Identification of significant pathways regulated by PRL-3.

<p>(A) Significant KEGG pathways identified by DAVID when PRL-3 was overexpressed in HCT116 cells. The left-side ordinate values are -lg (Benjamini-Hochberg p-value), while the right-side ordinate values are the ratios of phosphorylated proteins in the total significantly regulated phosphoproteins. (B) Heatmaps of several pathways to show the phosphoproteins regulated by PRL-3, including ErbB signaling pathway, Focal adhesion, MAPK signaling pathway and Chemokine signaling pathway. The red color represents the phosphorylation of the protein was increased in PRL-3 overexpressing cells, while the blue color represents the phosphorylation of the protein was decreased in PRL-3 overexpressing cells.</p

IL-1α participates in PRL-3 induced cell migration.

<p>(A) and (B) IL-1α was regulated by PRL-3 through NF-κB and Jak2-STAT3 signaling pathways. (A) The treatment of HCT116 cells with 10 μM BAY11-7082 for 24 hours reduced PRL-3-increased p-p65 (S536), and the treatment of HCT116 cells with 2 μM AG490 for 24 hours reduced PRL-3-increased p-STAT3 (Y705) while the treatment of HCT116 cells with 100 ng/mL IL-6 for 24 hours increased p-p65 (S536) and p-STAT3 (Y705). (B) The treatment of HCT116 cells with 10 μM BAY11-7082 or 2 μM AG490 for 24 hours reduced PRL-3-increased IL-1α, and the treatment of HCT116 cells with 100 ng/mL IL-6 for 24 hours enhanced PRL-3-induced IL-1α secretion. (C) The treatment of HCT116 cells with 5 ng/mL IL-1RN for 48 hours decreased PRL-3-induced HCT116 cell migration. (D) Inhibition of IL-1α by siRNA interference decreased PRL-3-induced HCT116 cell migration. The values are the mean and standard deviation, * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3.</p

Antibody Array Revealed PRL-3 Affects Protein Phosphorylation and Cytokine Secretion

<div><p>Phosphatase of regenerating liver 3 (PRL-3) promotes cancer metastasis and progression via increasing cell motility and invasiveness, however the mechanism is still not fully understood. Previous reports showed that PRL-3 increases the phosphorylation of many important proteins and suspected that PRL-3-enhanced protein phosphorylation may be due to its regulation on cytokines. To investigate PRL-3’s impact on protein phosphorylation and cytokine secretion, we performed antibody arrays against protein phosphorylation and cytokines separately. The data showed that PRL-3 could enhance tyrosine phosphorylation and serine/threonine phosphorylation of diverse signaling proteins. Meanwhile, PRL-3 could affect the secretion of a subset of cytokines. Furthermore, we discovered the PRL-3-increased IL-1α secretion was regulated by NF-κB and Jak2-Stat3 pathways and inhibiting IL-1α could reduce PRL-3-enhanced cell migration. Therefore, our result indicated that PRL-3 promotes protein phosphorylation by acting as an ‘activator kinase’ and consequently regulates cytokine secretion.</p></div

Significantly regulated cytokines by PRL-3.

<p>(A) Significantly regulated cytokines were all increased in PRL-3 overexpressing HCT116 cells. (B) The mRNA levels of several regulated cytokines were increased in PRL-3 overexpressing HCT116 and LoVo cells. (C) Relative levels of GDF-15, IL-1α and NPY in the supernatants of PRL-3 overexpressing HCT116 and LoVo cells and corresponding control cells. The values are the mean and standard deviation, *p <0.05, **p <0.01; n = 3.</p

The top 20 phosphoproteins experiencing changes in phosphorylation in the PRL-3 overexpressing cells.

<p>The top 20 phosphoproteins experiencing changes in phosphorylation in the PRL-3 overexpressing cells.</p

Overexpression of PRL-3 widely increased protein phosphorylation.

<p>(A) Confirmation of PRL-3 overexpression in HCT116 and LoVo cells. (B) Several phosphoproteins increased by PRL-3 in HCT116 and LoVo cells. (C) PRL-3’s wide impact on the whole protein phosphorylation, including tyrosine phosphorylation and serine/threonine phosphorylation in HCT116 and LoVo cells. (D) PRL-3’s impacts on phosphorylations of EGFR, p65, and ERK in eight colorectal cancer tissues.</p