. 1B). When 1 FM 3'P-labeled phosphorylated Raytide was used as a substrate in the trapping reaction, -1.0% of the PTPase was radiolabeled. Although this represents a low level of 3'P in- corporation, it should be noted that at this concentration of Raytide, the enzyme was not saturated with substrate. The trapping of the phosphoenzyme also appeared to be highly specific and could be inhibited by sodium vanadate, a PTPase inhibitor, andpNPP (Fig. 1C). The formation of phosphoprotein was transient (Fig. 1D). The phosphoprotein was appar- ent by SDS-PAGE at the earliest time point (10 s) examined. The intensity of the labeled protein diminished with time, and it was almost undetectable after 20 min of incubation with 32P-labeled substrate. Following completion of the reaction, analysis of the products demonstrated that free phosphate was the only observed 32P-labeled product. The loss of '"P label from the protein was unusually slow if the protein was forming an intermediate that rapidly breaks down. The fact that "P-labeled substrate concentrations are below the K,,, may complicate these observations. Additional efforts to document the presence of an intermediate by stop-flow kinetic analysis will be required to support the idea that we have trapped a true intermediate on the reaction pathway.

To trap the phosphoprotein more effectively, [3'P]pNPP was synthesized. PTPU323 has a K,,, of 2.7 mM for pNPP.4 At 20 mM [3'P]pNPP, 0.27 mol of phosphate was incorporated per mol of enzyme. Fig. 2 shows the chromatographic sepa- ration of the radiolabeled protein from 3'P-labeled substrate. SDS-PAGE analysis of the radiolabeled material is shown for each respective fraction. The presence of radiolabeled protein with the anticipated molecular weight is evident in fractions 19, 21, 22; and a trace of radiolabeled protein is evident in fraction 24 (visible upon longer exposures). No radiolabeled protein is evident in fractions 26-28. These observations demonstrate that a significant mole percent of the PTPase can be trapped as a 3'P-labeled protein.'

C215S Mutant PTPase Does Not Form 32P-Labeled Phos- phoprotein-Several of the mutants discussed previously were examined to determine if they would form the 3'P-labeled phosphoprotein. All mutants were able to form the phospho-

T. S. Ingebritsen, unpublished results.

protein except C215S (Fig. 3) when "P-labeled protein was analyzed by SDS-PAGE. It is interesting to note that mutant H214Q formed a 32P-labeled phosphoprotein almost as effectively as did the wild-type PTPase, whereas it displayed only a few percent of the wild-type enzyme activity. This observation suggests that the H214Q substitution does not influ- ence formation of the 32P-labeled phosphoprotein. The de- crease of phosphatase activity of mutant H214Q may be due to a slower rate of hydrolysis of the phosphate from the phosphoprotein. The PTPase composed of only 284 amino acids also formed the phosphoenzyme and displayed the anticipated molecular weight by SDS-PAGE analysis (Fig. 3, lane 4 ) . Mutant C215S was unable to form phosphoprotein even when a 4-fold increase in enzyme (lune 3 ) was used, whereas mutant C121S readily formed the phosphoprotein (data not shown). Collectively, this evidence suggests that Cys215 is the active-site residue that is involved in the catalysis and that a phosphoenzyme intermediate appears to be important in catalysis.

Evidence for Thiol Phosphate Bond-The phosphoamino acid linkage of the denatured "'P-labeled protein was shown to be consistent with a thiol phosphate bond by several criteria. The phosphoprotein displayed maximal instability in the pH range 2.5-3.5, whereas it was relatively stable at very low pH (pH t2.0). Furthermore, the 3'P-labeled phosphoprotein was relatively stable within pH 7-10. This bell-shaped stability profile (Fig. 4) for the 3'P-labeled protein is distinct from a phosphoester linkage involving serine, threonine, or tyrosine, which were stable throughout pH 1-10 (39). The pH stability profile does not resemble a phosphoramidate linkage (i.e. histidine or lysine), which shows increased lability as pH decreases from 7 to 0 (40), as has been found in prostatic acid phosphatase (31). The bell-shaped pH stability profile is also

1 2 3 4 5

FIG. 3. Effect of mutations on formation of 32P-labeled phosphoprotein. Lane 1,0.5 pg of PTPU323 H214Q; lanes 2 and 3, 0.5 and 2 pg of PTPU323 C215S, respectively; lane 4 , 0.5 pg of PTPU285; lane 5,0.5 pg of PTPU323.

0 I I 0 2 4 6 8 1 0

- ""- PH

FIG. 4. pH stability of phosphate on 32P-labeled phosphoprotein. The 32P-labeled protein was incubated a t 37 "C for 45 min at the indicated pH in the buffers specified under "Experimental Procedures." The percentage of hydrolysis of the "'P-labeled protein (0) was compared with a model compound, butyl thiophosphate (O), taken from Herr and Koshland (42).

17030 PTPase Catalysis: A Cysteine-Phosphate Intermediate

FIG. 5. Stability of S2P-labeled phosphoprotein to iodine, bromine, hydroxylamine, and pyridine. The stability of the 3zP- labeled phosphoprotein was examined in the presence of the individual chemical for 10 min at 37 "C as described under "Experimental Procedures." Lane 1, control (no additions); lane 2, 1.0 pM 12; lane 3, 10 p M 12; lane 4, 100 g M ID; lane 5, 1 mM 12; hne 6, 10 p M Br2; lane 7, 100 g~ Br2; lane 8,l mM Br2; lane 9,50 mM hydroxylamine; lane 10, 50 mM pyridine.

different from that representative of an acylphosphate linkage, which shows marked lability at both high (>lo) and low ((1) (41). The pH stability profile resembles that of thiol phosphate linkage seen in model compounds such as butyl thiophosphate (42) or cystamine thiol phosphate (43) as well as the thiol phosphate linkage in thioredoxin (44). A comparison of the pH stability profiles for the model compound butyl thiophosphate with that of the denatured 32P-labeled PTPase is shown in Fig. 4. Both demonstrate bell-shaped profiles with maximum breakdown near a pH of -3.0. The existence of a thiol phosphate linkage in proteins has been reported in bacterial thioredoxin (44) and mannitol carrier-specific enzyme I1 (45). The biological function of the thiol phosphate intermediate in thioredoxin is unclear. A phosphocysteine intermediate proposed for the mannitol carrier-specific enzyme in bacteria may be required for phosphate transfer from phosphoenolypyruvate to mannitol (45).

In addition to the unique bell-shaped pH stability profile, the thiol phosphate linkage is extremely sensitive to hydrolysis by iodine or bromine at neutral pH (43). The reaction involves the formation of a sulfenyl iodine-phosphate (or bromine-phosphate) intermediate. When the 32P-labeled phosphoenzyme was treated with either iodine or bromine (Fig. 5), it rapidly liberated its radioactive label. The phosphate was quantitatively lost in the presence of 10 p~ iodine (see "Experimental Procedures" for conditions). This is further evidence that the 32P-labeled phosphoprotein is formed via a thiol phosphate bond. The effectiveness of hydroxylamine and pyridine to hydrolyze phosphate esters has been used as a criterion to identify acylphosphates. The phosphoenzyme intermediate described here was not labile to either hydroxylamine or pyridine treatment (Fig. 5).

Evidence presented here along with the work of others (7, 28, 33, 34) support the idea that phosphate ester hydrolysis proceeds by at least three distinct phosphoenzyme interme- diates. Alkaline phosphatase proceeds via a phosphoserine intermediate (32), whereas some acid phosphatases form a phosphohistidine intermediate (31). The PTPase studied here, having the highly conserved residues HCXAGXXR surrounding the essential Cys215, formed a thiol phosphate bond. The fact that other PTPases from pathogenic bacteria (28) to humans also harbor this conserved sequence a t their

active sites suggests that this family of enzymes proceed by a similar catalytic mechanism.

Acknowledgments-We wish to thank Dr. Thomas S. Ingebritaen for thiol-phosphorylated RCMM lysozyme affinity resin, Drs. Mark Hermodson and Henry Weiner for stimulating discussions, and Dr. Robert L. VanEtten for reading and commenting on the manuscript. We would also like to acknowledge the advice of Drs. VanEtten and Z.-Y. Zhang in preparation of radiolabeledpNPP.

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