Thioredoxin-related protein 32 (TRP32) specifically reduces oxidized phosphatase of regenerating liver (PRL)

Abstract

PRL family constitutes a unique class of phosphatases associated with metastasis. The phosphatase activity of PRL has been reported to be important for promoting metastasis, and it is inactivated by reversible oxidation of its catalytic cysteine. Here, we show that TRP32 specifically reduces PRL. Reduction of oxidized PRL in cells is inhibited by 2,4-dinitro-1-chlorobenzene, an inhibitor of TRX reductase. In vitro assays for the reduction of PRL show that only TRP32 can potently reduce oxidized PRL, whereas other TRX-related proteins linked to TRX reductase show little or no reducing activity. Indeed, TRP32 knockdown significantly prolongs the H2O2-induced oxidation of PRL. Binding analyses reveal that the unique C-terminal domain of TRP32 is required and sufficient for its direct interaction with PRL. These results suggest that TRP32 maintains the reduced state of PRL and thus regulates the biological function of PRL.

FIGURE 1.

Sensitive oxidation of PRL by H₂O₂. A, COS7 cells were transfected with the indicated expression constructs and then treated with the indicated concentrations of H₂O₂ for 10 min. Cell lysates were subjected to nonreducing or conventional (reducing) SDS-PAGE followed by immunoblot analysis with anti-Myc antibody. Area between arrows, oxidized proteins containing intermolecular disulfide bonds; arrowhead, reduced proteins; asterisk, oxidized proteins containing intramolecular disulfide bonds. B, HEK293 cells were treated with the indicated concentrations of H₂O₂ for 10 min, and cell lysates were subjected to anti-PRL immunoblot analysis. C, HEK293 cells were transfected with the indicated constructs (without any tags) and then treated with 500 μm H₂O₂ for 10 min. Cell lysates were subjected to anti-PRL immunoblot analysis. D, COS7 cells were transfected with the indicated constructs and then treated with 500 μm H₂O₂ for 10 min. Cell lysates were subjected to anti-Myc immunoblot analysis. E, COS7 cells were transfected with Myc-PRL3 (upper panels) or Myc-PTEN (lower panels) and then treated with the indicated concentrations of H₂O₂ for 10 min. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. F, HEK293 cells were treated with the indicated concentrations of H₂O₂ for 10 min, and cell lysates were subjected to immunoblot analysis with the indicated antibodies.

FIGURE 2.

DNCB inhibits the reduction of oxidized PRL. A and B, COS7 cells expressing Myc-PRL3 (A) or HEK293 cells (B) were treated with 200 μm H₂O₂ for the indicated time intervals, and cell lysates were subjected to nonreducing or conventional (reducing) SDS-PAGE followed by immunoblot analysis with the indicated antibodies. Area between arrows, oxidized proteins containing intermolecular disulfide bonds; arrowhead, reduced proteins; asterisk, oxidized proteins containing intramolecular disulfide bonds. C and D, COS7 cells expressing Myc-PRL3 (C) or HEK293 cells (D) were treated with 50 μm DNCB for 30 min or 100 μm BSO for 16 h and then treated with 200 μm H₂O₂ for the indicated time intervals. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.

FIGURE 3.

TRP32 reduces oxidized PRL. A, purified His-PRL3 and various GST-cleaved proteins were subjected to SDS-PAGE and Coomassie Brilliant Blue R-250 staining (left). The GST-cleaved proteins were subjected to a reduction assay by using insulin as the substrate. The assay mixture lacking GST-cleaved proteins served as the control. Insulin reduction was monitored by changes in absorbance at 405 nm (right). OD, optical density. B–D, His-PRL3 was incubated in the assay mixture containing the indicated GST-cleaved proteins at a final concentration of 5 μm (B and C) or 0.1–5 μm (D) and then subjected to nonreducing or conventional (reducing) SDS-PAGE followed by immunoblot analysis. The amounts of reduced PRL3 were quantified and are indicated as a percentage of those of total PRL3 (D). Area between arrows, oxidized proteins containing intermolecular disulfide bonds; arrowhead, reduced proteins; asterisk, oxidized proteins containing intramolecular disulfide bonds. E, lysates of HEK293 cells (10 μg) and the indicated amounts of recombinant GST-cleaved TRX or TRP32 proteins of human origin were subjected to immunoblot analysis with the indicated antibodies (right). The purity of the recombinant proteins is also shown (left).

FIGURE 4.

Prolonged oxidation of PRL by TRP32 knockdown. U2OS cells were transfected with siRNA against TRP32 (siTRP32) and then treated with 200 μm H₂O₂ for the indicated time intervals. Cell lysates were subjected to nonreducing or conventional (reducing) SDS-PAGE followed by immunoblot analysis with the indicated antibodies (upper panels). The amounts of reduced PRL3 were quantified and indicated as a percentage of those of total PRL3 (lower panel). Arrowhead, reduced proteins; asterisk, oxidized proteins containing intramolecular disulfide bonds. siControl, siRNA negative control.

FIGURE 5.

PRL specifically binds to TRP32. A, various GST fusion proteins immobilized on beads were mixed with His-PRL3 proteins. The bound proteins were detected by Coomassie Brilliant Blue R-250 staining. B, schematic illustration of TRP32. The TRX domain and DUF1000 domain are indicated with gray color. C, GST fusion WT or deletion mutants (N and C) of TRP32 were subjected to pulldown assays with His-PRL3 proteins as in A. D, His-PRL3 was incubated in the assay mixture containing the indicated GST-cleaved proteins at a final concentration of 5 μm and then subjected to nonreducing or conventional (reducing) SDS-PAGE followed by immunoblot analysis. The amounts of reduced PRL3 were quantified and are indicated as a percentage of those of total PRL3.