Structure, inhibitor, and regulatory mechanism of Lyp, a lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases
Edited by Arthur Weiss, University of California School of Medicine, San Francisco, CA, and approved October 25, 2007
Abstract
The lymphoid-specific tyrosine phosphatase (Lyp) has generated enormous interest because a single-nucleotide polymorphism in the gene (PTPN22) encoding Lyp produces a gain-of-function mutant phosphatase that is associated with several autoimmune diseases, including type I diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus. Thus, Lyp represents a potential target for a broad spectrum of autoimmune disorders. Unfortunately, no Lyp inhibitor has been reported. In addition, little is known about the structure and biochemical mechanism that directly regulates Lyp function. Here, we report the identification of a bidentate salicylic acid-based Lyp inhibitor I-C11 with excellent cellular efficacy. Structural and mutational analyses indicate that the inhibitor binds both the active site and a nearby peripheral site unique to Lyp, thereby furnishing a solid foundation upon which inhibitors with therapeutic potency and selectivity can be developed. Moreover, a comparison of the apo- and inhibitor-bound Lyp structures reveals that the Lyp-specific region S35TKYKADK42, which harbors a PKC phosphorylation site, could adopt either a loop or helical conformation. We show that Lyp is phosphorylated exclusively at Ser-35 by PKC both in vitro and in vivo. We provide evidence that the status of Ser-35 phosphorylation may dictate the conformational state of the insert region and thus Lyp substrate recognition. We demonstrate that Ser-35 phosphorylation impairs Lyp's ability to inactivate the Src family kinases and down-regulate T cell receptor signaling. Our data establish a mechanism by which PKC could attenuate the cellular function of Lyp, thereby augmenting T cell activation.
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Protein tyrosine phosphorylation mediates multiple signal transduction pathways that play key roles in innate and acquired immunity (1, 2). The level of tyrosine phosphorylation is controlled by the coordinated action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The importance of the PTKs in the immune system are well recognized and widely appreciated. However, the functional significance of the PTPs in regulating various immune responses is far from clear. The lymphoid-specific tyrosine phosphatase (Lyp) has received enormous attention because of the finding that a single-nucleotide polymorphism (SNP) in the gene (PTPN22) encoding Lyp is associated with several autoimmune diseases, including type I diabetes (3), rheumatoid arthritis (4, 5), Graves disease (6), and systemic lupus erythematosus (7). Lyp is a 110-kDa protein consisting of an N-terminal PTP domain and a noncatalytic C-terminal segment with several Pro-rich motifs (8, 9). Lyp belongs to a subfamily of PTPs, which include PTP-PEST (PTPN12), PTP-HSCF/BDP1 (PTPN18), and Lyp/PEP (PTPN22) (10). Biochemical studies suggest that Lyp inhibits T cell activation, likely through dephosphorylation of the T cell receptor (TCR)-associated Lck and ZAP-70 kinases (9, 11, 12). Interestingly, the disease-causing SNP (a C-to-T substitution at position 1858 in the coding region of Lyp) produces an amino acid substitution (R620W) within the first Pro-rich region in the C terminus, thereby impairing Lyp binding to the Src homology 3 (SH3) domain of Csk (3, 4). Moreover, it has been shown that the autoimmune-predisposing variant of Lyp is actually a gain-of-function mutation, generating a more active phosphatase that is more effective in inhibiting T cell signaling than the wild-type enzyme (13).
Given the strong association of the C1858T polymorphism with various autoimmune disorders and the elevated phosphatase activity associated with the resultant Lyp/R620W variant, Lyp represents a potential target for a broad spectrum of autoimmune diseases. Small-molecule Lyp inhibitors may have therapeutic value for treating these disorders. In addition, specific Lyp inhibitors will be useful in delineating the mechanism of Lyp in T cell signaling, development, and differentiation. To aid the design of Lyp inhibitors and gain insight into the regulatory mechanism for Lyp, we have determined the crystal structures of the Lyp PTP domain either alone or in complex with a selective small-molecule inhibitor. Structural analysis together with mutational studies led to the identification of molecular determinants that can be exploited for the acquisition of more potent and selective Lyp inhibitors. In addition, the structures revealed a unique flexible region in Lyp, which harbors a consensus protein kinase C (PKC) phosphorylation site (Ser-35). We demonstrated that Lyp can be phosphorylated by PKC on Ser-35, which impairs the Lyp-mediated substrate dephosphorylation and signaling in T cell. This finding establishes a mechanism whereby signaling through PKC may directly influence the cellular processes regulated by Lyp.
Results and Discussion
Identification of a Lyp-Specific Inhibitor.
Despite the promise of Lyp-directed therapeutics for autoimmune diseases, no Lyp inhibitor has been reported. Because PTPs share a high degree of structural conservation in the active site, i.e., the pTyr binding pocket, designing active site-directed inhibitors with both high affinity and selectivity for these enzymes is quite a challenge. Fortunately, it has been shown that pTyr alone is not sufficient for high-affinity binding and residues flanking the pTyr are important for PTP substrate recognition (14). These studies point to a unique paradigm for PTP inhibitor design, namely bidentate ligands that can engage both the active site and an adjacent less-conserved subpocket for enhanced affinity and selectivity. As an initial effort to develop Lyp inhibitors, we screened an 80-member focused library that was designed to bridge the PTP active site and an adjacent peripheral site. The ability of the compounds to inhibit the Lyp-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was assessed at pH 7 and 25°C. Compound I-C11 (Fig. 1) was identified as the most potent and selective Lyp inhibitor from the library with an IC50 of 4.6 ± 0.4 μM. Kinetic analysis indicated that I-C11 is a reversible and competitive inhibitor for Lyp with a Ki of 2.9 ± 0.5 μM. To examine the specificity of I-C11 for Lyp, its inhibitory activity toward a panel of PTPs including cytosolic PTPs, PTP1B, SHP2, HePTP, PTP-Meg2, and FAP1, the receptor-like PTPs, CD45, LAR, and PTPα, and the dual specificity phosphatase VHR, was determined. As shown in Table 1, 1-C11 is reasonably selective for Lyp, exhibiting, with one exception, >7-fold selectivity against all PTPs examined. The sole exception is PTP1B, which is more related to Lyp. A 2.6-fold preference for Lyp was observed in the latter instance.
Fig. 1.

Table 1.
PTP | IC50, μM |
---|---|
Lyp | 4.6 ± 0.4 |
PTP1B | 11.8 ± 1.8 |
SHP2 | 36.0 ± 3.8 |
HePTP | 32.1 ± 3.2 |
PTP-Meg2 | 31.8 ± 4.6 |
FAP-1 | 36.8 ± 8.8 |
VHR | 103 ± 27 |
CD45 | 186 ± 14 |
LAR | No inhibition at 100 μM |
PTPα | No inhibition at 100 μM |
All measurements were made by using pNPP as a substrate at pH 7.0, 25°C, and ionic strength of 0.15 M.
Crystal Structures of Lyp.
To map the architecture of Lyp and determine the molecular basis for Lyp inhibition by I-C11, we crystallized the PTP domain of Lyp (residues 1–294) with and without I-C11. The 3D structures of Lyp alone or bound to I-C11 were solved by molecular replacement using the coordinates of PTP1B (15) as a search model and refined to 3.0- and 2.8-Å resolution, respectively. The details of the crystals and structure solution are summarized in [supporting information (SI) Table 3]. While this work was in progress, coordinates of the PTP domains of Lyp and BDP1 were deposited in the Protein Data Bank (ID codes 2P6X and 2OC3, respectively. Like other PTPs, the Lyp PTP domain adopts a compact α+β structure comprising a central eight-stranded β-sheet surrounded by six α-helixes on one side and two α-helixes on the other (Fig. 2A). The PTP signature motif (H226CSAGCGR233) forms a loop (P loop) at the base of the active-site pocket. The overall structures of Lyp in the ligand free and bound forms are quite similar, with significant conformational differences confined to the WPD loop (residues 193–204), which contains the catalytically important general acid Asp-195, and an insert (residues 35–42) between α2′ and α1 that is unique to the Lyp subfamily of PTPs (Fig. 2). While the WPD loop exits in a half-open conformation in the apo-Lyp structure, it is fully open in the ligand-bound form to accommodate the inhibitor in the active site. Interestingly, the Lyp-specific insert can either adopt a loop conformation in the apo-structure or a helical conformation in the Lyp·I-C11 complex (Fig. 2A). As will be shown below, this conformational flexibility may be important for Lyp substrate/ligand recognition.
Fig. 2.

A comparison with published PTP structures using DALI (16) reveals that the Lyp structure is most similar to the catalytic domain of PTP1B with a Z score of 33.3 and a rmsd of 1.9 Å for all Cα pairs. In general, the core elements of Lyp superimpose well with those of PTP1B (Fig. 2A). However, several surface loops surrounding the active site display substantial differences between the Lyp structures and that of PTP1B. For example, Lyp has a longer loop between α1 and β1 (residues 75–80). Both the WPD loop and the loop between α3 and β8 (residues 216–222) of Lyp assume conformations that are different from those in PTP1B. Finally, the most striking difference between Lyp and PTP1B resides in the Lyp-specific insert (residues 35–42), which is absent in other PTP subfamilies. These structural variations in the surface loops surrounding the active site may contribute to the distinct PTP substrate/ligand binding specificity.
Molecular Basis of Lyp Inhibition by I-C11.
Fig. 3A depicts the overall structure of Lyp showing electron density for I-C11 contoured at 2.0 σ in the simulated annealing Fo − Fc OMIT map. The structure reveals that I-C11 interacts with both the active site and a nearby peripheral site. Consistent with the ability of salicylic acid derivatives to serve as effective pTyr surrogates (17, 18) and the observed competitive mode of Lyp inhibition by I-C11, the benzofuran salicylic acid moiety occupies the Lyp active-site pocket. The remarkable potency and selectivity of I-C11 for Lyp are the results of numerous specific interactions (Fig. 3B). The benzofuran salicylic acid engages in both polar and hydrophobic interactions with the Lyp active site. The carboxylic acid of the inhibitor forms hydrogen bonds with the main-chain amide of Ala-229, the side chains of Cys-227 and Cys-129, and charge–charge interactions with Arg-233 and Lys-138. The adjacent hydroxyl group makes additional hydrogen bonds with Glu-133, which further strengthens polar interactions with the active site. In addition to the polar interactions, the benzofuran ring participates in aromatic–aromatic stacking interactions with Tyr-60 in the pTyr binding loop and van der Waals contacts with the aliphatic side chains of Gln-274 in the Q loop, Ala-229 and Ser-228 in the P loop, and Lys-138 in the loop between β3 and β4. Besides interactions with the Lyp active site, the triazolidin ring in the linker makes van der Waals contacts with side-chain atoms (Cδ, Oε1, and Nε2) of Gln-274. In addition, the distal naphthalene ring in I-C11 binds a peripheral site defined by Phe-28, Leu-29, and Arg-33 (Fig. 3B). Interestingly, these residues, which are located in α2′, border a binding pocket equivalent to the second aryl phosphate-binding site previously identified in PTP1B (19). This pocket (Fig. 2), with Arg-254 and Gly-259 (Arg-266 and Ser-271 in Lyp) in the bottom and surrounded by Arg-24, Met-258, and Gln-262 (Lys-32, Pro-270, and Gln-274 in Lyp), is important for PTP1B substrate recognition (20, 21) and has been targeted for PTP1B inhibitor development (22).
Fig. 3.

To further investigate the structural basis for Lyp inhibition by I-C11, we mutagenized several amino acids in Lyp, including Cys-129, Lys-138, Phe-28, Leu-29, and Arg-33, that were implicated in I-C11 binding and evaluated the effect on Lyp activity and inhibitor binding affinity. As shown in Table 2, no significant differences in the kinetic parameters for pNPP hydrolysis were observed for the wild-type and mutant Lyps, indicating that the mutations did not perturb the catalytic site's integrity. In support of the structural observations, replacement of Cys-129 and Lys-138 with an Ala reduced Lyp's affinity for I-C11 by 1.8- and 4.3-fold, respectively. Similarly, removal of the side chain at Phe-28, Leu-29, and Arg-33 also led to a reduction in I-C11 binding affinity, with a 7.6-, 2.6-, and 2.7-fold increase in the IC50 values for F28A, L29A, and R33A, respectively. Collectively, our structural and mutational analyses of the interactions between Lyp and I-C11 showed that the benzofuran salicylic acid occupies the active site, whereas the distal naphthalene ring makes hydrophobic interactions with a region close to the second aryl phosphate-binding site. It is worth noting that many of the residues that are in direct contact with I-C11, especially those in the peripheral site, are unique to Lyp, which provide a structural basis for Lyp inhibitor selectivity. The atomic-level information on the architecture of Lyp furnishes a solid foundation for the design of more potent and selective Lyp-based small-molecule therapeutics.
Table 2.
Lyp | kcat, S−1 | Km, mM | IC50 for I-C11, μM |
---|---|---|---|
WT | 0.70 ± 0.03 | 4.2 ± 0.4 | 4.6 ± 0.4 |
pLyp35 | 0.64 ± 0.03 | 4.7 ± 0.3 | 48 ± 7.4 |
F28A | 0.67 ± 0.07 | 4.0 ± 0.4 | 35 ± 3.8 |
L29A | 0.56 ± 0.05 | 3.9 ± 0.3 | 11.8 ± 0.9 |
R33A | 0.61 ± 0.03 | 3.8 ± 0.2 | 12.5 ± 2.9 |
S35E | 0.59 ± 0.8 | 4.5 ± 0.6 | 21 ± 3.1 |
T36E | 0.37 ± 0.03 | 2.4 ± 0.2 | 2.8 ± 0.2 |
S35E/T36E | 0.89 ± 0.04 | 6.9 ± 0.6 | 250 ± 45 |
C129S | 0.73 ± 0.6 | 2.4 ± 0.2 | 8.1 ± 0.9 |
K138A | 0.81 ± 0.07 | 2.8 ± 0.3 | 19 ± 2.5 |
All measurements were made by using pNPP as a substrate at pH 7.0, 25°C, and ionic strength of 0.15 M.
The Lyp-Specific Insert Harbors a PKC Phosphorylation Site.
Although Lyp is implicated as a negative regulator of TCR signaling, little is known about how Lyp is regulated. Interestingly, sequence analysis revealed that residues K32RQSTKYK39 in Lyp correspond to a consensus PKC phosphorylation motif, (K/R)XX(S/T)X(K/R) (23). To determine whether Lyp can be phosphorylated by PKC, we incubated the Lyp catalytic domain with recombinant PKCδ in the presence of [γ-32P] ATP. Lyp phosphorylation was monitored by the incorporation of radioactive γ phosphate into the protein and found to be time- and dose-dependent (Fig. 4A). In addition, Lyp phosphorylation was saturable with a stoichiometry of 1.08 ± 0.11 mol of phosphate/mol Lyp. The PKCδ-catalyzed Lyp phosphorylation was also examined by electrospray mass spectrometry. Under the same conditions, an increase of 81 Da in mass was observed for phospho-Lyp, consistent with a single residue being phosphorylated (data not shown). To identify the site of phosphorylation, we determined the effect of S35E or T36E mutation on the PKCδ-catalyzed Lyp phosphorylation. As shown in Fig. 4B, although Lyp/T36E could still be phosphorylated by PKCδ, replacement of Ser-35 by Glu abolished Lyp phosphorylation. The results suggest that PKCδ phosphorylates Lyp exclusively on Ser-35. We further demonstrated that the PKCδ-mediated Lyp phosphorylation follows Michaelis–Menten kinetics, with a kcat of 10.6 ± 0.4 min−1 and Km of 0.59 ± 0.07 μM. These values are within the normal range observed for PKC substrates (24, 25), indicating that Lyp/Ser-35 is an efficient substrate for PKCδ. We should note that the corresponding Ser-39 in PTP-PEST could also be phosphorylated by PKC (26). To show that Lyp is phosphorylated at Ser-35 in vivo, Jurkat T cells transfected with Lyp, Lyp/S35E, and Lyp/S35A were treated with phorbol 12-myristate 13-acetate. Western blot analysis with a phospho-specific antibody that recognizes PKC substrates with the motif (R/K)X(phosphoS)(Hyd)(R/K) revealed that PKC activation in T cells leads to Lyp phosphorylation (Fig. 4C).
Fig. 4.

Potential Role of Ser-35 Phosphorylation in Lyp Substrate Recognition.
The observation that Lyp is phosphorylated exclusively at Ser-35 by PKC raises the possibility that the activity of Lyp may be regulated by Ser-35 phosphorylation. Remarkably, the PKC phosphorylation site Ser-35 is located within the Lyp-specific insert (S35TKYKADK42) that exists in two discrete conformations in the apo- and ligand-bound Lyp structures (Fig. 2A). In our apo-Lyp structure, the insert assumes a loop conformation projecting out from the protein and making crystal packing contacts with residues Tyr-94, Met-245, Lys-248, and Leu-252 in the neighboring Lyp. In the Lyp·I-C11 complex, residues S35TKYKADK42 adopt a helical conformation and become part of α2′. The Lyp-specific insert is also helical in the apo-Lyp structure (Protein Data Bank ID code 2P6X) crystallized at a higher pH (7.9 vs. 5.9), indicating conformational flexibility in this region. In the helical form, Ser-35 faces the interior of the structure and makes a H-bond with the conserved Arg-266 at the bottom of the second aryl-phosphate binding pocket. Moreover, the side chain of Tyr-38 participates in favorable edge to face stacking interactions with the phenyl rings of Tyr-44 and Tyr-66. These interactions are important for the stabilization of the helical conformation of the Lyp-specific insert. Thus, phosphorylation at Ser-35 may destabilize the helical conformation of the Lyp insert region. Interestingly, in the helical conformation, residues Phe-28, Lys-32, Ser-35, and Lys-39 form part of the second aryl-phosphate binding pocket, which, in the case of PTP1B, has been shown to be important for substrate recognition (20, 21). Given the potential importance of the helical conformation to Lyp substrate recognition and the role of Ser-35 in maintaining this helical conformation, we hypothesized that Lyp phosphorylation by PKC at Ser-35 may regulate the Lyp-mediated substrate dephosphorylation.
Ser-35 Phosphorylation Impairs Lyp-Mediated Src Dephosphorylation.
To examine the possibility that Lyp activity may be regulated by PKC, we initially studied the effect of Ser-35 phosphorylation on Lyp-catalyzed hydrolysis of a small-molecule substrate pNPP. No significant changes in the kcat and Km values were observed for the pNPP reaction catalyzed by phospho-Lyp (Table 2). Thus, Ser-35 phosphorylation does not affect the intrinsic Lyp phosphatase activity. We then assessed the effect of Lyp phosphorylation on more physiologically relevant substrates. Previous studies suggest that Lyp is a potent negative regulator immediately downstream of TCR (27) and identify the Src family kinase Lck as a cellular substrate for Lyp (9, 12). We were unable to prepare sufficient quantities of recombinant phospho-Lck for kinetic analysis. As an alternative, we turned to Src, which has almost identical residues surrounding the regulatory tyrosine phosphorylation sites and shares >60% of overall sequence identity with Lck.
The activity of the Src family kinase is regulated by phosphorylation at two distinct tyrosine residues. Autophosphorylation of Tyr-416 (Tyr-394 in Lck) in the kinase domain is required for Src activation. In contrast, phosphorylation of Tyr-527 (Tyr-505 in Lck) in the C-terminal tail by Csk inactivates Src because of an intramolecular pTyr-SH2 interaction (28, 29). We prepared doubly phosphorylated Src and measured Src dephosphorylation by purified Lyp, pLyp35, and Lyp/S35E by using phosphospecific pSrc416 and pSrc527 antibodies. As shown in Fig. 5A, the level of pSrc416 decreased by >75% when Src was incubated with Lyp for 5 min, whereas no significant change was detected in pSrc527. This result is consistent with the finding that Lyp negatively modulates the activity of Lck (9, 11, 27). Strikingly, <20% of pSrc416 was hydrolyzed by pLyp35 or Lyp/S35E under the same conditions. Time-course analysis showed that the rate of pSrc416 dephosphorylation was 8-fold slower for the pLyp35-catalyzed reaction (Fig. 5B). Apparently, Ser-35 phosphorylation inhibits Lyp activity toward pSrc416. Similar to pLyp35, mutation of Ser-35 to Glu had no effect on the Lyp-catalyzed pNPP hydrolysis (Table 2) but impeded pSrc416 dephosphorylation by 4-fold (Fig. 5B). Consequently, we conclude that Glu-35 mimics the effect of Ser-35 phosphorylation. Our results with Src highlight the importance of using physiologically relevant substrates in mechanistic characterization of the PTPs.
Fig. 5.

Phosphorylation at Ser-35 Suppresses Lyp Function in T Cell.
To further investigate the role of Ser-35 phosphorylation as a potential regulatory mechanism for Lyp function, we measured the status of Lck phosphorylation in human Jurkat T cells expressing either Myc-Lyp or Myc-Lyp/S35E, which functions as constitutively Ser-35 phosphorylated Lyp (Fig. 6A). Lck activation is an early event in TCR signaling, and stimulation of T cell with anti-CD3 antibody for 5 min led to a dramatic increase in Lck Tyr-394 phosphorylation. As expected, overexpression of Lyp resulted in a 2.4-fold decrease in pLck394 phosphorylation, consistent with a negative role for Lyp in T cell signaling. In accord with the reduced capacity of Lyp/S35E to dephosphorylate pSrc416, we found that Lyp/S35E also failed to dephosphorylate pLck394 inside the cell.
Fig. 6.

To provide further evidence that Ser-35 phosphorylation impairs Lyp's ability to down-regulate TCR signaling, we next examined two major pathways downstream of Lck, the Ras-Raf-MAPK module important for T cell proliferation, and the nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1), two critical transcription factors involved in TCR-induced IL-2 production (13, 18). In agreement with the effect on Lck, a 3.5-fold decrease in ERK1/2 activity was observed in the Lyp cells when compared with the vector control, whereas no appreciable change in ERK1/2 phosphorylation was observed as a result of Lyp/S35E overexpression (Fig. 6A). In addition, wild-type Lyp could reduce the TCR-induced NFAT/AP-1 transcriptional activation by nearly 3-fold, whereas Lyp/S35E was unable to attenuate NFAT/AP-1 activation (Fig. 6B). Taken together, the results support the conclusion that Ser-35 phosphorylation abrogates Lyp's ability to inactivate the Src family kinases and PKC plays a role in augmenting T cell activation by inhibiting Lyp.
The loss-of-function phenotype associated with pLyp35 or Lyp/S35E may result from a phosphorylation-induced structural change in the Lyp-specific insert (S35TKYKADK42), which in the helical conformation forms part of a presumed substrate recognition pocket (the second aryl phosphate-binding site). Because the distal naphthalene ring in I-C11 also binds the same area, one would predict that structural alterations at Ser-35 (phosphorylation or phosphorylation mimicking substitutions) should also reduce I-C11 binding affinity. Indeed, the IC50 values of I-C11 for pLyp35, Lyp/S35E, and S35E/T36E are 10.4-, 4.6-, and 54-fold higher than that of the wild-type Lyp (Table 2). These results further support the notion that Ser-35 phosphorylation obliterates a major substrate-binding element in Lyp and thus interferes with Lyp substrate recognition.
Inhibition of Lyp by I-C11 Enhances TCR Signaling.
Given the observed selectivity of I-C11 for Lyp, we proceeded to evaluate its ability to inhibit Lyp inside the cell. Jurkat T cells expressing either wild-type Lyp or the inhibitor insensitive Lyp/S35E were incubated with 20 μM I-C11 for 1 h and subsequently treated with or without anti-CD3 antibody for 5 min. As expected, I-C11 increased the TCR-stimulated Lck394 and ERK1/2 phosphorylation by 1.8- and 2.9-fold, respectively, in T cells expressing wild-type Lyp (Fig. 7). Again, Lyp/S35E was inefficient in down-regulating TCR signaling. No appreciable changes in pLck394 and pERK1/2 were observed when the Lyp/S35E-expressing cells were treated with the same concentration of I-C11.
Fig. 7.

Conclusions
We have identified a salicylic acid-based Lyp inhibitor from a focused library that binds with micromolar affinity and is relatively specific among the PTPs tested. Crystal structures of Lyp solved in the presence and absence of the I-C11 reveal that the inhibitor interacts with both the catalytic site and a cluster of unique resides adjacent to the active site. I-C11 is a Lyp specific inhibitor that exhibits cellular activity, and our studies provide a platform to generate future inhibitors that display therapeutic potency and selectivity. Further comparison of the apo- and I-C11-bound Lyp structures indicate that the Lyp-specific insert S35TKYKADK42 could adopt either a loop or helical conformation. Interestingly, the Lyp-specific insert harbors a PKC phosphorylation site, raising the possibility that the conformation of the insert region may be controlled by phosphorylation. Biochemical analyses show that Ser-35 is phosphorylated by PKC both in vitro and in vivo. We provide evidence that the status of Ser-35 phosphorylation dictates the conformational state of the insert region and thus Lyp substrate recognition. We further demonstrate that Ser-35 phosphorylation impairs Lyp's ability to inactivate the Src family kinases and down-regulate TCR signaling. Given the importance of PKC in TCR signaling (30), our data suggest a unique mechanism by which PKC could negatively regulate the cellular function of Lyp, thereby augmenting T cell activation.
Materials and Methods
Materials.
pNPP was purchased from Fluke. [γ-32P]-ATP was from Perkin–Elmer. The monoclonal anti-Myc antibody was from Upstate Biotechnology. Anti-Src, anti-Src/pY416, and anti-Src/pY527 antibodies were from Biosource Interantional. Polyclonal anti-ERK1/2, anti-phospho-ERK1/2, and anti-phospho(Ser) PKC substrate antibodies were purchased from Cell Signaling. Anti-CD3 (OKT3) was from eBioscience. All other reagents were obtained from Sigma.
Kinetics and Inhibition of Lyp-Catalyzed Substrate Dephosphorylation.
Initial rate measurements for the Lyp-catalyzed pNPP hydrolysis in the absence and presence of small-molecule inhibitors were determined as described (15). All assays were carried out at 25°C in 50 mM 3,3-dimethylglutarate (pH 7.0) buffer, containing 1 mM DTT and 1 mM EDTA, with an ionic strength of 0.15 M adjusted with NaCl. Recombinant Src protein phosphorylated at both Tyr-416 and Tyr-527 was used as a physiological substrate for Lyp. The Lyp-catalyzed Src dephosphorylation was carried out under the same conditions used for pNPP. The reaction was quenched by adding 1 mM pervanadate and the SDS buffer. The extent of the reaction was analyzed by Western blot and quantitated by densitometry.
Cell Culture, Transfection, Immunoblotting, and Luciferase Assay.
Jurkat T cells were grown at 37°C under an atmosphere of 5% CO2 in RPMI medium 1640 supplemented with 10% FBS. Full-length Lyp and Lyp/S35E mutant were subcloned into the pcDNA4/mycHis plasmid, and the resulting vectors were introduced into Jurkat T cells by electroporation. Forty-eight hours after transfection, the cells were treated with 5 μg/ml anti-CD3 antibody (OKT3; eBioscience) or medium for 5 min. Subsequently, cells were lysed in 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 50 mM NaF, 10 mM pyrophosphate, 5 mM iodoacetate, 1 mM sodium orthovanadate, 1 mM PMSF, and the protease inhibitor mixture. Cell lysates were subjected to SDS/PAGE and transferred electrophoretically to nitrocellulose membrane, which was immunoblotted by appropriate antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The luciferase assay was carried out as described (31). In general, 1 × 107 cells were transfected by electroporation with 2 μg of the NFAT/AP-1-luc plasmid, 50 ng of the Renilla-TK luciferase plasmid, and full-length Lyp plasmids or pcDNA4 vector. Forty-eight hours after transfection, cells were stimulated with OKT3 (5 μg/ml) or left untreated for 6 h. Dual luciferase activity was measured according to Promega's instruction, and NFAT/AP-1-luciferase activity was normalized by Renilla activity.
Details on expression and purification of Lyp catalytic domain, crystallization, data collection, structure determination, Lyp phosphorylation by PKC, and inhibition by I-C11 in Jurkat cells are provided in SI Text.
Data Availability
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2QCJ and 2QCT).
Acknowledgments
We thank Jim Hurley (National Institutes of Health, Bethesda) for the baculovirus pGEX-PKCδ expression vector, Robert Stahelin for advice with PKC assays, and Millie Georgiadis for assistance with crystallographic data analysis. This work was supported by National Institutes of Health Grant CA69202.
Supplementary Material
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© 2007 by The National Academy of Sciences of the USA.
Data Availability
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2QCJ and 2QCT).
Submission history
Received: July 5, 2007
Published online: December 11, 2007
Published in issue: December 11, 2007
Keywords
Acknowledgments
We thank Jim Hurley (National Institutes of Health, Bethesda) for the baculovirus pGEX-PKCδ expression vector, Robert Stahelin for advice with PKC assays, and Millie Georgiadis for assistance with crystallographic data analysis. This work was supported by National Institutes of Health Grant CA69202.
Notes
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0706233104/DC1.
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The authors declare no conflict of interest.
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Structure, inhibitor, and regulatory mechanism of Lyp, a lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases, Proc. Natl. Acad. Sci. U.S.A.
104 (50) 19767-19772,
https://doi.org/10.1073/pnas.0706233104
(2007).
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