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Originally published In Press as doi:10.1074/jbc.M412211200 on December 20, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8180-8187, March 4, 2005
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Crystal Structure of the PTPL1/FAP-1 Human Tyrosine Phosphatase Mutated in Colorectal Cancer

EVIDENCE FOR A SECOND PHOSPHOTYROSINE SUBSTRATE RECOGNITION POCKET*

Fabrizio Villa{ddagger}§, Maria Deak§, Graham B. Bloomberg||, Dario R. Alessi§, and Daan M. F. van Aalten, Supported by a Wellcome Trust Senior Research Fellowship and the EMBO Young Investigator Programme{ddagger}**

From the {ddagger}Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, DD1 5EH, Scotland, the §MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, DD1 5EH, Scotland, and the ||Department of Biochemistry, School of Medical Science, University of Bristol, Bristol BS8 1TD, United Kingdom

Received for publication, October 28, 2004 , and in revised form, December 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Protein-tyrosine phosphatase-L1 (PTPL1, also known as FAP-1, PTP1E, PTP-BAS, and PTPN13) is mutated in a significant number of colorectal tumors and may play a role in down-regulating signaling responses mediated by phosphatidylinositol 3-kinase, although the precise substrates are as yet unknown. In this study, we describe a 1.8 Å resolution crystal structure of a fully active fragment of PTPL1 encompassing the catalytic domain. PTPL1 adopts the standard PTP fold, albeit with an unusually positioned additional N-terminal helix, and shows an ordered phosphate in the active site. Interestingly, a positively charged pocket is located near the PTPL1 catalytic site, reminiscent of the second phosphotyrosine binding site in PTP1B, which is required to dephosphorylate peptides containing two adjacent phosphotyrosine residues (as occurs for example in the activated insulin receptor). We demonstrate that PTPL1, like PTP1B, interacts with and dephosphorylates a bis-phosphorylated insulin receptor peptide more efficiently than monophosphorylated peptides, indicating that PTPL1 may down-regulate the phosphatidylinositol 3-kinase pathway, by dephosphorylating insulin or growth factor receptors that contain tandem phosphotyrosines. The structure also reveals that four out of five PTPL1 mutations found in colorectal cancers are located on solvent-exposed regions remote from the active site, consistent with these mutants being normally active. In contrast, the fifth mutation, which changes Met-2307 to Thr, is close to the active site cysteine and decreases activity significantly. Our studies provide the first molecular description of the PTPL1 catalytic domain and give new insight into the function of PTPL1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Protein-tyrosine phosphatase-L1 (PTPL1)1 is a non-receptor protein-tyrosine phosphatase and is the largest of the known 107 protein-tyrosine phosphatases (PTPs), comprising 2485 residues (1). The N terminus contains a kinase non-catalytic C-lobe (KIND) domain (residues 3–190), a new protein module identified recently (2), and a four-point-one/ezrin/radixin/moesin (FERM) domain (residues 568–781). PTPL1 also contains five PSD-95/Drosophila disc large/zonula occludens (PDZ) domains located between residues 1102 and 1990 and a protein-tyrosine phosphatase domain at its C terminus (residues 2087–2485). PTPL1 is the only tyrosine phosphatase possessing this domain organization (1). The non-catalytic region of PTPL1 is likely to regulate its cellular localization and/or interaction of PTPL1 with other regulators and/or substrates (reviewed in Ref. 3). The non-catalytic domain of PTPL1 does not appear to control PTPL1 activity directly as the isolated catalytic domain of PTPL1 possesses the same activity as full-length PTPL1 (4).

Recent studies have provided initial evidence that PTPL1 may play a role in regulating PI 3-kinase-dependent signaling pathways that regulate cell growth and survival responses. This was first based on the finding that overexpression of PTPL1 in a breast cancer cell line induced the dephosphorylation of the insulin receptor substrate protein-1 (IRS1), resulting in inhibition of PI 3-kinase-regulated cell growth and survival responses (5). Subsequently, the cellular localization of PTPL1 was also found to be controlled by PI 3-kinase, through a PDZ domain-mediated interaction of PTPL1 with the phosphatidylinositol 3,4 bisphosphate (PtdIns(3,4)P2) binding adaptor protein, TAPP1 (4). Binding of TAPP1 to PTPL1 localizes PTPL1 in the cytosol, and following stimulation of cells with agonists, which elevate the level of PtdIns(3,4)P2, PTPL1 is recruited to the plasma membrane.

More recently, a mutational analysis of colorectal cancers identified 19 different somatic mutations in PTPL1; eight mutations deleted the entire catalytic phosphatase domain, six point mutations were observed outside the catalytic domain, and five point mutations were located within the phosphatase domain (6). The effects that these mutations had on phosphatase activity were not analyzed in this study but nevertheless suggest that PTPL1 plays a role in regulating cancer-relevant growth and/or survival responses. Although numerous proteins have been proposed to interact with PTPL1 (reviewed in Ref. 3), and certain proteins such IRS1 (5), ephrin B (7), and IkB (8) have been suggested to comprise substrates based on mainly in vitro or overexpression studies, further analysis is required to establish the physiological significance of these findings.

Apart from PTP1B, the x-ray crystallographic structure of the catalytic domains of several PTPs (e.g. TCPTP (9), Yop51 (10), SHP1 (11), SHP2 (12), (PTPµ (13), PTP{alpha} (14), and LAR (15)) have been characterized. All PTPs possess a common catalytic conformation, following the fold of PTP1B, the first PTP structure to be characterized (16). This is composed of a single domain, with the polypeptide chain organized into nine {alpha}-helices (termed {alpha}1', {alpha}2' at the N terminus followed by {alpha}1–7) and 12 {beta}-strands (termed {beta}1–12). The 12 {beta}-strands adopt a highly twisted conformation spanning the entire length of the molecule and are located in the core of the structure, surrounded by the {alpha}-helices (16). All PTPs possess a catalytic nucleophilic cysteine that forms a covalent intermediate with the phosphate group. The catalytic cysteine is located in a region termed the PTP signature motif or phosphate binding loop and lies within a conserved His-Cys-Ser-Xaa-Gly-Xaa-Gly-Arg-Thr/Ser-Gly sequence (where Xaa is any amino acid). This phosphate binding loop is located at the N terminus of the {alpha}4-helix in the core of the PTP fold. Another key region of the catalytic domain, the "WPD loop," contains the catalytic acid (Asp-181), which protonates the phosphoester bond in the first step of the reaction (17). This is located between {beta}11 and {alpha}3, positioned near the catalytic cysteine. The third key catalytic motif of PTPs, termed the Q loop, possesses a Gln residue that positions the nucleophilic water molecule appropriately to attack and hydrolyze the cysteine-phosphate intermediate. The Q loop is found between {alpha}5 and {alpha}6 and is located opposite the WPD loop.

The best studied non-receptor tyrosine phosphatase is the PTP1B tyrosine phosphatase, which contributes to the down-regulation of the insulin signaling pathway by dephosphorylating and inactivating the insulin receptor. PTP1B possesses a catalytic domain and, in contrast to PTPL1, no other modular domain. In vitro, PTP1B dephosphorylates the insulin receptor efficiently (18), and mice lacking PTP1B are sensitized to insulin (19). The insulin receptor is activated by phosphorylation of 2 adjacent tyrosine residues (1162 and 1163), and structural analysis of the PTP1B catalytic domain (18) has revealed that PTP1B possess two phosphotyrosine binding sites that are required for PTP1B to bind and dephosphorylate the insulin receptor efficiently. One of the phosphotyrosine binding sites is located within the active site, and the other is located in a nearby positively charged pocket (18). Interestingly, only PTP1B and the highly related TCPTP possess this second phosphotyrosine binding site. This observation suggests that PTP1B and TCPTP have evolved specifically to dephosphorylate the receptors for insulin and growth factors that contain two adjacent phosphotyrosine residues. To learn more about the potential roles of PTPL1, we have determined and analyzed the structure of the catalytic domain of PTPL1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Protease-inhibitor mixture was from Roche Applied Science, p-nitrophenyl phosphate (pNPP) and lysozyme were from Sigma, glutathione-Sepharose was from Amersham Biosciences, and BIOMOL Green reagent was from BIOMOL research laboratory (Plymouth Meeting, PA). The PreScission protease with an N-terminal glutathione S-transferase (GST) tag was expressed by the protein production group of the Division of Signal Transduction Therapy Unit (University of Dundee, Scotland, UK) from a cDNA clone kindly provided by John Heath (University of Birmingham, UK). The mono-, bis-, and tris-phosphorylated peptides derived from the insulin receptor activation loop sequence TRDIYETDYYRK, in which phosphorylated residues are underlined, were synthesized using standard methodologies.

General Methods and Buffers—Restriction enzyme digests, DNA ligations, site-directed mutagenesis, and other recombinant DNA procedures were performed using standard protocols. Site-directed mutagenesis was performed using the QuikChange method (Stratagene). All DNA constructs were verified by DNA sequencing, which was performed by the Sequencing Service, School of Life Sciences, University of Dundee using DYEnamic ET terminator chemistry (Amersham Biosciences) on Applied Biosystems automated DNA sequencers. Protein concentration was determined using the Bradford assay with bovine serum albumin employed as the standard. Lysis buffer contained 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.27 M sucrose, 1 mM EGTA, 1 mM EDTA, 0.1% (v/v) {beta}-mercaptoethanol, 1 mg/ml lysozyme, 1 mg/ml DNase, and Complete proteinase inhibitors mixture (Roche Applied Science; one tablet/25 ml). Buffer A is 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.27 M sucrose, 1 mM EGTA, 1 mM EDTA, 0.1% (v/v) {beta}-mercaptoethanol, and Buffer B is 50 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.27 M sucrose, 1 mM EGTA, 1 mM EDTA, 0.1% (v/v) {beta}-mercaptoethanol.

Cloning, Expression, and Purification—The cDNA encoding full-length human PTPL1 (NCBI accession number NP_006255 [GenBank] ) was kindly provided by Dr C. H. Heldin (Ludwig Institute for Cancer Research, Biomedical Centre, Uppsala, Sweden) and utilized as template to amplify by PCR reaction a number of different DNA fragments encoding residues of PTPL1 that encompass the catalytic domain that were tested for crystallization. For example, the construct that successfully crystallized encompassed residues 2152–2485 was amplified using primers (5'-ggatccatgaatgggaagttatcagaagagaga-3' and 5'-gcggccgctcacttcagaagctgaggctgctgtttttg-3'). The resulting PCR product was ligated into pCR2.1 cloning vector (Invitrogen) and then subcloned further as a BamHI-NotI fragment into pGex-6-P1 expression vector (Amersham Biosciences). This encodes for the Escherichia coli expression of the catalytic fragment of PTPL1 with an N-terminal GST affinity purification tag that can be cleaved following digestion with the PreScission protease. BL21 E. coli cells were transformed with the pGEX6-PTPL1 vector cultured in Luria-Bertani broth with 100 µg of ampicillin/ml and were grown at 37 °C until the A600 reached 0.8, 0.25 mM isopropyl 1-thio-{beta}-D-galactopyranoside was added, and the culture was grown at 26 °C for 16 h. Cells derived from 4 liters of culture were harvested by centrifugation at 5000 x g for 30 min, lysed by suspension in 200 ml of ice-cold lysis buffer, and after incubation on ice for 30 min, were subjected to six cycles of sonication for 30 s each. The lysate was centrifuged at 20,000 x g for 30 min, and the supernatant was incubated for 1 h at 4 °C with 4 ml of glutathione-Sepharose. The beads were then washed three times with 40 ml of Buffer A, washed three times with 40 ml of Buffer B, and finally washed twice with 40 ml of Buffer A. The resin was resuspended to a final volume of 15 ml of Buffer A, the amount of protein bound to the resin was estimated, and 50 µg of GST-tagged PreScission protease/mg of protein bound to the glutathione-Sepharose was added. After overnight (16 h) incubation at 4 °C in which the resin was gently mixed, eluted PTPL1 in the supernatant fraction was passed over 1 ml of glutathione-Sepharose beads previously equilibrated in Buffer A to remove any remaining GST-PTPL1 or GST-PreScission protease. The eluted PTPL1 was then further purified on a Superdex 200 26/60 gel filtration column equilibrated against 25 mM Tris/HCl, pH 7.5, 15 mM NaCl, and 5 mM dithiothreitol. PTPL1 purified in this manner was homogeneous as assessed by SDS-PAGE and electrospray mass spectrometry.

Crystallization, Structure Solution, and Refinement—The purified PTPL1 was concentrated with a VivaScience concentrator until a concentration of 10 mg/ml was reached. Crystals were grown by the vapor diffusion method. 0.75 µl of protein solution was mixed with 0.75 µl of reservoir solution consisting of 0.1 M CAPS, pH 11, 1.7 M Na2HPO4, 0.4 M KH2PO4, and 0.2 M Li2SO4. Plate-shaped crystals grew in 16–20 h to a size of 0.1 x 0.1 x 0.50 mm. The crystals were cryoprotected with 3 M Li2SO4 before freezing. X-ray diffraction data were collected at The European Synchrotron Radiation Facility, Grenoble, France beamline BM14 and were processed with the HKL suite (19) (Table I).


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  		TABLE I
Crystallographic data and refinement statistics Values in parenthesis are reflections from the highest resolution shell. All data were included in the refinement.

 
The PTPL1 structure was solved by molecular replacement using AMoRe (20) and the PTP1B structure (Protein Data Bank code 1AAX [PDB] (21)) as a search model against 8–4 Å data. A single solution was found with an R-factor of 0.49 and a correlation coefficient of 0.26. This solution was then used for automatic model building with warpNtrace (22), which was able to build 292 out of 334 residues. Further refinement was performed using CNS (23) interspersed with model building in O (24) (Table I). The figures were made with PyMOL and GRASP (25)

Kinetic Analysis of PTL1 Activity—Phosphatase activity was measured by monitoring hydrolysis of pNPP using a spectrophotometric assay that detects formation of the reaction product para-nitrophenol by absorbance of 405 nm (26) or by monitoring the dephosphorylation of the insulin receptor peptide substrate (TRDIYETDYYRK) phosphorylated at the indicated underlined tyrosine residues, equivalent to Tyr-1158, Tyr-1162, and Tyr-1163 on the insulin receptor. For the pNPP assay, 50 ng of purified PTPL1 was incubated in a 50-µl solution containing 20 mM Tris, pH 7.4, 0.1 mg/ml bovine serum albumin, 150 mM NaCl, 3–100 mM pNPP, and 10 mM dithiothreitol. Reactions were terminated after 15 min by the addition of 100 µl of 1 M NaOH, and absorbance was measured at 405 nm, using a Versa max absorbance plate reader with Softmax Pro 4.0 software (Molecular Devices). Rates or amounts of para-nitrophenol production were calculated using a standard curve generated from known amounts of para-nitrophenol. For the insulin receptor peptide assay, a 50-µl reaction was set up containing 10 ng of PTPL1 in a solution of 20 mM Tris, pH 7.4, 0.1 mg/ml bovine serum albumin, 150 mM NaCl, 10 mM dithiothreitol, and 3–500 µM the indicated tyrosine-phosphorylated peptide. After 10 min at room temperature, the reactions were terminated by the addition of 100 µl of BIOMOL Green reagent, and after an additional 20 min, the amount of phosphate liberated was quantitated by absorbance at 620 nm and measured against inorganic phosphate standards. In all assays, we ensured that the rate of para-nitrophenol formation or the dephosphorylation of the insulin receptor peptide was linear with time and that less than 10% of the substrate was consumed in the assay. The steady state kinetics parameters Km and kcat were determined from a direct fit of the data to the Michaelis-Menten equation using the non-linear curve fitting Prism program (version 3, GraphPad Software, Inc.). All points were performed in triplicate, and experiments were repeated at least two times to ensure that consistent results were obtained.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
The PTPL1 Structure—To identify the boundaries of a soluble fragment of the catalytic domain of PTPL1, a limited proteolysis approach was performed in which a fragment of PTPL1 encompassing residues 2087–2485 was digested with trypsin. This resulted in a proteolytically stable fragment comprising residues 2152–2485 (data not shown), encompassing the PTP domain with an additional 40 residues at the N terminus. We cloned and expressed this fragment of PTPL1 in E. coli and, following purification to homogeneity, it showed good stability and specific activity when assayed with either pNPP or the insulin receptor peptide substrate as compared with either full-length PTPL1 or the 2087–2485 fragment. This fragment of PTPL1 was readily crystallized using a high (>2 M) concentration of phosphate as the precipitant. Synchrotron diffraction data were collected to 1.85 Å resolution, and the structure was solved by molecular replacement followed by refinement to a final R-factor of 0.177 (Rfree = 0.205) (Table I). The structure contains two disordered loops encompassing the first 18 residues (2152–2170) and the final 8 residues (2477–2485). The PTPL1 phosphatase domain displays the classic PTP1B fold (16) described in the Introduction (Fig. 1). The only difference in secondary structure between PTPL1 and PTP1B is an additional {alpha}-helix (termed {alpha}0) located at the N terminus (Fig. 1A). The PTPL1 phosphate binding loop, containing the catalytic cysteine (Cys-2408), is located between {beta}12 and {alpha}4, the WPD loop is located between {beta}11 and {alpha}3, and the Q-loop is located between {alpha}5 and {alpha}6. These key catalytic residues are located at equivalent positions to all other PTPs crystallized to date.



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  		FIG. 1.
Comparison of the structure of the catalytic domain of PTPL1 and PTP1B. A, overall ribbon structure of the PTPL1 catalytic domain (residues 2152–2485) phosphate complex (left panel). A 2 Fo - Fc, {varphi}calc electron density map for the phosphate molecule is drawn in red and displays well ordered density. The key features of the structure that are described under the Introduction are indicated in magenta. The N-terminal {alpha}0 helix that replaces the {alpha}7 helix on PTP1B is displayed in yellow. Shown in stick representation are Cys-2408, Asp-2378, Arg-2205, Ile-2458, and Met-2307. The electrostatic potential of the surface of PTPL1 and the location of the positively charged primary and secondary phosphotyrosine binding pockets are indicated (right panel). The blue areas (+6kT) represent highly positively charged residues, and the red areas (-6kT) represent highly negatively charged residues. B, overall ribbon structure of the PTP1B catalytic domain (residues 1–298, left panel). The electrostatic potential of the surface of PTP1B, calculated without the peptide bound to the enzyme, complexed to the phosphorylated insulin receptor peptide phosphorylated at residues equivalent to Tyr-1162 and Tyr-1163 on the insulin receptor that are located in the primary and secondary phosphotyrosine binding pockets, respectively is shown in the right panel.

 
Interestingly, from the early stages of the refinement, well ordered, tetrahedral-shaped, electron density was observed near the Cys-2408 S{gamma} atom (Fig. 1A). As the crystallization mother liquor contained 2.1 M phosphate, the density was refined as a phosphate ion, and the structure is therefore likely to represent a product complex. The phosphate is situated at the amino terminus of {alpha}4. The three phosphate oxygen atoms facing the PTPL1 core form hydrogen bonds to the backbone nitrogens of residues Ala-2410, Ile-2412, Gly-2413, and Arg-2414, as well as to the guanidinium group of Arg-2414. The fourth oxygen projects toward the solvent and hydrogen-bonds with the catalytic acid Asp-2378 located 2.6 Å away, which is presumably protonated, like the equivalent residue in other PTPs such as PTP1B (17). To our knowledge, none of the receptor or non-receptor PTPs reported to date possess a phosphate at the catalytic site. Several PTPs have been crystallized with the phosphate mimetics vanadate and sulfate that are localized at a similar position to the phosphate group on PTPL1 (27–30). It is believed that the WPD loop in PTPs moves into a "closed" conformation when substrates bind (31). Comparison of the structure of PTPL1 with PTP1B complexed to the insulin receptor substrate peptide (18) suggests that the WPD loop in the PTPL1 structure is in the closed substrate-bound conformation.

Comparison with PTP1B, Helix Swapping—Although the overall fold of PTPL1 is similar to that of PTP1B, there is one remarkable difference. In PTP1B, the {alpha}7 helix is located at the C terminus of the protein and buries helix {alpha}3 (Fig. 1B). In PTPL1, however, the situation is markedly different. Helix {alpha}7 does not exist (the PTPL1 structure ends with helix {alpha}6), but the PTPL1 {alpha}0 helix, located at the N terminus, occupies a topologically equivalent position to helix {alpha}7 in PTP1B, also interacting with helix {alpha}3. For instance, residue Leu-192 on helix {alpha}3 of PTP1B forms a hydrophobic interaction with residue Trp-291 on helix {alpha}7, whereas the equivalent Leu-2389 on helix {alpha}3 in PTPL1 interacts with Tyr-2466 in helix {alpha}0. Structure-based sequence alignment of PTPL1 with PTP1B and three other structurally characterized PTPs shows that the N-terminal residues of PTPL1 that form {alpha}0 do not align with the other PTPs (Fig. 2).



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  		FIG. 2.
Structure-based sequence comparison of PTPL1 with other PTPs. A, structure-based sequence alignment of the catalytic domain of PTPL1 with the indicated PTPs, the structures of which have been solved. PTP1B and the highly related TCPTP possess a secondary phosphotyrosine pocket, whereas PTPalphaD1 and LAR do not. Conserved {alpha}-helical regions are shown in red, the non-conserved {alpha}-helix is in yellow, and the conserved {beta}-strands are in blue. The numbering of the residues is based on PTPL1, and the position of the catalytic PTP phosphate binding loop, WPD loop, and Q-loop are shown. The catalytic cysteine is marked with a red star, and the residues lining the secondary phosphotyrosine pocket on PTPL1 are marked with a blue circle. The residues on PTPL1 that have been found to be mutated in human colorectal tumors are marked with a green triangle The Protein Data Bank entries are as follows: PTP1B, 2HNQ [PDB] ; TCPTP, 1L8K; PTPaplhaD1, 1YFO; and LAR, 1LAR. The alignment is graphically represented with the computer program ALINE (see Footnote 2). B, PTP activity of the indicated forms of PTPL1 was assessed with the tris-phosphorylated insulin receptor peptide TRDIY(P)ETDY(P)Y(P)RK (upper panel)or pNPP (lower panel). Each point represents the mean specific activity ± S.D. of two independent experiments each assayed in triplicate. 2 µg of each form of PTPL1 was subjected to SDS-polyacrylamide gel electrophoresis, which was stained with Coomassie Blue to demonstrate similar expression the different forms of PTPL1.

 
To investigate the importance of the {alpha}0 helix, we compared the catalytic activities of the PTPL1 construct that was crystallized (residues 2152–2485) with a fragment of PTPL1 lacking the {alpha}0 helix, termed {Delta}{alpha}0-PTPL1 (residues 2209–2485) (Fig. 2B). Interestingly, although this form of PTPL1 was well expressed, it possessed the same very low catalytic activity as a PTPL1 mutant in which the catalytic Cys-2408 had been mutated to Ser (Fig. 2B). These findings are consistent with the notion that {alpha}0 helix plays a crucial role in stabilizing PTPL1 in a correctly folded and active conformation. 2

A Second Phosphotyrosine Binding Pocket—All residues that contact the ordered phosphate in the PTPL1 active site are conserved with PTP1B (16, 18) (Fig. 2). As discussed in the Introduction, structural analysis of PTP1B complexed with the insulin receptor peptide substrate phosphorylated at 3 tyrosines has revealed that PTP1B possesses a secondary phosphotyrosine binding site in a positively charged pocket next to the active site (18). Mutational analysis established that this second phosphotyrosine binding site was required for PTP1B to efficiently dephosphorylate the insulin receptor peptide phosphorylated at two adjacent tyrosine residues. In PTP1B, the second phosphotyrosine binding pocket comprises Arg-24, Arg-254, Met-258, and Gly-259 (Fig. 3). The Arg residues make specific ionic interactions with the phosphotyrosine phosphate, whereas the Met does not interact with the phosphotyrosines (18). The Gly-259 residue lying at the base of the pocket is thought to comprise the structurally most important residue of this site as its small size and flexibility is required to accommodate phosphotyrosine (32). Unexpectedly, surface analysis of the PTPL1 structure suggests the presence of a similar, positively charged pocket, located next to the active site (Figs. 1 and 3). The residues that comprise this pocket are Gln-2221, Arg-2444, His-2448, and Gly-2449. Furthermore, a structure-based sequence alignment of PTPL1 with PTP1B shows that these residues align with Arg-24, Arg-254, Met-258, and Gly-259, which form the secondary phosphotyrosine binding site in PTP1B, as well as the equivalent residues in TCPTP (Fig. 2A). Although the PTP1B Arg-254 is conserved in all PTPs, Arg-24 is not conserved in the structurally characterized PTP{alpha}D1 and LAR phosphatases that do not possess a secondary phosphotyrosine binding site. Moreover, the residues in the PTP{alpha}D1 and LAR that are equivalent to Gly-259 in PTP1B/Gly-2449 in PTPL1 are bulkier Gln and Tyr residues, respectively (Fig. 2A).



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  		FIG. 3.
Comparison of the catalytic and secondary phosphotyrosine binding site of PTPL1 and PTP1B. A, a ribbon drawing of the catalytic center of PTPL1 displaying the electron density of the phosphate molecule shown in red. Shown in stick representation are Cys-2408; Asp-2378 in the WPD loop; Gln-2452 from the Q-loop. Also shown are the His-2448, Gln-2221, Gly-2449, and Arg-2444 that make up the secondary phosphotyrosine binding pocket. Superimposed on the structure is a model of how the phosphorylated insulin receptor peptide (ETDY(P)Y(P)R) might interact with PTPL1 based on the structure of this peptide with PTP1B. B, a ribbon drawing of the catalytic center of PTP1B complexed to the phosphorylated insulin receptor peptide (ETDY(P)Y(P)R). Shown in stick representation are Ser-215 (replacing the catalytic cysteine in trapping mutant PTP1B-C215S); Asp-181 in the WPD loop; Gln-262 from the Q-loop. Also shown are the Arg-24, Met-258, Gly-259, and Arg-254 in the secondary phosphotyrosine binding pocket. Note that His-2448 located on the loop between {alpha}5 and {alpha}6 in PTPL1 is structurally replacing Arg-24 in PTP1B located on the {alpha}2'-helix.

 
Structural comparison of PTPL1 with the PTP1B-peptide complex reveals further (structural) conservation in the second phosphotyrosine binding site (Fig. 3). Although in PTP1B, Arg-24 (on helix {alpha}2') interacts with the second phosphotyrosine (pTyr-1163) in the peptide complex, this arginine is not present at the equivalent position in the PTPL1 sequence (Figs. 2 and 3). Strikingly, however, a histidine in PTPL1 (His-2448), located in a different position in the PTP fold, performs the same role, projecting down from the {alpha}5-{alpha}6 loop and positioning its imidazole group close to the putative second phosphotyrosine site (Fig. 3). This suggests that the PTPL1 substrate binding site could be structurally compatible with peptides carrying two neighboring phosphotyrosines.

Catalytic Analysis of Wild Type and Mutant PTPL1—PTPL1 dephosphorylates the insulin receptor peptide TRDIY(P)ETDY(P)Y(P)RK efficiently with a Km value of 21 µM and a kcat value of 13.1 s-1 (Fig. 4), not dissimilar to PTP1B, which dephosphorylates this peptide with a Km value of 8 µM and a kcat value of 11.3 s-1 (18). It should be noted that we assay PTPL1 at pH 7.5, which we have found to be the optimal pH (data not shown), whereas PTP1B is generally assayed at its pH optimum of pH 5.5. To investigate the presence of a secondary phosphotyrosine binding pocket, we compared the rates at which PTPL1 dephosphorylated the tris-phosphorylated TRDIY(P)ETDY(P)Y(P)RK, bis-phosphorylated TRDIYETDY-(P)Y(P)RK, and mono-phosphorylated TRDIYETDYY(P)RK and TRDIYETDY(P)YRK peptides. We found that the bis-phosphorylated TRDIYETDY(P)Y(P)RK peptide was dephosphorylated by PTPL1 with the same efficiency as tris-phosphorylated TRDIY(P)ETDY(P)Y(P)RK, indicating that phosphorylation of the first tyrosine (Tyr-1158) in these insulin receptor peptides does not affect the catalytic parameters of PTPL1. This is similar to the situation reported for PTP1B and suggests that there is no binding site on the phosphatase for this third phosphotyrosine. In contrast, both mono-phosphorylated peptides were much poorer substrates for PTPL1 than the bisphosphorylated peptide. Kinetic analysis revealed that the decrease in catalytic efficiency resulted from a 10-fold increase in Km for the mono-phosphorylated peptides rather than a change in kcat (Fig. 4).



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  		FIG. 4.
Evidence that PTPL1 possesses a second phosphotyrosine pocket. The PTPL1 catalytic domain (residues 2152–2485) was assayed with the indicated concentrations of the phosphorylated insulin receptor peptides (upper panel). Each point represents the mean specific activity ± S.D. of three independent experiments each assayed in triplicate. This data were employed to calculate the Km and kcat values from nonlinear regression plots as described under "Experimental Procedures" (lower panel).

 
These findings are consistent with the notion that PTPL1 possesses a secondary phosphotyrosine binding pocket. To confirm that this corresponds to the region on the PTPL1 crystal structure, we mutated the residues that comprise this pocket and determined how this affected the ability of PTPL1 to dephosphorylate the bis-phosphorylated peptide. Mutation of Arg-2444 (equivalent to Arg-254 in PTP1B) to Lys, Gln, or Glu virtually abolished PTPL1 activity toward both the insulin receptor peptide as well as pNPP, which would be expected to only interact with the primary pocket. These findings suggest that the mutations induce conformational changes that disrupt the active site, although the mechanism by which this occurs is not clear. Interestingly, mutation of Arg-254 in PTP1B also inactivates the phosphatase (18). Similar results were obtained when substituting Gly-2449 (equivalent to Gly-259 in PTP1B) with the bulkier valine, with the aim of reducing the size of the second phosphotyrosine pocket. The PTPL1 G2449V mutant not only showed a significant reduction in both activity and affinity against the pTyr peptide but also against pNPP and mono-phosphorylated forms of the insulin receptor peptides. It has recently been suggested that the equivalent mutation in PTP1B significantly affects the activity due to a reduction in flexibility of the Q-loop, which carries the glutamine (Gln-262) involved in coordinating the hydrolytic water molecule (33). As shown in Fig. 3, the equivalent Gln-2252 in PTPL1 is located at the same position as in PTP1B, and mutation of Gly-2449 in PTPL1 could thus affect the position of Gln-2252 similarly. Mutation of Gln-2221 and His-2448, either individually or together, resulted in a 2–3-fold increase in the Km values for the bis-phosphorylated peptide, whereas not affecting kcat. Importantly however, the Gln-2221 and His-2448 PTPL1 mutants dephosphorylated pNPP or the mono-phosphorylated insulin receptor peptide with similar efficiency to wild type PTPL1 (Fig. 5). This observation supports the notion that the Gln-2221 and His-2448 residues contribute to a secondary phosphotyrosine binding site that enhances the rate at which PTPL1 dephosphorylates the insulin receptor peptide. Despite considerable effort, we were unable to co-crystallize the catalytic domain of PTPL1 with the TRDIYETDY(P)Y(P)RK peptide. As the PTPL1 catalytic domain only crystallized in the presence of 2.1 M phosphate, the high ionic strength and competition of the excess of phosphate for the active site may hinder binding of the insulin substrate peptide to PTPL1.



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  		FIG. 5.
Mutational analysis of residues making up the secondary phosphotyrosine pocket in PTPL1. The wild type and indicated mutants of the PTPL1 catalytic domain (all encompassing residues 2152–2485) were assayed with the indicated concentrations of the bisphosphorylated insulin receptor peptide TRDIY(P)ETDY(P)Y(P)RK (upper panel). Each point represents the mean specific activity ± S.D. of three independent experiment, each assayed in triplicate. These data were employed to calculate the Km and kcat values shown in Row 1 from non-linear regression plots as described under "Experimental Procedures" (lower panel). The data shown in Rows 2–4 were obtained as above except that the indicated pNPP or mono-phosphorylated forms of the insulin receptor peptides were employed.

 
PTPL1 and Cancer—As described in the Introduction, five PTPL1 point mutations (D2154H, E2474D, R2205W, I2458V, and M2307T), lying within the PTP domain, have been identified in patients with colorectal cancers (6). Two of these mutations, D2154H and E2474D, lie at the extreme termini of the crystallized PTP domain and are disordered. Arg-2205 and Ile-2458 lie on the surface of the catalytic domain remote from the active site (Fig. 1A). Met-2307 is fully conserved in all tyrosine phosphatases (reviewed in Ref. 34) and is positioned immediately behind the catalytic cysteine (Fig. 1A), contributing to the hydrophobic core between the {beta}-sheet {beta}3-{beta}4 and helix {alpha}1. The PTPL1(D2154H), PTPL1(E2474D), PTPL1(R2205W), and PTPL1(I2458V) mutants possessed similar catalytic properties to wild type PTPL1 when assayed with the pNPP substrate (Table II). This is not unexpected from a structural analysis of the catalytic domain of PTPL1 as these residues are not located nearby to any catalytic motifs. As these residues on PTPL1 lie on the surface, they could affect cellular function by disrupting the binding of PTPL1 to protein partners, perhaps even substrates. Consistent with the Met-2307 being located nearby the active site, the PTPL1(M2307T) mutant showed significantly reduced catalytic efficiency possessing a 7-fold higher Km and 2-fold lower in kcat (Table II). It is possible that replacement of this residue with a threonine disrupts the packing behind the catalytic cysteine. Attempts at crystallizing the PTPL1(M2307T) mutant were unsuccessful.


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  		TABLE II
Kinetic parameters of PTPL1 mutants found in colorectal cancer The wild type and indicated mutants of the PTPL1 catalytic domain (all encompass residues 2152–2485) were assayed with pNPP, and kinetic parameters were calculated employing the same approach described in the legend for Fig 5. The data shown are the mean specific activity ± S.D. and calculated from data of three independent experiment each assayed in triplicate.

 
Conclusions—The most interesting and unexpected feature of the PTPL1 catalytic domain structure is the presence of a second phosphotyrosine binding pocket adjacent to the catalytic core. We demonstrate that this plays a similar role to the equivalent pocket on PTP1B and enables PTPL1 to dephosphorylate the bis-phosphorylated insulin receptor peptide with similar catalytic efficiency to PTP1B. The finding that overexpression of PTPL1 in cells down-regulates PI 3-kinase signaling responses (5) also provides functional evidence that PTPL1 may be dephosphorylating tyrosine kinase receptors and/or their substrates. Apart from the insulin receptor, the IGF1 receptor also contains two adjacent tyrosine residues (Tyr-1280/Tyr-1281) (35) and many growth factor receptors, and the substrates they phosphorylate contain clusters of phosphotyrosine residues that may be dephosphorylated more efficiently by a tyrosine phosphatase containing multiple phosphotyrosine recognition pockets. Interestingly, the platelet-derived growth factor-{alpha} (PDGF{alpha}) receptor contains a phosphorylated Ser residue (Ser-767) adjacent to Tyr-768 that is phosphorylated (36). It would be interesting to investigate whether a phosphorylated Ser residue could interact with the secondary phosphotyrosine site on PTPL1, thereby enhancing dephosphorylation of Tyr-768. The findings that PTPL1 is associated with the plasma membrane through its FERM domain (37) and that the cytosolic PTPL1-TAPP1 complex can translocate to the plasma membrane in response to agonists that generate PtdIns(3,4)P2 (4) also support the notion that PTPL1 could down-regulate insulin growth factor-mediated signaling processes. As (PtdIns(3,4)P2) is generated as a breakdown product of phosphatidylinositol (3,4,5)trisphosphate (PtdIns(3,4,5)P3), the TAPP1-mediated recruitment of PTPL1 to the plasma membrane could serve as a simple mechanism to switch off PI 3-kinase signaling pathways.

PTP1B plays a major role in dephosphorylating the insulin receptor, although it is located in the endoplasmic reticulum rather than the plasma membrane (38). PTP1B can therefore only inactivate receptors that are brought to this location by cellular trafficking processes. It is possible that PTPL1 may play a role in inactivating the plasma membrane-localized pool of the insulin receptor or other receptors and/or their substrates. The observation that insulin signaling processes are still inactivated in PTP1B-deficient mice provides genetic evidence that PTPs other than PTP1B must be capable of inactivating the insulin receptor in vivo. The generation of a PTPL1-deficient mouse would be required to obtain more definitive evidence as to whether PTPL1 functions as a negative regulator of the insulin-signaling pathway. If PTPL1 does play a role in down-regulating the insulin signaling pathway, then inhibitors of PTPL1 might be useful for the treatment of diabetes. The availability of a crystal structure of the catalytic core of this enzyme would greatly aid the process of identifying compounds that target this enzyme. Analysis of the sequences of the 107 PTPs indicates that 11 have a glycine residue at the position equivalent to Gly-259 in PTP1B/Gly-2449 in PTPL1. This suggests that a significant number of these enzymes may also possess a secondary phosphotyrosine binding pocket.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1WCH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by grants from the Association for International Cancer Research (to D. R. A.), Diabetes UK (to D. R. A. and D. M. F. v. A.), the Medical Research Council (to D. R. A.), the Moffat Charitable Trust (to D. R. A.), and the pharmaceutical companies supporting the Division of Signal Transduction Therapy (Astra-Zeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck & Co. Inc, Merck KGaA, and Pfizer). 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. Back

Supported by a Diabetes UK studentship. Back

** To whom correspondence should be addressed. Fax: 44-1382-345764; E-mail: dava{at}davapc1.bioch.dundee.ac.uk.

1 The abbreviations used are: PTPL1, protein-tyrosine phosphatase-L1; PtdIns(3,4)P2, phosphatidylinositol 3,4 bisphosphate; pNPP, p-nitrophenyl phosphate; GST, glutathione S-transferase; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PI, phosphatidylinositol; TCPTP, T cell protein tyrosine phosphatase; LAR, leukocyte antigen related. Back

2 C. S. Bond, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank David Komander for helpful advice and discussion. We also thank the Sequencing Service (School of Life Sciences, University of Dundee) for DNA sequencing, Nick Morrice and David Campbell for mass spectroscopy analysis, the protein production and antibody purification teams (Division of Signal Transduction Therapy (DSTT), University of Dundee) coordinated by Hilary McLauchlan and James Hastie for expression and purification of PreScission protease and the European Synchrotron Radiation Facility, Grenoble for the time at beamline BM14. We are grateful to Charlie Bond for providing us with the ALINE program.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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