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Figure 1 | Cell Division

Figure 1

From: The DNA repair component Metnase regulates Chk1 stability

Figure 1

Metnase, itself a target of Chk1 phosphorylation, stabilizes Chk1 half-life. A, B- Western analysis showing Metnase stabilizes Chk1 after cycloheximide, and this is largely dependent on Chk1 phosphorylation of S495, as the S495A Metnase species, which cannot be phosphorylated, does not extend Chk1 protein half-life nearly as much. Total Chk1 levels were normalized to Chk1 present at time 0, before cycloheximide exposure. C,D- Western analysis of p296 Chk1 half-life in cells with or without Metnase (pCAPP). The presence of wt Metnase enhances the stability of phosphorylated Chk1. Phosphorylated Chk1 levels were normalized to time 0, before cycloheximide. E,F- Metnase promotes Chk1, and ATR-mediated p317 Chk1 induction after exposure to the replication stressor hydroxyurea (HU). The quantitation in panel F is the ratio of Chk1 or pChk1 in siRNA-Metnase cells divided by WT Metnase-expressing cells. This implies that the increase in phosphorylated Chk1 is mainly due to the increase in total Chk1 protein stability, consistent with D. G- Metnase reduces Chk1 interaction with DDB1, and reduces Chk1 ubiquitination. 293 T-pCAPP (which do not express Metnase due to T antigen) and 293 T-MET were transiently transfected with FLAG-Chk1, and treated with HU, and then FLAG was immunoprecipitated. This immunoprecipitate was probed for DDB1 and ubiquitin. Metnase over-expression reduces association of Chk1 with DDB1. HU induces Chk1 ubiquitination, but this is largely blocked by wt Metnase over-expression. A longer exposure of the ubiquitin autoradiogram is also presented. H- ATR phosphorylates Chk1 S317 upon replication stress. Phosphorylated Chk1 phosphorylates S495 of Metnase. Phosphorylated Metnase decreases the interaction of DDB1 with Chk1, reducing Chk1 targeting to the Cul4a E3 ubiquitin ligase, increasing Chk1 stability.

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