Structure, inhibitor, and regulatory mechanism of Lyp, a lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases

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 S(35)TKYKADK(42), 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.

Fig. 1.

The chemical structure of I-C11.

Fig. 2.

Structural features of Lyp. (A) Superposition of the structures of apo (blue), I-C11 bound Lyp (green), and PTP1B (red; Protein Data Bank ID code 1G1H). (Right) Superposition of the overall structures. Besides the WPD loop, the most significant difference is the Lyp-specific insert (S³⁵TKYKADK⁴²) between α2′ and α1. This region can exist either as an extension of α2′ in the Lyp·I-C11 complex or a loop in the native structure. (Left) Superposition of the residues corresponding to the “second aryl phosphate-binding site” of PTP1B and the Lyp-specific insert region. Residues in apo-Lyp, Lyp·I-C11 complex, and PTP1B are depicted in blue, green, and red, respectively. (B) Sequence alignment of the Lyp-specific insert and α5-Q-loop with corresponding regions from PTP-PEST, BDP1, LAR, PTPα, SHP2, and PTP1B. The Lyp-specific insert is highlighted in red with the PKC phosphorylation site marked by a star. Residues involved in defining the second aryl phosphate-binding site are shown in blue. Lyp residues involved in binding the distal naphthalene ring in I-C11 are shown in green.

Fig. 3.

Structure of Lyp in complex with I-C11. (A) Overall structure of Lyp showing electron density of I-C11 contoured at 2.0 σ in the F_o − F_c simulated annealing OMIT map. (B) Detailed interactions between Lyp and I-C11. Hydrogen bonds and charge–charge interactions are depicted as black or red dotted lines, respectively. Residues involve in hydrophobic interactions were shown with a cutoff distance of 5 Å .

Fig. 4.

Lyp is phosphorylated at Ser-35 by PKC. (A) (Upper) Four micrograms of (His)₆-tagged Lyp catalytic domain was incubated with 40 ng of PKCδ with or without [γ-³²P]-ATP at 25°C for 20 min. (Lower) Four micrograms of Lyp was incubated with 40 ng of PKCδ with [γ-³²P]-ATP at 30°C and quenched at different time points. (B) Identification of the phosphorylation site in Lyp. Four micrograms of (His)₆-tagged wild-type, S35E, and T36E mutant Lyps were incubated with or without 40 ng of PKCδ, at 30°C for 40 min. The phosphorylation of Lyp was analyzed by autoradiography, and total Lyp protein was detected with anti-(His)₆ antibody. (C) Lyp phosphorylation by PKC in Jurkat cells, analyzed by an anti-phospho(Ser) PKC substrate antibody.

Fig. 5.

Dephosphorylation of pSrc416 by Lyp. (A) Lyp prefers pSrc416 over pSrc527, and Ser-35 phosphorylation or S35E substitution impairs Lyp-catalyzed hydrolysis of pSrc416. (B) Time course of pSrc416 dephosphorylation by Lyp (■), S35E (▴), and phospho-Lyp (●). Doubly phosphorylated Src (200 nM) was incubated with 10 nM Lyp, S35E, or phospho-Lyp. The dephosphorylation of pSrc416 or pSrc527 was monitored by Western blotting with specific antibodies.

Fig. 6.

Effect of Lyp and the phosphorylation mimicking Lyp/S35E on T cell activation. (A) Effects on T cell signaling. Jurkat T cells were transfected with the indicated plasmids and treated with medium or anti-CD3 antibody for 5 min. The level of Myc-tagged Lyp protein, Lck phosphorylation at both Tyr-394 and Tyr-505, total Lck protein, phosphorylated ERK1/2, and total ERK1/2 protein were detected by specific antibodies. (B) Effect on TCR-mediated transcriptional activation. Activation of an NFAT/AP-1-driven luciferase reporter gene in Jurkat T cells cotransfected with the indicated plasmids was measured after treatment with medium (empty bars) or anti-CD3 antibody (filled bars) for 6 h. The activity of cotransfected Renilla luciferase was used for normalization. The data are presented as mean ± SD.

Fig. 7.

Inhibition of Lyp by I-C11 enhances T cell signaling. Jurkat T cells expressing either Lyp or Lyp/S35E were treated with 20 μm IC-11 followed by anti-CD3 stimulation. The effects of IC-11 on T cell signaling were evaluated with specific antibodies.