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. 2013 Mar 22;288(12):8762-8771.
doi: 10.1074/jbc.M112.414524. Epub 2013 Feb 5.

"V体育ios版" The tumor suppressor Mst1 promotes changes in the cellular redox state by phosphorylation and inactivation of peroxiredoxin-1 protein

Sonali Jalan Rawat  1 , Caretha L Creasy  2 , Jeffrey R Peterson  2 , Jonathan Chernoff  3
Affiliations

"VSports最新版本" The tumor suppressor Mst1 promotes changes in the cellular redox state by phosphorylation and inactivation of peroxiredoxin-1 protein

Sonali Jalan Rawat et al. J Biol Chem. .

Abstract (V体育安卓版)

The serine/threonine protein kinases Mst1 and Mst2 can be activated by cellular stressors including hydrogen peroxide. Using two independent protein interaction screens, we show that these kinases associate, in an oxidation-dependent manner, with Prdx1, an enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. Mst1 inactivates Prdx1 by phosphorylating it at Thr-90 and Thr-183, leading to accumulation of hydrogen peroxide in cells. These results suggest that hydrogen peroxide-stimulated Mst1 activates a positive feedback loop to sustain an oxidizing cellular state. VSports手机版.

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Figures

FIGURE 1.
FIGURE 1.
Both Mst1 and Mst2 undergo stress-inducible interaction with Prdx1. A, HEK-293 cells were transfected with the indicated combinations of plasmids. Cells were stimulated with H2O2 for 30 min. Cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The resulting immunoprecipitated proteins were immunoblotted (IB) with anti-Myc antibodies. B, HEK-293 cells were transfected either with EV or with SBP-Prdx1. Cells were stimulated with H2O2 for 30 min. Cell lysates were immunoprecipitated with streptavidin beads (IP-strep beads). The immunoprecipitated proteins were immunoblotted with anti-Mst1 antibodies. C, HEK-293 cells were transfected either with EV or with SBP-Mst1. Cell lysates were immunoprecipitated with streptavidin beads. The immunoprecipitated proteins were immunoblotted with anti-Prdx1 antibodies. Endog-Prdx1, endogenous Prdx1. D, time-dependent SH-Mst1 expression. Cells were treated with 500 ng/ml tetracycline for different time points as indicated. Expression of SH-Mst1 and endogenous Mst1 (Endo Mst1) was monitored by immunoblotting using anti-Mst1 antibodies. E, Mst1-Flp-In 293 cells were treated with 500 ng/ml tetracycline (Tet) for 4 h followed by treatment with H2O2 for 30 min. The cell lysates were immunoprecipitated with streptavidin beads, and the resulting immunoprecipitated proteins were probed with anti-Prdx1 antibodies. F and G, HEK-293 cells were transfected with the indicated combination of plasmids. Cells were stimulated with H2O2. The cell lysates were immunoprecipitated with anti-HA antibodies and probed with Mst1 antibodies. C52/173S, C52S/C173S. H, HEK-293 cells were transfected with the indicated combinations of plasmids. Cells were stimulated with H2O2 for 30 min. Cell lysates were immunoprecipitated with anti-HA antibodies. The resulting immunoprecipitated proteins were immunoblotted with anti-Mst2 antibodies.
FIGURE 2.
FIGURE 2.
Prdx1 is not required for Mst1 activation by H2O2. HEK-293 cells were transfected with control or Prdx1-specific siRNA pools. Seventy-two h after transfection, the cells were exposed to H2O2 for 30 min. The cells were then lysed, and Mst1 was immunoprecipitated with anti-Mst1 antibodies. Cell lysates were analyzed for expression of Prdx1 and Mst1, and the Mst1 immunoprecipitates were analyzed for kinase activity using myelin basic protein (MBP) as substrate.
FIGURE 3.
FIGURE 3.
Prdx1 is phosphorylated by Mst1 predominantly at Thr-18, Thr-90, and Thr-183. A, recombinant Prdx1 (2 μg) was subjected to in vitro kinase assay with Mst1 in the presence of [γ-32P]ATP and increasing concentration of H2O2. Phosphorylated Prdx1 and phospho-Mst1 were visualized by autoradiography. B, mass spectrometry results showing all the phosphorylation sites identified for Prdx1 and the corresponding phosphopeptides. C, sequence alignment of Prdx1 from different species. D–F, both single and combination alanine mutants of Prdx1 were incubated with Mst1 in the presence of [γ-32P]ATP at 30 °C for 30 min. Proteins were separated by SDS-PAGE; phosphorylated Prdx1 was visualized by autoradiography, and total Prdx1 by Coomassie Blue stain.
FIGURE 4.
FIGURE 4.
Phosphorylation of Prdx1 by Mst1 results in its inactivation. A, following kinase assay, the phosphorylated and unphosphorylated proteins were separated by anion exchange as described under “Experimental Procedures.” p-Prdx1, phosphorylated Prdx1; IB, immunoblot; p-Thr, phosphothreonine. B, peroxidase activity of Prdx1 and phosphorylated Prdx1 was monitored by measuring absorbance at 340 nm for 200 s. C, Prdx1 activity was calculated from plot in B, and the results are presented as mean ± S.E. for three independent experiments.
FIGURE 5.
FIGURE 5.
Phosphorylation at Thr-90 and Thr-183 inactivates Prdx1. A and C, peroxidase activities of WT-Prdx1, T90A, T90D, T183A, and T183D mutants of Prdx1 were determined by measuring decrease in absorbance (Ab) of NADPH at 340 nm for 300 s. B and D, peroxidase activities of WT-Prdx1, T90A, T90D, T183A, and T183D mutants of Prdx1 were calculated from the graph in A and C. The results are represented as mean ± S.E. for three independent experiments.
FIGURE 6.
FIGURE 6.
Phosphorylation of Prdx1 at Thr-183 increases H2O2 levels and DNA damage in cells. A, Western blot showing expression of WT, T183A, and T183D Prdx1 in Prdx1−/−WT, Prdx1−/−T183A, and Prdx1−/−T183D MEFs, respectively. B, Prdx1−/−EV, Prdx1−/−WT, Prdx1−/−T183A, and Prdx1−/−T183D MEFs were stimulated with 5 ng/ml PDGF for 10 min and were stained with 10 μm carboxy-H2DCFDA for 30 min. DCFDA fluorescence was measured by flow cytometry. The curve represents the DCFDA fluorescence for 10,000 events. % of Max, percent of maximum. C, mean fluorescence values ± S.E. of triplicate samples calculated for Prdx1−/−EV, Prdx1−/−WT, Prdx1−/−T183A, and Prdx1−/−T183D MEFs. D, Prdx1−/−EV, Prdx1−/−WT, Prdx1−/−T183A, and Prdx1−/−T183D MEFs were treated with different concentrations of H2O2 for 2 h. DNA damage was measured by performing immunoblot using phospho-H2AX (pH2AX) (Ser-139) antibodies.
FIGURE 7.
FIGURE 7.
Proposed model for Mst1 regulation by H2O2. A, crystal structure of Prdx1 showing relative positions of Thr-183 and Glu-171. B, under oxidative stress conditions, Mst1 associates with Prdx1. Mst1 interaction with Prdx1 induces Prdx1 phosphorylation at several sites. Phosphorylation (indicated by P) at Thr-90 and Thr-183 leads to Prdx1 inactivation and hence increase in hydrogen peroxide level in cells. This increase in H2O2 may result in further activation of Mst1 by a feedback loop and induction of apoptosis by Mst1 under oxidative stress conditions.
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