PTPN2 associates with Src and modulates phosphorylation pattern of Src.

<p>(A) Control (□) or PTPN2-WT (■) cells were pre-incubated with Src inhibitor (SU6656) (10 μM) or vehicle for 2 hr and then stimulated with LPS for 4 hr. IL-1β, IL-6 and TNF-α mRNA expression levels were examined by qRT-PCR and normalized with cyclophilin. Data represent the means ± S.D. of three independent experiments. **, <i>p</i>< 0.01 (Student <i>t</i> test). (B) 293T cells were transiently cotransfected with HA-Src and control, Myc-PTPN2-WT or Myc-PTPN2-MT. After 36 hr post transfection, equal amount of protein lysates was immunoprecipitated with an anti-Myc antibody, followed by immunoblotting with anti-HA (Src) and anti-Myc antibodies. Total cell lysates were immunoblotted with anti-HA and anti-Myc antibodies. (C) Recombinant GST or GST-PTPN2 was incubated with equal amounts of His–Src fusion protein. After pulldown with GSH-beads, the bound proteins were immunoblotted with an anti-His antibody. GST and GST-PTPN2 were detected by immunoblotting with an anti-GST antibody. (D) HA-Src and control, Myc-PTPN2-WT or Myc-PTPN2-MT were transiently cotransfected into the 293T cells. After 36 hr post transfection, protein lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with specific antibodies against phosphor-Src at different residues. (E) To confirm the direct regulation of Src by PTPN2, an <i>in vitro</i> phosphorylation assay was performed. Recombinant Src and PTPN2 were incubated for 1 hr in the presence of ATP, and the phosphorylated status of Src was checked with phosphor-specific antibodies. (F-G) PTPN2-knockdowned cells (F) or overexpressed cells (G) were stimulated with LPS (1 μg/ml) for several times. Protein lysates were analyzed with related specific antibodies to confirm the phosphorylation pattern of Src.</p

Overexpression of PTPN2 enhances LPS-stimulated cytokine genes.

<p>(A) The expression of PTPN2-WT and PTPN2-MT (substrate trapping mutant) was detected with anti-Myc antibody and control cell was confirmed by GFP expression. (B-C) GFP, PTPN2-WT and PTPN2-MT cells were treated with LPS (1 μg/ml) for 4 hr to check the mRNA expression or for 24 hr to measure the cytokine secretion. IL-1β, IL-6 and TNF-α mRNA expression levels were examined by qRT-PCR (B) and secreted cytokines were measured with ELISA (C). Data represent the means ± S.D. of triplicate experiments. *, <i>p</i> < 0.05 and **, <i>p</i> < 0.01 (Student <i>t</i> test).</p

Protein Tyrosine Phosphatase N2 Is a Positive Regulator of Lipopolysaccharide Signaling in Raw264.7 Cell through Derepression of Src Tyrosine Kinase

<div><p>T cell protein tyrosine phosphatase N2 (PTPN2) is a phosphotyrosine-specific nonreceptor phosphatase and is ubiquitously expressed in tissues. Although PTPN2 functions as an important regulator in different signaling pathways, it is still unclear what is specific target protein of PTPN2 and how is regulated in lipopolysaccharide (LPS)-induced inflammatory signaling pathway. Here, we found that PTPN2 deficiency downregulated the expression of LPS-mediated pro-inflammtory cytokine genes. Conversely, overexpression of PTPN2 in Raw264.7 cells enhanced the expression and secretion of those cytokines. The activation of MAPK and NF-κB signaling pathways by LPS was reduced in PTPN2-knockdowned cells and ectopic expression of PTPN2 reversed these effects. Furthermore, we found that PTNP2 directly interacted with Src and removed the inhibitory Tyr527 phosphorylation of Src to enhance the activatory phosphorylation of Tyr416 residue. These results suggested that PTPN2 is a positive regulator of LPS-induced inflammatory response by enhancing the activity of Src through targeting the inhibitory phosphor-tyrosine527 of Src.</p></div

PTPN2 deficiency downregulates LPS-induced pro-inflammatory signaling.

<p>(A) PTPN2 mRNA and protein expression level in PTPN2-knockdowned cells. (B) The heat map represents the pro-inflammatory cytokine profiling of PTPN2-knockdowned cells relative to parental control Raw264.7 cells after treatment with LPS (1 μg/ml) using cytokine array. (C) The mRNA expression of IL-1β, IL-6 and TNF-α in control and PTPN2-knockdowned cells was examined by quantitative real-time PCR. (D) Secreted cytokines were measured with ELISA after 6 or 24 hr treatment of LPS. Data represent the means ± S.D. of three independent experiments. *, <i>p</i> < 0.05 and **, <i>p</i> < 0.01 (Student <i>t</i> test).</p

PTPN2 promotes LPS signaling through activation of MAPK and NF-κB.

<p>(A-B) Scramble and PTPN2-knockdowned cells (A) or control, PTPN2-WT and MT cells (B) were stimulated with LPS (1 μg/ml) for various durations. Immunoblotting was performed with specific antibodies to detect the phosphorylation of MAPK and NF-κB. The β-actin was used as the internal control. (C) MAPK inhibitors (U or SB, 10 μM) and NF-κB inhibitor (BMS, 10 μM) were pre-incubated for 2 hr, followed by the treatment of LPS (1 μg/ml) for 4 hr. IL-1β, IL-6 and TNF-α mRNA expression levels were determined by qRT-PCR. Data represent the means ± S.D. of three independent experiments. *, p < 0.05 and **, p < 0.01 (Student <i>t</i> test).</p