Fig. 7.

ITCH ubiquitinates WWOX upon DNA damage. (A) HEK293 cells were transfected with HA-Ub, GST-WWOX, and FLAG-ITCH or FLAG-ITCHC830A plasmids. At 24 h, cells were mock or KU55933 treated overnight followed by NCS (200 ng/mL) treatment of 1 h. Cell lysates were blotted against the indicated antibodies. Pulled down complexes were blotted with anti-HA (Ub), anti-GST (WWOX), and anti-ITCH antibodies. (B) Control- and ITCH-depleted HEK293 cells were transfected with GST-WWOX and HA-Ub. At 24 h, cells were incubated with NCS (200 ng/mL) for an additional 1 h. Cell lysates and pulled down complexes were treated as in A. (C) Itch-KO and WT MEFs were treated with NCS (200 ng/mL) for the indicated times. Lysates were blotted using the indicated antibodies. (D) Hypothetical model of WWOX action in DDR. WWOX deficiency leads to an increased number of DNA strand breaks after DNA damage. After DNA damage, ATM positively enhances ITCH-mediated ubiquitination and translocation of WWOX into the nucleus. Nuclear WWOX physically interacts with ATM and mediates ATM monomerization and activation in a positive feed-forward loop manner. When WWOX is lost, ATM function is hampered.