Disease association, structure representation, and sequence alignment of 3 Lyp variants S201F, R263Q and R266W.

<p>(A) Association analysis of S201F, R263Q and R266W in T1D patients and association of R263Q with RA, TB, UC and SLE patients. (B) Structural representation of Lyp variants S201F, R263Q and R266W. The figure was prepared with Pymol using the coordinates from PDB code 3OLR. P-loop, WPD-loop and Q-loop are depicted in red, blue and green, respectively. (C) Structure model of S201F. The bulky Phe instead of hydrophilic Ser at position 201 may affect WPD loop movement. The figure was prepared with Pymol using the coordinates for PDB code 3OLR and 2QCJ. (D) Structure representation of R263. R263 faces to Lyp specific insert, and the guanine group of R263 interacts with the side chain of Q34 through hydrogen bonds. The figure was prepared with Pymol using the coordinates for PDB code 3OLR. (E) Structure model of R266. R266 resides in the α5 helix adjacent to Q loop. The side chain of R266 forms hydrogen bonds with the main-chain amides of L272 and V273, and the side chain of S35. R266 also makes hydrophobic interaction with L31 and V273. The figure was prepared with Pymol using the coordinates for PDB code 3OLR. (F) Sequence alignment of α2′-Lyp-specific insert, WPD-loop-α3, and α5-Q-loop with corresponding regions from PTPN12, PTPN18 and PTPN1. S201, R263 and R266 are highlighted in red. Interacting residues are represented in green. The unconservative residues are shown in blue.</p

Dephosphorylation of pSrc416 by Lyp wild type and mutants.

<p>(A) Time dependence of pSrc416 dephosphorylation by Lyp wild type, S201F, R263Q and R266W. The phosphorylation level of the Src protein at 416 position was monitoted by Western blotting. (B) The dephosphorylation levels were quantified with Image J software, and plotted against the different time. (C) Statistical analysis of Lyp catalyzed pSrc416 dephosphorylation at 10 min. All experiments were repeated in triplicate. *P<0.05; ** P<0.01.</p

Lyp-W266 variant loses its function in T cell activation and its pH profile of enzyme activity.

<p>(A) Comparison of Lyp wild type and W266 variant on TCR-induced AP-1 transcriptional activity. (B) pH-Kcat profile of the pNPP hydrolysis catalyzed by Lyp wild type and R266W mutant.</p

Kinetic analysis of the phosphatase activity of Lyp catalytic domain wild type and S201F, R263Q and R266W mutants toward pNPP and phosphor-peptide.

<p>(A) pNPP hydrolysis with Lyp catalytic domain domain and 3 mutants. Data were fitted to the Michaelis-Menten equation. (B–C) Kinetics parameters for the wild-type and the mutants of Lyp toward pNPP. All experiments were repeated four times. * represents P<0.05; ** represents P<0.01. (D) IC50 values of 5- sulfosalicylic acid toward Lyp wild type and variants. (E) Lyp catalyzed phosphate release of a 9 amino acid phosphor peptide derived from Lck 394 phosphorylation site (EDNEpYTARE). Data were fitted to the Michaelis-Menten equation. (F–G) Kinetics parameters for the wild-type and the mutants of Lyp toward EDNEpYTARE. Data showed the mean of three independent experiments. *P<0.05; ** P<0.01.</p

Effect of Lyp and 3 variants on T cell signaling.

<p>(A) Jurkat T cells were transfected with the His/Myc tagged full-length Lyp plasmids or mutated variants, and stimulated with medium or anti-CD3 (OKT3) antibody for 5 min. The level of full-length Lyp protein was detected by anti-Myc antibody. The phosphorylation of Lck394 and phosphorylation of ERK1/2 were monitored by immunoblotting with specific antibodies. Actin was detected as loading control. (B–C) Statistical analysis of Lck394 phosphorylation and ERK1/2 phosphorylation in Jurkat T cells overexpressed with Lyp wild type and variants. ** represents P<0.01 compared to the signals in control T cells stimulated with anti-CD3. ## represents P<0.01 compared to the stimulated signals in T cells overexpressed with wild-type Lyp.</p