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 Lypspecific 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.crystal structure ͉ enzyme regulation ͉ Lyp inhibitor ͉ phosphorylation

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

“…1). Previous studies showed that Lyp preferentially dephosphorylates Lck at Tyr(P) 397 in the activation loop over the autoinhibitory Tyr(P) 505 at its C terminus (11,22). Consistent with this finding, the k cat /K m value for the Lck Tyr 394 peptide (Ac-ENDEpYTARE-NH 2 ) is 10.3-fold higher than that of the Tyr 505 peptide (Ac-TEPQpYQPGE-NH 2 ) (Table 1).…”

“…Crystallization, Data Collection, and Structure Determination-The N-terminal His 6 -tagged catalytically inactive Lyp/C227S (residues 1-294) mutant was crystallized with the phosphopeptides under conditions similar to those reported previously (22). The co-crystals of Lyp/C227S with the consensus or the SKAP-HOM peptide appeared after 3 days and grew to 0.1 ϫ 0.2 ϫ 0.2 mm after 5 days.…”

“…Protein Expression and Purification-Expression and purification of the His-tagged Lyp catalytic domain (residues 1-294) were performed as described previously (22). For substrate trapping, the Lyp catalytic domain was subcloned into the pGEX-2T vector, and the GST-tagged Lyp protein was expressed in BL21 Escherichia coli.…”

“…Phosphatase Assay-Initial rate measurements for the Lypcatalyzed p-nitrophenyl phosphate hydrolysis were carried out as described (22,23). The Lyp-catalyzed hydrolysis of Tyr(P)-containing peptides was continuously monitored at 305 nm for the increase in tyrosine fluorescence with excitation at 280 nm (18 Peptide Synthesis and Characterization-The synthesis and characterization of the inverse alanine phosphopeptide library were described previously (18).…”

Protein Tyrosine Phosphatases and Type 1 Diabetes: Genetic and Functional Implications of PTPN2 and PTPN22

“…Yu et al have demonstrated that the phosphatase function of LYP is regulated by phosphorylation at Ser-35. The phosphorylation at this residue alters the conformational state of LYP and impairs its phosphatase activity resulting in an augmentation of TCR signal transduction [13]. The role of LYP as a regulator of TCR signaling is also bolstered by the development of a salicylic acid-based LYP inhibitor which binds to the catalytic site and enhances TCR signaling [13].…”